Radiance MP

Radiance2000 MP Operating Manual
Radiance Operating Manual
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Table of Contents Preface and Warnings ................................................................................................. 6 LaserSharp2000 Software - limited use licence conditions....................................... 6 Software copyright................................................................................................... 6 Software use ........................................................................................................... 6 Unauthorized use of software .................................................................................. 6 Software support limitations..................................................................................... 6 Other products referred to in this manual................................................................. 6 Manual part number ................................................................................................ 6 Laser Safety ............................................................................................................ 7 1.8.1 Laser warnings ................................................................................................. 7 1.8.2 Laser cautions .................................................................................................. 7 1.8.3 Laser safety labels............................................................................................ 8 1.8.3.1 Identification and Certification Labels ........................................................... 8 1.8.3.2 Warning Logotype Label............................................................................... 9 1.8.3.3 Aperture Label............................................................................................ 10 1.8.3.4 Labels for Non-interlocked Housing ............................................................ 10 1.8.4 Microscope Interface Specification ................................................................. 11 1.9 Electrical safety..................................................................................................... 12 1.9.1 Electrical warnings.......................................................................................... 12 1.9.2 Electrical cautions .......................................................................................... 12 1.10 System Cooling ..................................................................................................... 12 1.10.1 Cooling warnings ............................................................................................ 12 1.11 System Management............................................................................................. 12 1.12 Comments............................................................................................................. 12 1.13 Symbols and conventions...................................................................................... 12 2. The system at a glance.............................................................................................. 14 2.1.1 Scan head ...................................................................................................... 15 2.1.2 Instrument Control Unit................................................................................... 16 2.1.3 Beam Conditioning unit .................................................................................. 17 2.1.4 Direct detectors .............................................................................................. 18 2.1.5 Computer and software .................................................................................. 18 2.1.6 Z-drive............................................................................................................ 18 2.1.7 Transmission detector .................................................................................... 18 3. Getting started........................................................................................................... 19 3.1 Switching on ancilliary equipment.......................................................................... 19 3.2 Switching on the femtosecond pulsed laser ........................................................... 19 3.3 Switching on the system ........................................................................................ 19 3.4 Starting the LaserSharp2000 software ................................................................... 23 3.5 Switching off the system........................................................................................ 23 3.6 A brief introduction to the software ........................................................................ 24 4. Factors Which Effect Optical Performance................................................................ 30 4.1 Objective lenses.................................................................................................... 30 4.1.1 Care and maintenance of objective lenses ..................................................... 33 4.2 Confocal aperture size........................................................................................... 34 4.3 Telecentric adjustment .......................................................................................... 35 4.4 Visible laser considerations ................................................................................... 36 4.5 Femtosecond pulsed laser considerations ............................................................. 36 4.6 Alignment of the Beam Conditioning Unit .............................................................. 37 4.7 Filters .................................................................................................................... 39 4.7.1 Filter configuration For Radiance2000 MP AG-2............................................. 39 4.7.2 Filter configuration For Radiance2000 MP K-2 ............................................... 40 4.7.3 Filter configuration For Radiance2000 MP AGR-3 .......................................... 41 4.7.4 Filter configuration For Radiance2000 MP KR-3............................................. 42 4.7.5 Filter configuration For Radiance2000 MP AGR-3 (Q) .................................... 43 4.7.6 Inserting custom emission filters..................................................................... 44 4.7.6.1 Physical insertion of filters.......................................................................... 44 4.7.6.2 Updating the firmware ................................................................................ 46 4.8 Direct detectors ..................................................................................................... 47 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8
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1.
4.8.1 Introduction .................................................................................................... 47 Direct detectors coupled to an Olympus BX50WI upright microscope .................... 48 Direct detectors coupled to a Nikon TE300 inverted microscope............................ 48 4.8.2 Optical configuration....................................................................................... 49 4.8.3 Filter configurations ........................................................................................ 50 4.8.3.1 Bi-Alkali/Bi-Alkali Configuration (BB) .......................................................... 50 4.8.3.2 Bi-Alkali/Multi-Alkali Configuration (BM) ..................................................... 50 4.8.4 Selecting filter cubes ...................................................................................... 51 4.8.5 Changing filter cubes...................................................................................... 51 4.8.6 Using the Direct Detectors .............................................................................. 52 4.8.6.1 Nikon E600FN............................................................................................ 52 4.8.6.2 Olympus BX50WI....................................................................................... 55 4.8.6.3 Nikon TE300 .............................................................................................. 56 4.9 Fluorophores ......................................................................................................... 57 4.9.1 Fluorophores excited by 1-photon excitation................................................... 57 4.9.2 Fluorophores excited by 2-photon excitation................................................... 61 4.10 Dynamic range ...................................................................................................... 62 4.11 Monitor adjustment................................................................................................ 63 4.12 Sampling resolution............................................................................................... 64 4.13 Signal strength and noise ...................................................................................... 65 5. Sample preparation suggestions for confocal and muti-photon imaging ..................... 66 5.1 Fixatives for biological tissue................................................................................. 66 5.2 Bleaching and anti-fade agents ............................................................................. 66 5.3 Mounting media..................................................................................................... 66 5.4 Autofluorescence................................................................................................... 67 6. Software reference .................................................................................................... 68 6.1 Menu bar............................................................................................................... 68 6.1.1 File Menu ....................................................................................................... 68 6.1.1.1 6.1.1.2 6.1.1.3 6.1.1.4 Create a New Experiment Open an existing Experiment ................................................................ 68 ........................................................... 68
.................................................. 69 Close a currently Open Experiment Save an Experiment................................................................................... 69
6.1.1.5 Print an image .................................................................................. 69 6.1.1.6 Log Out ...................................................................................................... 69 6.1.1.7 A list of recently opened Experiments......................................................... 69 6.1.1.8 Exit the application..................................................................................... 69 6.1.2 Edit menu....................................................................................................... 69 6.1.3 Methods menu................................................................................................ 69 6.1.3.1 Save Method ..................................................................................... 70 6.1.3.2 Edit... (Method) .......................................................................................... 70 6.1.4 Method Wizard ............................................................................................... 71 6.1.5 Acquire menu ................................................................................................. 76 6.1.5.1 6.1.5.2 6.1.5.3 6.1.5.4 6.1.5.5 6.1.5.6 Live Scan ....................................................................................... 76 ........................................................................ 76 ................................................................... 77 ........................................................ 78
Sequential live scan XY (Z-Series) collection
X-Z (vertical section) collection
X-T (line scan) collection .................................................................. 79 Pixel Size (depth)....................................................................................... 80

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6.1.6 Image menu ................................................................................................... 80 6.1.6.1 Adjust contrast ........................................................................................... 81 6.1.7 Tools menu .................................................................................................... 82 6.1.7.1 System Setup............................................................................................. 82 6.1.7.2 User set up................................................................................................. 84 6.1.7.3 Objective lens set up.................................................................................. 85 6.1.8 Script Menu .................................................................................................... 87 6.2 Image display window............................................................................................ 88 6.3 System control panels ........................................................................................... 90 6.3.1 Microscope control ......................................................................................... 90 6.3.2 Channels control............................................................................................. 94 6.3.3 Focus motor control........................................................................................ 95 6.3.4 Optics panel ................................................................................................... 97 6.3.5 Mixer control panel ......................................................................................... 98 6.4 Image operators .................................................................................................. 100 6.4.1 Arithmetic..................................................................................................... 101 6.4.2 Histogram..................................................................................................... 102 6.4.3 Line Profile ................................................................................................... 103 6.4.4 Merge........................................................................................................... 104 6.4.5 Projection ..................................................................................................... 105 6.4.5.1 Single view............................................................................................... 105 6.4.5.2 Multiple Views .......................................................................................... 106 6.4.5.3 Projection method (type) .......................................................................... 107 6.4.5.4 Source data.............................................................................................. 108 6.4.5.5 Two Pass Projections ............................................................................... 109 6.4.6 Seed Fill....................................................................................................... 110 6.4.7 Importing and exporting files ........................................................................ 111 7. Technical details...................................................................................................... 112 7.1 Filter specifications.............................................................................................. 112 7.1.1 Radiance2000 MP Dichroic filters................................................................. 112 7.1.2 Radiance2000 MP Emission filters ............................................................... 115 7.1.3 Radiance2000 MP Blocking filters ................................................................ 121 7.2 System calibration............................................................................................... 122 7.3 Switching the scanhead between microscopes .................................................... 123
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1. PREFACE AND WARNINGS

Bio-Rad Microscopy Division April 1999 Radiance2000 MP Operating manual, Issue 2 Copyright 1999 Bio-Rad Microscience Ltd All rights reserved. Customers may only copy this manual for their own use.
1.1 LaserSharp2000 Software - limited use licence conditions

The software described in this manual is supplied under a limited use licence agreement.
1.2 Software copyright

The LaserSharp2000 software is the sole and exclusive property of Bio-Rad. The customer undertakes not to copy any part of the software without written permission from Bio-Rad except for a back-up copy for security purposes.
1.3 Software use

The software may only be used on the machine for which it was originally supplied.
1.4 Unauthorized use of software

The customer agrees to protect the software from unauthorized use.
1.5 Software support limitations

The LaserSharp2000 software will only be supported when run on a Bio-Rad recommended computer (at the time of purchase).
1.6 Other products referred to in this manual

Windows NT and Windows 2000 are registered trademarks of Microsoft Corporation. The Trademarks of all fluorochromes and probes are recognised.
1.7 Manual part number

Further copies of this manual may be obtained by quoting Manual Part Number 9MRC60UM04 issue 2.
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1.8 Laser Safety

Lasers are potentially hazardous to the eyes and skin and you should, for your own safety and that of your co-workers, read and comply with the following warnings and cautions.
1.8.1 LASER WARNINGS

WARNING: VISIBLE AND INVISIBLE LASER RADIATION IS EMITTED FROM THE MICROSCOPE OBJECTIVE. AVOID EXPOSURE TO DIRECT OR SPECULARLY REFLECTED RADIATION.
WARNING:
ENSURE THAT EACH POSITION OF THE MICROSCOPE TURRET CONTAINS AN OBJECTIVE LENS, AN ALIGNMENT PRISM OR A BLANKING COVER. NO PORTS MAY BE LEFT OPEN.
During operation it is possible to have access to laser radiation at the objective which can pose a skin hazard. This is only true when the system is scanning.
WARNING:
AVOID EYE OR SKIN EXPOSURE WHEN PLACING OR REMOVING SAMPLES FROM THE MICROSCOPE STAGE.
The white BCU LED is illuminated when laser radiation is present.

WARNING: VIEWING OF THE MICROSCOPE OBJECTIVE WITH EXTERNAL OPTICAL INSTRUMENTS COULD BE HAZARDOUS WHEN THE SYSTEM IS SCANNING.
A special laser blocking filter can be obtained which fits over the optical instrument (for example, a microscope for observing patch clamps and micro-manipulators). Contact your local Bio-Rad office for details.
WARNING:
USE OF CONTROLS OR ADJUSTMENTS OR PERFORMANCE OF PROCEDURES OTHER THAN THOSE SPECIFIED HEREIN MAY RESULT IN HAZARDOUS VISIBLE AND/OR INVISIBLE RADIATION EXPOSURE.
WARNING: EYE PROTECTION MUST BE WORN WHEN THE SYSTEM IS SCANNING.
1.8.2 LASER CAUTIONS

Improper use of this equipment could damage the equipment. The system carries BRH recommended interlocks and warning labels. Ensure that all personnel read and follow these warnings. The Scan Head cover, Instrument Control Unit covers and BCU cover should only be removed by Bio-Rad authorised technicians and staff.
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1.8.3 LASER SAFETY LABELS 1.8.3.1 Identification and Certification Labels
Primary Identification and Certification Label (fitted at rear of Instrument Control Unit)
Secondary Identification and Certification Label (fitted inside front door of Instrument Control Unit)
AMERICAN LASER CORPORATION 1832 SOUTH 3850 WEST SALT LAKE CITY, UTAH 84104 USA
MODEL #: IS19BR12 SERIAL #: DATE MFD: VOLTAGE: CURRENT:
REV: Hz:
Identification label for Ion Secondary Laser

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FOR EXPORT OR OEM USE ONLY

THIS LASER PRODUCT IS DESIGNATED FOR USE SOLELY AS A COMPONENT (OR REPLACEMENT) IN AN ELECTRONIC PRODUCT AND THEREFORE DOES NOT COMPLY WITH THE APPROPRIATE REQUIREMENTS OR DHHS REGULATIONS No 21 CFR 1040.10 AND 1040.11
Identification label for Ion Secondary Laser
1.8.3.2 Warning Logotype Label
Warning Logotype Label (fitted to the BCU)
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1.8.3.3 Aperture Label
Aperture Label (fitted to the scan head focus ring)
1.8.3.4 Labels for Non-interlocked Housing
DANGER
VISIBLE AND INVISIBLE LASER RADIATION WHEN OPEN AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION
(Fitted to base of Scan Head adj. to cover screws and to ICU on l/hand side of electronics unit towards rear of ICU)fitted to Tube supports (2off), periscope mirror covers (2off), power meter pocket, BCU connector panel and laser connecting tubes (2off).)
(Fitted to the ICU on both sides of the 4-axis laser launcher, to the fibre interface at each detector module and also to the Scan Head chassis beneath the cover.)
DO NOT OPEN
For SERVICE ACCESS only by BIO-RAD trained personnel. In case of instrument malfunction contact BIO-RAD or an authorised representative.
(Fitted to the ICU on the l/hand side of the electronics unit and on the right-hand side of the secondary gas laser module.)
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1.8.4 MICROSCOPE INTERFACE SPECIFICATION

The Radiance scan heads attach to the photoport of a conventional optical microscope. The optical microscope must have been originally designed, or modified by Bio-Rad, so that it is impossible for laser light to be directed into the binocular viewer. Bio-Rad staff are instructed not to proceed with the installation if this requirement is not met.
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1.9 Electrical safety

Electricity is potentially hazardous and you should, for your own safety and that of your coworkers, read and comply with the following warnings and cautions.
1.9.1 ELECTRICAL WARNINGS

POTENTIALLY LETHAL VOLTAGES ARE PRESENT IN THE INSTRUMENT CONTROL UNIT WHEN EITHER OF THE MAINS SUPPLIES ARE PLUGGED IN.
1.9.2 ELECTRICAL CAUTIONS

Ensure that the electrical system earth (ground) is connected at all times during operation. Ensure that the mains supplies (outlets) are sufficiently rated and correctly fused.
1.10 System Cooling

Effective cooling (air flow) is essential for the correct operation of the equipment and to ensure the expected operating lifetime of lasers in particular.
1.10.1 COOLING WARNINGS

ENSURE THAT THE INSTRUMENT CONTROL UNIT AND THE LASER COOLING FANS ARE POSITIONED SO THAT ADEQUATE AIR FLOW IS ENSURED. WHEN TURNING OFF THE SYSTEM ENSURE THAT THE SHUT-DOWN PROCEDURE, PARTICULARLY WITH REGARD TO COOLING THE GAS LASER HEAD, IS RIGOROUSLY FOLLOWED.
1.11 System Management

It is recommended that one person is given the job of System Manager. The System Manager should be responsible for understanding how to use and align the system. The System Manager should control access to the system, and should also keep alignment tools safely out of the reach of unqualified personnel.
1.12 Comments
Written comments on the contents of this manual should be addressed to: Marketing Group Bio-Rad Microscience Ltd Bio-Rad House Maylands Avenue HEMEL HEMPSTEAD Herts HP2 7TD UK Fax: +44 (0)181 3282500 email: [email protected]
1.13 Symbols and conventions

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<Enter> indicates the identification of a specific key on the computer keyboard. File|Open... indicates a menu title on the menu bar followed by a menu item choice within that menu. Drop-down menus: When you click on a menu title a drop-down menu appears. Dropdowns: Click on the down arrow to select an option from a dropdown menu. This can also be an option in a drop-down menu, which produces a second menu. Pop-up menus: These context sensitive menus offer functionality relevant to the mouse position. They are accessed by right-clicking. Spin box : Click on the left or right arrow to move the slider left or right. Alternatively, you can enter a value directly in the box provided alongside the slider or grab the slider with the mouse.
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2. THE SYSTEM AT A GLANCE
Figure 1 Schematic view of system The system consists of six main components: - Femtosecond pulsed laser - Beam Conditioning Unit - Scan Head - Instument Control Unit - Direct detectors - Computer and software and two optional ancilliary components: - Z-drive - Transmission detector
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2.1.1 SCAN HEAD

The miniaturised scan head is permanently connected to the Instrument Control Unit via a single umbilical conduit (bottom left). This conduit carries electrical control signals to the motorised devices inside the scan head e.g. the dichroic filter wheels (B), the visible laser illumination input fibre (A) and the output signal fibres (C) which route the optical signal to the remote detector modules (Section 2.1.2). Additionally, the pulsed laser is directly coupled into the scan head (D) where it is then combined with the visible beam using a polarisation and chromatically dependent beam splitter (E). An optional Multi-photon Demagnification Lens (MDL) (F) can be switched into the emission beam path to enhance collection of scattered light when imaging in multi-photon mode. Figure 2 Schematic internal view of scan head
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Each collector module has its own switchable polarising analyser (F) and continuously variable confocal aperture (G).
Figure 3 Schematic view of collector module
2.1.2 INSTRUMENT CONTROL UNIT

The Instrument Control Unit houses either a combination of an Argon laser (A), green Helium Neon laser (B) and the red laser diode (C), or a 2-line Krypton/Argon laser (A) plus the red laser diode (C). All lasers are combined, passed through an AOTF (D) and then into the same single mode fibre as shown in the scan head diagram above (section 2.1.1)
Figure 4 Internal view of Instrument Control Unit
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On the opposite side of the unit an elctronics enclosure houses the remote detector modules (E) and system control electronics.
Figure 5 Electronics enclosure internal view
2.1.3 BEAM CONDITIONING UNIT
The Beam Conditioning Unit (BCU) contains all the components to control the infra-red beam: 1. 2. 3. 4. 5. 6. Beam steering (alignment) mirrors Beam collimator module (manual or motorised) Diagnostic pick-off beamsplitter 50:50 beam splitter Spectrum analyser Near-point alignment window
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7. 8. 9. 10. 11.
Far-point alignment window Pockels cell (alternatively Neutral density filters) Power meter pick-off beam splitter Power meter Safety shutter
2.1.4 DIRECT DETECTORS

Direct detectors (also known as external or non-descanned (NDS) detectors) for the Radiance2000 MP system are available in three basic models: Two channel manually operated Two channel motorised (software controlled) Four channel motorised (software controlled) These detectors are designed to collect scattered light (typically from deep within tissue) highly efficiently. They do this by picking-off the emitted fluorescence from immediately behind the objetive lens in the microscope and diverting it to one or more photo-multiplier tubes. Because the 2-photon excitation process inherently generates an optical section there is no need to pass the signal through a (confocal) detection aperture and hence the beam doesn t need to be descanned. This optically simple method of detection has the added advantage that shorter wavelength light 350 < ? > 700 can also be detected.
2.1.5 COMPUTER AND SOFTWARE

The system computer is a PC with a powerful processor, large hard disk, integrated networking hardware and a CD-ROM drive. Optional data storage devices such as recordable CD-ROM drives (CD-R) and JazTM or ZipTM drives from Iomega are available. The operating system is MS Windows NT4.0TM.
2.1.6 Z-DRIVE
Z-drives are available for most microscopes from the key microscope manufacturers; Zeiss, Nikon and Olympus. In all cases a 2000 half-step stepping motor is used to drive the microscopes focus mechanism. For the Nikon and Olympus microscopes the fine focus knob is driven directly giving a focusing resolution of 50nm (0.05 microns). For the Zeiss microscopes the stepper motor output is geared down to drive the coarse focus knob, also resulting in a resolution of 50nm.
2.1.7 TRANSMISSION DETECTOR

As above, transmission detectors are available for most microscopes from the key microscope manufacturers; Zeiss, Nikon and Olympus. The detector is situated between the body of the microscope and the trans-illumination lamp housing. In operation a motorised and computer controlled mirror is translated into the path of the transmitted scanned laser light and is focused into a multi-mode optical fibre. The optical signal is relayed to either a photodiode detector module or a PMT detector module housed in the Instrument Control Unit (Section 2.1.2)
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3. GETTING STARTED 3.1 Switching on ancilliary equipment

If you have a conventional epi-fluorescence lamp attached to the host microscope, switch this on before anything else. Refer to the manufacturers documentation regarding the switch-on sequence for devices such as external disk drives and printers.
3.2 Switching on the femtosecond pulsed laser

Refer to the laser manufacturer s documentation regarding turning on and off the femtosecond pulsed laser. It will generally be necessary to allow the pulsed laser several tens of minutes to warm up and reach stable operating conditions, so it will usually be advisable to turn this laser on before any other parts of the system. However, the pulsed laser can be turned on or off at any stage.
3.3 Switching on the system

1. Make sure that the mains supplies are connected to the ICU, the PC, the monitor and any other ancilliary equipment. WARNING AVOID ELECTRICAL SURGES FROM OTHER EQUIPMENT, EITHER ATTACHED TO THE SYSTEM OR IN THE VICINITY. IF POSSIBLE, USE A CLEAN ISOLATED MAINS SUPPLY TO AVOID SURGES AND EARTH LOOPS.
IF YOU HAVE A CONVENTIONAL EPI-FLUORESCENCE LAMP ATTACHED TO THE HOST MICROSCOPE, SWITCH THIS ON BEFORE ANYTHING ELSE.
2. Turn ON the laser mains breaker switch (A) on the rear of the Instrument Control Unit (Figure 6 Rear panel of ICU). If desired (and if this does not contravene local regulations), this mains breaker can be left turned ON, but it should be noted that mains voltage will be present in the Instrument Control Unit whenever the breaker is ON.
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Figure 6 Rear panel of ICU
3. Switch on the computer and monitor. The computer will boot directly into Windows NT and you will be presented with the desktop. An icon named LaserSharp2000 will be found on the desktop. Do not start the LaserSharp software until all steps in this section have been completed. It is important that the ICU is not powered on when the PC boots up because spurious serial commandsare sent out during boot up which can corrupt the ICU s firmware status. 4. Turn ON the mains switch (A) on the Instrument Control Unit switch panel (Figure 7 ICU switch panel). The adjacent green LED power indicator (B) will then be lit.
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A B F E G D H C
Figure 7 ICU switch panel
5. Check that the green LED power indicators on the scan head (A, Figure 8) and on the BCU (Figure 9 - A) are lit. 6. Turn the laser key switch (C) clockwise 90 . This switch enables all three visible lasers. 7. Press the button(s) (D, E & F) for the required visible laser(s). The Argon or Argon/Krypton laser has two associated LED indicators; the blue LED (G) indicates that the laser has been turned on and the orange LED (H) indicates that the external cooling fan is running. As soon as the button is pressed an intermittent beeping , lasting approximately thirty seconds, will be heard from the ICU. When the beepingstops the Argon laser will be emitting. To reach full stability the Argon laser should be allowed to run for thirty minutes, but in practice image acquisition can commence immediately. The green Helium Neon laser and the red laser diode each have a single LED indicator to show that the lasers have been turned on. There will be a brief delay of a few seconds (less than ten) before these lasers emit.
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Figure 8 Scan head LED indicators
Figure 9 BCU LED indicators
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3.4 Starting the LaserSharp2000 software

To start the software double click the LaserSharp2000 icon:
The software will initially prompt you to Login. The system will be supplied with the Default user s password set to 1 .
Once you have successfully logged in, the system will proceed with an initialisation phase during which firmware is downloadedto microprocessors distributed around the system and system communication protocols are checked.
3.5 Switching off the system

It is important to turn off the computer and the Instrument Control Unit in the prescribed manner. Failure to shut down the computer properly may cause disruption to the operating system. Failure to turn off the gas laser properly may cause its lifetime to be shortened. 1. Exit LaserSharp2000. Either use the single-click exit icon at the top right of the application or select Exit from the File menu. 2. Shut down Windows NT. Select Shut down from the Start button. 3. Turn the laser keyswitch anti-clockwise 90 . All lasers will immediately cease to emit. The Green Helium Neon laser and the red laser diode indicator LEDs will immediately extinguish. The blue gas laser indicator LED will immediately extinguish but the orange cooling fan runningindicator LED will remain lit until the gas laser tube has been sufficiently cooled. Once the orange indicator LED is extinguished the mains switch on the Instrument Control Unit switch panel can be switched OFF. 4. If desired, or if required by local regulations, the mains breaker on the rear of the Instrument Control Panel can be switched OFF. 5. Turn off the computer and monitor.
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3.6 A brief introduction to the software

The LaserSharp software is run under Windows NT4.0TM operating system. It is a unified application which controls acquisition and provides functionality for processing and analysis of images and data. The following text should be read by all users before attempting to use the system.
The user interface has been designed to be used with a minimum screen resolution of 1280 x 1024 pixels. The main menu bar and tool bars will initially appear in the top left hand corner as below:
But they can be repositioned in almost any way desired by clicking the double vertical bar at the left of each bar:
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On the raghthand side of the screen the instrument control panel will be displayed:
As can be seen, this panel controls the scanning, the detectors and the focus motor.
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One or more image display windows can be open at any one time:
Each image display window has its own display controls in the toolbar and an animation slider control below the image.
An experiment browser for the management of images is provided:
This will show all your open experiments. You will notice that within each experiment there can be one Raw Data set, there could be several imported data sets, and for any of those data sets there can be further data resulting from operations upon them. All of this data is arranged in a hierarchy with just one experiment name e.g. C:\Experiments\Neurons.
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Control of the optical system set up is achieved through the Optics/Filter setup configuration dialog:
To show this dialog press the Opticbutton in the Microscope control panel:
Mix button Optic button
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To display the Mixer control dialog press the Mixbutton.
The Mixer controls enable the additive combination of multiple detectors into one data channel. To show the Direct Detector setup dialog press the Direct Detectorsbutton in the Optics panel.
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Image processing and analysis functions are accessed by right clicking on the image to show a pop-up menu:
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4. FACTORS WHICH EFFECT OPTICAL PERFORMANCE 4.1 Objective lenses

There are five key properties to consider when choosing a lens for use on a confocal or multiphoton microscope:
Magnification The magnification of the objective lens determines the maximum field of view.
Numerical aperture The numerical aperture of an objective lens is the refractive index of the (immersion) medium times the sine of the semi-angle of the included cone. The latter means the angle to the optical axis of the extreme rays, those which only just get into the lens. The expression is normally written: N.A. = n sin The N.A. of a lens is a measure of both its light collecting and resolving capabilities. As is well known, the performance of any imaging system is dependent on the numerical aperture (NA) of the objective lens - this particularly so for confocal imaging systems. The larger the numerical aperture, the better the spatial resolution of the system. A generally accepted expression for the lateral resolution (Rlat ) in a confocal microscope given by:
Rlat =
0.61 N . A.
in multi-photon mode the lateral resolution is given by:
Rlat =
0.61 1 N . A. 2
where is the wavelength of the light and N.A. is the numerical aperture of the lens. At first sight it may appear that the lateral resolution in multi-photon mode is better than for confocal, but one should remember that the multi-photon wavelength will be approximately 2 times greater than that used in confocal (1-photon) excitation.
Note: The effective wavelength in fluorescence imaging is a function of the excitation wavelength and the emission wavelengths - a practical approach is to use the mid value of the emission band pass filter or the peak emission wavelength of the fluorophore. So it is seen that the lateral resolution is inversely proportional to the N.A. and the axial resolution is inversely proportional to the square of the N.A.
Immersion medium
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If the lensimmersion medium and the sample s mountant have different refractive indices, the light will be brought to a focus at a point displaced from the expectedposition. Medium Air Water Glycerol Oil Glass Refractive Index 1.00 1.333 1.473 1.515 to 1.517 1.525
The result of this unexpectedrefraction will be to distort the image s aspect ratio when viewed either in the XZ or YZ orientation. The distortion will be seen either as an elongation or a compression depending upon the refractive indices of the media. The LaserSharp software applies a correction factor to the calibration of the Z-axis using an algorithm devloped by White et al described in the publication: Aberration control in quantitative imaging of botanical specimens by multidimensional fluorescence microscopy N.S. White, R.J. Errington, M.D. Fricker & J.L. Wood. Journal of Microscopy, Vol. 181, Pt 2, February 1996, pp. 99-116 The table below shows a selection of correction factors for various immersion media and mountant combinations for a few different N.A.s, but the algorithm works for all lenses and refractive index values. It can be seen that for very low N.A.s the correction factor is closely related to the ratio of the refractive idices of the mountant and the immersion medium (M/I), but that this changes somewhat with higher N.A. It will also be noticed that when the immersion medium and the mountant have identical refractive indices the correction factor is unity. This is the ideal arrangement as mismatched refractive indices introduce spherical abberation which can adversely effect resolution and sensitivity. This spherical aberration can also be seen in the point spread funcion of the system and is an important consideration where image deblurring (or deconvolution) is planned.
Immersion Mountant Air Glycerol Air Water Equal Oil Glycerol Glycerol Water Oil Water
Ratio M/I 1.473 1.333 1 0.972277 0.904956 0.879868
0.2 1.49 1.34 1 0.97 0.905 0.88
N.A. of objective lens 0.5 0.75 1 1.6 1.685 1.685 1.425 1.49 1.49 1 1 1 0.97 0.965 0.96 0.89 0.87 0.855 0.865 0.835 0.82
1.4 1 0.96 0.86 0.82
This correction factor is automatically written into the file notes and applied to the z-axis calibration value. The correction factor can be viewed by selecting View|Notes to display the dialog box below:
Chromatic and field corrections To achieve results with the highest possible spatial integrity planapochromatic lenses with a high numerical aperture should be used. These are highly chromatically corrected across the visible spectrum, and have a very flat field. It should be remembered that in a confocal microscope chromatic correction between the illumination wavelength and the emission wavelength is very important - if these two wavelengths are not brought to the same focus by
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the lens then there will be significant signal loss. Chromatic correction between different emission wavelengths is also very important when imaging multiple labelled specimens particularly if accurate 3D colocalisation measurements are to be made. However, there may be applications where colour correction and flatness of field are not of such great importance and in these cases lenses optimised for light throughput (e.g. Fluor or Fluar) should be considered. The user should be aware that the highly corrected lenses typically contain many lens elements held together with optical cement - this inevitably leads to transmission losses. The fluor/fluar type lenses are often very much simpler constructions with accordingly higher throughput.
Transmission charactersitics of infra red light To achieve deep penetration in scattering samples it is often necessary to deliver relatively high power to the sample. This clearly makes it important that the objective lens transmits well in the infra red part of the spectrum this is not true for many lenses. Below is some transmission information for a limited number of objectives from different manufacturers: Nikon CApo40x/WI, greater than 65% from 700 to 1000nm. PlanApo60x/WI, greater than 60% from 700 to 1000nm. PlanApo60x/oil, greater than 60% from 700-800nm, less than 50% from 900-1000nm.
Olympus LUMPLFL40xIR-2, dipping lens, good to 1000nm, greater than 65%. LUMPLFL60xIR-2, dipping lens, good to 1000nm, greater than 65%. PlanApo40x/oil, greater than 70% to 800nm, less than 20% between 900-1000nm. UPlanfl100x/oil, greater than 70% to 800nm, less than 20% between 900-1000nm. PlanApo100x/oil, greater than 70% to 800nm, less than 20% between 900-1000nm. PlanApo60x/oil, greater than 70% to 800nm, between 30-40% between 900-1000nm.
Zeiss PlanApo63x, linear drop from 80% at 700nm to 30% at 1000nm. PlanApo100x, linear drop from 80% at 700nm to 30% at 1000nm. CApo63x/WI, greater than 60% at 800nm, no information above. CApo40x/WI, linear drop from 80% at 700nm to 40% at 1000nm. Achroplan40x/WI, dipping, 90% at 700nm, no information above.
Currently, our strongest recommendations are for the following lenses:

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Nikon Olympus
Zeiss
CApo40x/WI and PlanApo60x/WI LUMPLFLIR-2, 40x and 60x, dipping and PlanApo60x/oil
CApo40x/WI
Summary of objective lens specifications In summary, lenses should generally have the following properties: High N.A./magnification ratio Flat field (Plan) Good chromatic correction (Achromat or Apochromat) for confocal imaging High working distance/N.A. ratio High transmission at required wavelengths particularly for IR illumination in MP imaging
4.1.1 CARE AND MAINTENANCE OF OBJECTIVE LENSES

It is certainly the case that the objective lenses on the microscope are the most vulnerable accessible components on the system - they are also expensive! The following points should be carefully borne in mind: Microscope objective lenses should be kept dust and grease-free and should be kept in their protective cases when not in use. Objective lenses often become accidentally contaminated with inappropriate immersion oil or mounting medium. Should this occur, refer to the manufactures literature or contact the objective lens manufacturer for advice about cleaning with solvents. This is important, because the glue or mountant which holds the lens in place may dissolve in some solvents. Oil should be removed from oil immersion lenses after use. This is for two reasons: 1. Oil remaining on the lens can accumulate dust particles which reduce image quality. 2. Oil can harden on to the objective lens after a while which also reduces the imaging performance. Once most of the oil has been removed with clean tissue, a piece of lens tissue should be placed over the immersion end of the lens. A drop of recommended solvent should be administered, and the tissue gently drawn across the lens surface. This should be repeated (with a clean piece of lens tissue each time) as often as is necessary to attain total cleanliness. A magnifiying lens or a dissecting microscope is useful for assessing this. Coverslips on top of specimens should also be kept dust and grease-free. Water or condensation should never be allowed to remain on the surface of the coverslip during imaging. In general, ethanol or acetone can be used to clean the surface of a coverslip once most of the water or oil has been removed with a tissue. Care should be taken however to ensure that the cleaning solvent does not contact any mounting medium or sealant which may dissolve in it. The lens tissue should be placed over the coverslip and a small drop of solvent placed on top. The lens tissue should be slowly drawn across the coverslip. This should be repeated (with clean lens tissue each time) until the coverslip is clean.
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4.2 Confocal aperture size

Confocal aperture size is, of course, only relevant when using the system in confocal mode. In multi-photon mode the confocal aperture should be opened to its full extent. In common with the MRC series of instruments the Radiance systems employ macroscopic (large), continuously variable, circular apertures. The aperture should be set to Airy disk diameter to achieve theoretically optimal sectioning performance. To calculate the Airy disk diameters in the Radiance systems the following formula can be used:
Diameter =
73.2 mag obj N . A.
where is the wavelength (in fluorescence the mid value of the band pass emission filter should be used) magobj is the magnification of the objective and N.A. is the numerical aperture of the objective lens There is a button in the User Interface which ca be used to set the aperture to its optimal size as defined by the equation above. When an aperture is set to its optimal size the optical section thickness will be reported in the Z Info box.
Z Info box
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4.3 Telecentric adjustment

The Radiance systems are designed to work optimally with a wide range of different microscopes and objective lenses. To achieve this a small degree of adjustment in the relative position of the eyepointand the final scanning mirror in the system is provided. This variation in eyepoint position occurs in conventional microscopes but most users are oblivious to the small change caused when low power objective lenses are used - we simply move our head to the correct position relative to the eyepiece on the microscope. In the Radiance systems it is important that this adjustment is checked and corrected, if necessary, when using low power objective lenses (< 20X). Failure to make this adjustment can result in reduced field uniformity. The system will have been set optimally for lenses of magnification 20 X and higher as described in section 7.3. Before making any adjustment you should familiarise youself with this procedure so that you are confident in your ability to return the system to its normal configuration. To adjust the system specifically for a low power objective lens follow the steps below: 1. 2. 3. 4. 5. 6. Select the chosen objective Image a uniform sample - for example a piece of fluorescent plastic Release the focus locking screw Rotate the focussing ring until the most even field illumination can be achieved Tighten the focus locking screw After imaging has been completed return the system to its normal configuration
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4.4 Visible laser considerations

The three visible lasers used in the Radiance systems have outputs as shown below. Laser Argon ion Argon/Krypton Green HeNe Red laser diode Wavelength & power 514 nm (11 mW) 568 nm (5 mW)
488 nm (14 mW) 488 nm (5 mW) 543 nm (1.5 mW) 638 nm (5 mW)
It is immediately obvious that there is less power in the green HeNe laser s single line than there is in any of the lines from the other lasers. For this reason it is quite normal to use a much higher percentage of the available power from the HeNe laser. One should always use the minimum possible power to minimise the illumination dose to the sample - in the case of fixed samples this will minimise bleaching and in the case of live cells it will prolong their life.
4.5 Femtosecond pulsed laser considerations

The Radiance2000 MP can be configured with one of any of the following lasers: Coherent/Microlase Bio Light DPM1000 Coherent Vitesse Coherent Vitesse XT Spectra Physics Mai Tai Spectra Physics Tsunami/Millenia Coherent Mira/Verdi 1047 nm 800 nm 700-760 nm or 760-860 nm 750-850 nm 690-1050 nm 690-1050 nm
As with lasers for confocal imaging, one should always use the minimum possible power to image the sample. However, particularly when imaging deep (several hundred microns) it will be necessary to use comparatively high laser powers. It has been suggested by several researchers that there is often a well defined damage threshold for a particular set of circumstances. This threshold will be heavily dependent upon the sample and laser wavelength and will be found experimentally for different samples.
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4.6 Alignment of the Beam Conditioning Unit

The beam conditioning unit has four alignment screws that should be used in conjunction with the near-point and far-point alignment indicators to re-align the beam following tuning of the IR laser. The extent to which the beam needs to be re-aligned will be dependent upon the particular laser being used.
Far-point X
Far-point Y
Near-point X
Near-point Y
Side view
Shutter
Near point indicator Far point indicator Plan view
To re-align the beam follow these steps. 1. Open the shutter to allow the IR beam to hit the alignment targets - turn the knob so that it is aligned aong the Y axisof the BCU. 2. Adjust the near-point X and near-point Y adjusters so that the IR beam is centred in the near-point indicator window. 3. Adjust the far-point X and far-point Y adjusters so that the IR beam is centred in the farpoint indicator window. 4. Iterate around steps 2 and 3 until the beam is accurately centred in both indicator windows.
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5. Close the shutter. Following these simple steps it should be possible to quickly re-align the IR beam onto the microscope axis. It is important that the beam is aligned on the microscope axis so that: The intensity of the beam through the lens is maximised The field illumination is even The MP image is co-aligned with the confocal image
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4.7 Filters
Filters for the Radiance2000 MP systems are fitted as shown in the following schematics:
4.7.1 FILTER CONFIGURATION FOR RADIANCE2000 MP AG-2
Dichroic 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Dichroic 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6
Plane mirror 440DCLPXR 500DCLPXR 560DCLPXR Open window -
Plane mirror 560DCLPXR 650DCLPXR Open window -
Det 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8
Emission filters Open HQ390/70 HQ450/80 D488/10 HQ500LP HQ515/30 HQ528/50 -
Blocking filters Open BGG22 E625SP -
Open E460LP HQ515/30 HQ528/50 E570LP HQ590/70 HQ600/50 E600LP
Open E525/150 HQ575/150 E625SP -
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4.7.2 FILTER CONFIGURATION FOR RADIANCE2000 MP K-2
Det 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8
Open E525/150 HQ575/150 E625SP -
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4.7.3 FILTER CONFIGURATION FOR RADIANCE2000 MP AGR-3

Det 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 3 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8
Open E525/150 HQ575/150 E625SP -
Open E570LP HQ590/70 HQ600/50 E600LP HQ660LP -
Open HQ575/150 -
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4.7.4 FILTER CONFIGURATION FOR RADIANCE2000 MP KR-3

Dichroic 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Dichroic 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Det 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 3 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Plane mirror 440DCLPXR 500DCLPXR 560DCLPXR Open window -
Plane mirror 560DCLPXR 650DCLPXR Open window Emission filters Open HQ390/70 HQ450/80 D488/10 HQ500LP HQ515/30 HQ528/50 Blocking filters Open BGG22 E625SP -
Open E525/150 HQ575/150 E625SP -
Open HQ575/150 -
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4.7.5 FILTER CONFIGURATION FOR RADIANCE2000 MP AGR-3 (Q)

Dichroic 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Dichroic 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Plane mirror 440DCLPXR 500DCLPXR 560DCLPXR Open window -
Det 1 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 2 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Det 3 Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8
Emission filters Open HQ390/70 HQ450/80 HQ485/30 D488/10 HQ500LP HQ515/30 HQ528/50
Open E460LP HQ515/30 HQ528/50 HQ545/40 E570LP HQ590/70 HQ600/50
Open E525/150 HQ575/150 E625SP -
Open HQ575/150 -
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4.7.6 INSERTING CUSTOM EMISSION FILTERS

The Radiance detector modules are designed so that users can insert their own emission filters. These standard size emission filters are available from Chroma and are simply refered to as 12.5 mm diameter filters . The filter thickness is 5.0 mm. These filters are normally supplied housed in a metal filter ringwhich has the Chroma Part Number 10-12.55.0 . There are two steps involved in inserting a custom emission filter : Physical insertion of the filter Update of firmware using a proprietary download utility Warning: Unless you feel confident in both of these steps you should not attempt to insert, remove or replace filters in the detector modules.
4.7.6.1 Physical insertion of filters

The procedure for inserting a filter is dependent upon the number of filters already in place in the filter wheel. If there are three or more filters (equivalent to four or more options in the Emission Filter drop down) follow Procedure 1 then the General Procedure, if there are two or less filters filters (equivalent to three or less options in the Emission Filter drop down) then follow Procedure 2 then the General Procedure.
Procedure 1 Warning: Do not open the filter access cover whilst the system is running - this is likely to cause permanent damage to the PMT. 1. With the system running select the filter position according to the table below: Number of filters currently in place (Not including OPEN position) 3 4 5 6 Set filter wheel position to OPEN Second position Third position Fourth position
2. This will have positioned the first free filter wheel space in line with the access port. 3. Turn the system off in the normal way. 4. Open the ICU door and using a 3.0 mm allen key (wrench) open the appropriate filter access cover by unscrewing (counter clockwise) the stainless steel screw marked Ain the figure below. The cover will slowly rise and then turn to reveal the filter wheel. Check that the filter position corresponds to the required position - the number is marked in white in the position shown by the black spot Bin the following figure.
Procedure 2 1. Turn off the system in the normal way.
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2. Open the ICU door and open the appropriate filter access cover by unscrewing (counter clockwise) the stainless steel screw marked Ain the figure below. The cover will slowly rise and then turn to reveal the filter wheel.
3. Rotate the filter wheel to the required position (marked on the wheel at B ) using a fine tool, such as a small screwdriver blade, by pushing gently either clockwise or counter clockwise in the slot marked Cin the figure above. Warning: The filter wheel is not free to rotate continuously. It can only move as follows: 5 6 7 8 1 2 3 4 Any attempt to move the filter wheel directly between position 4 and 5 may cause damage to or misalignment of the filter wheel assembly.
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General procedure 1. Firmly grasp the filter top-to-bottom with the tweezers at right angles to the face of the filter using the tweezers supplied. The filter will probably be marked on its edge with an arrow - the filter must be inserted with this arrow pointing out from the system (towards you). If the filter is not marked with an arrow then its orientation is not important. 2. From above, push the filter into the vacant slot until it clicks firmly home. Check that the filter is properly seated before closing the filter access cover by turning the screw A clockwise until the cover is completely closed and light-tight.
4.7.6.2 Updating the firmware

Information to follow.
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4.8 Direct detectors

4.8.1 INTRODUCTION
Multi-photon excitation only occurs at the focal plane thereby generating optical sectioning without the need for an emission aperture (pinhole) as used in confocal imaging. Therefore as much of the emitted fluorescence as possible must be collected. Some of this emitted light will travel directly back to the objective lens, but (increasingly so with depth) a proportion will be scattered on its return path. As it emerges from the back of the objective lens the scattered light will not be in a parallel beam as shown below:
Objective lens
Scattering event Sample
In order to collect as much of this scattered light as possible the direct detectors are designed with a large collection lens placed close to the pick-off beam splitter (chromatic reflector).
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Direct detectors coupled to an Olympus BX50WI upright microscope
Direct detectors coupled to a Nikon TE300 inverted microscope
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4.8.2 OPTICAL CONFIGURATION

The 2 channel direct detectors have a very simple optical configuration. The emitted light is split by a single filter cube and the light is sent into one or both of the PMTs.
Emission 1
Emission 2
PMT 2
PMT 1
The shortest wavelength light is reflected into PMT 1and the longer wavelength light passes through the long pass (LP) dichroic into PMT 2. The filter cube containing the dichroic and the emission filters is a standard Olympusblock. There are two different types of 2 channel direct detectors one has two bi-alkali PMTs (BB) and the other has one bi-alkali and one multi-alkali PMT.
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4.8.3 FILTER CONFIGURATIONS 4.8.3.1 Bi-Alkali/Bi-Alkali Configuration (BB)
Blue/Green (DAPI/ Fluorescein) UV/Visible (Serotonin/ Fluorescein) UV (Serotonin) Indo-1
Olympus filter cube Emission 1 Dichroic Emission 2 HQ450/80 DC500LP HQ515/30
UG11/IR
UV400DCLP
HQ575/150
Open HQ390/70
Open 440DCLPXR
UG11/IR HQ495/20
4.8.3.2 Bi-Alkali/Multi-Alkali Configuration (BM)
Blue/Green (DAPI/ Fluorescein) Blue/Red (DAPI/ Rhodamine) Green/Red (Fluorescein/ Rhodamine) UV/Visible (Serotonin/ Fluorescein) UV (Serotonin) Indo-1
Olympus filter cube Emission 1 Dichroic Emission 2 HQ450/80 DC500LP HQ515/30
HQ450/80
DC500LP
HQ620/100
HQ515/30
DC560LP
HQ620/100
UG11/IR
UV400DCLP
HQ575/150
UG11/IR HQ390/70
670UVDCLP 440DCLPXR
Open HQ495/20
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4.8.4 SELECTING FILTER CUBES

To select a filter cube the following procedure should be used: a. Remove the filter carousel wheel cover b. Rotate the carousel wheel to the desired filter position. c. Replace the filter carousel wheel cover. Filter carousel wheel Filter carousel wheel cover
4.8.5 CHANGING FILTER CUBES

To change a filter cube the following procedure should be used: a. Remove the filter carousel cover b. Slide the window to the left to reveal the filter blocks c. Loosen locking screw (maximum 2 turns) and slide the filter block toward you and out of the unit. d. Insert the new filter block (check correctly orientated) and secure locking screw. e. Slide back window and replace cover.
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4.8.6 USING THE DIRECT DETECTORS 4.8.6.1 Nikon E600FN

a. Depending on filters supplied, there will be a number of filter options entered into the software. b. To enter further filter options go to the Control Panel ToolBox - Optic Filter Setup and select Direct Detectors to reveal the Carousel Setup:
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c.
Under Filter Blocks, click on Add to enter a new filter block name (i.e. green/red). Please note that the filter selection in the direct detector unit must be visibly verified.
d. Under Filter Block Components, select Emission filter A, click on add and enter the filter (i.e. HG515/30). Note that the position of the filter is displayed on the adjacent diagram. Repeat with the Dichroic and Emission filter B. Please note that Blocking filter are now included in the emission filters and do not need to be added. e. Drag and drop the filter block diagram in Filter Blocks into the desired position in the optical diagram. (Please note that manually changing the position of the filters blocks in the unit will therefore invalidate the optical layout). f. Create a method with direct detectors selected. From the Optic Configuration diagram, click on which direct detectors are required. The filter block selection entered here will be saved in the method which is recorded in the experiment properties.
g. Select filter position 4 on the microscope epislider unit to divert the fluorescence from the sample to the direct detectors:
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Aperture to scan head and objective lens
Filter cube positions
The Epi-slider positions are as follows: 1 2 3 4 DDS - Direct Detectors (multi-photon only), 670UVDCLP INT - Internal Detectors (multi-photon, confocal and transmission), glass window EF1 - Fluorescence cube EF2 - Fluorescence cube
Position 4
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4.8.6.2 Olympus BX50WI

a f. As described above for Nikon E600FN h. Select filter position 4 on the microscope epislider carousel unit to divert the fluorescence from the sample to the direct detectors.
To Direct Detectors
1 2 3 4
EF1 - Fluorescence cube EF2 - Fluorescence cube INT - Internal Detectors (multi-photon, confocal and transmission), glass window DDS - Direct Detectors (multi-photon only), 670UVDCLP
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4.8.6.3 Nikon TE300

a f. As described above for Nikon E600FN g. Select filter position 4 on the microscope epislider unit to divert the fluorescence from the sample to the direct detectors.
Position 4 for direct detectors
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4.9 Fluorophores
The selection of appropriate fluorophores is clearly very important - the use of an inappropriate fluorophore, or combination of fluorophores, could seriously impair key imaging performance measures such as sensitivity and specificity. The choice of fluorophores for 1-photon imaging and multi-photon imaging should be considered separately.
4.9.1 FLUOROPHORES EXCITED BY 1-PHOTON EXCITATION

To judge whether a particular fluorophore is suitable for use with the system is fairly straightforward - one simply looks at excitation curve of the fluorophore. These are usually published by the manufacturers. If the peak of the excitation curve is close to one of the available laser lines then it should be possible to image it successfully - however, even if the peak is not close to an available line then it may still be possible to image the fluorophore well. This is particularly true when the laser line falls below the excitation peak - as in the case of Cy3.
0.9
0.8
0.7
0.6 Excitation 0.5 Emission Laser lines 0.4
0.3
0.2
0.1
0 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640 650 660 670 680 690 Wavelength (nm)
It can be seen that the peak excitation occurs at 554 nm, but that 15% excitation is achieved at 488nm, 49% 514 nm and 73% at 543 nm. It is important to remember that the excitation wavelength affects the amount of emitted light which can be collected; if the excitation peak and the emission peaks are close together then using a laser line very close to (or at a longer wavelength than) the peak excitation wavelength will result in loss of emitted signal because in blocking the reflected laser light the dichroic amd emission filters will necessarily cut off part of the emission. So, for single labeling one should choose a fluorophore with its excitation peak close to but preferably slightly longer than an available laser line. For double or triple labeled preparations there are slightly more complex considerations which are dependent upon the configuration of the system, the required data acquisition rate and the intended use of the resultant data. For the best separation of the different fluorophores (e.g. FITC Cy3 as shown below) one should collect the data sequentially.
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0.9
0.8
0.7
0.6 CY3 Ex 0.5 FITC Ex Lines 0.4
0.3
0.2
0.1
0 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
Wavelength (nm)
This approach ensures that there is almost negligible excitation of the green emitting fluorophore (FITC) when the red emitter (Cy3) is being excited at 543 nm - so even though FITC has an extended emission spectrum there will be virtually no bleed through into the red detector. In a multiple detector system it is possible to acquire two or three channels simultaneously. However, it is important to remember that sequential acquisition will still generate data in which the two channels are most clearly separated.
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The graph above clearly shows that although the green channel (515/30) exclusively detects signal from the emission of FITC the red channel (570LP) detects signal from Cy3 and a significant amount of signal from FITC. However, it should be noted that illumination intensity balancing with the AOTF can reduce bleedthrough very significantly. This bleed through can also be reduced by using Cy2 or Alexa 488 which both have shorter emission tailsthan does FITC. The following table gives the excitation and emission maxima for a wide range of fluorochromes and probes which might be used with the Radiance2000: Green emitting fluorochromes and stains Acridine Orange (DNA) BOBO-1 Bodipy-fl CY2 Dabsyl chloride DiOC6(3) DiOC7(3) DTAF ELF-97 Eosin Eosin F3S FITC GFP Hydroxystilbamidine (fluorogold) IANBD amide Lucifer yellow MitoFluor green MitoTracker green Oregon green 488 Oregon green 500 Oregon green 514 Rhodamine green Rhodol green SYTO 16 TOPRO-1 TOTO-1 YOPRO-1 YOYO-1 Green emitting fluorescent probes BCECF Bilirubin Bodipy FL brefeldin A Calcein Calcium green Carboxyfluorescein CellTracker Green Bodipy CFDA Cl-NERF DiOC5(3) DiOC16(3) DM-NERF Fluo-3 5 hexadecanoyl fluorescein JC-1 (low) Excitation maximum 487 462 505 489 335 484 482 493 345 524 535 494 395/470 385 478 428 489 490 493 503 511 502 499 488 515 514 491 491 439/490 452 503 494 506 492 522 494 518 484 484 515 506 497 514
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Emission maximum 520 481 513 506 536 501 504 518 530 544 542 518 509 536 541 533 517 516 520 522 530 527 525 518 531 533 509 509 530 525 510 517 533 517 529 517 544 500 501 542 526 519 529/590
LysoSensor Green DND-153 LysoTracker green DND-26 NBD-C6 ceramide Rhodamine -123 Stachyose fluorescein Orange emitting fluorochromes and stains Bodipy R6G Bodipy 530/550 Bodipy TMR Bodipy 558/568 Carboxyrhodamine 6G CY3 DiA Dichloro-dimethoxyfluorescein DiI JOE B-Phycoerythrin R-Phycoerythrin POPO-3 POPRO-3 Rhodamine 6G SYTO 25 TAMRA (carboxytetramethylrhodamine) TRITC Orange emitting fluorescent probes Calcium orange DiC18(3) DiC16(3) LysoTracker Yellow DND-68 MitoTracker Orange Octadecyl Rhodamine B Rhodamine B Hexyl ester SNARF (Acid) Tetramethylrosamine Red emitting fluorochromes and stains BoBo-3 Bodipy 576/589 Bodipy 589/617 BOPRO-3 Chromomycin A3 Cy3.5 Dihydroethidium DIOC2(5) DiQ Ethidium Bromide Ethidium homodimer-2 Hexidium iodide Lissamine rhodamine B MitoTracker red Propidium iodide Radiant redTM Rhodamine Red Texas red Texas red-X succinimidyl ester XRITC
442 504 466 505 491
505 511 536 533 516
528 534 542 558 520 550 491 522 547 525 546/565 480/546/565 534 539 525 521 544
550 554 574 569 546 565 590 550 565 555 575 578 570 567 555 556 572
549 549 534 551 556 556 490 550 570 576 589 575 458 581 518 579 562 510 528 518 570 578 536 500 570 587 583 572
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565 565 551 576 578 578 580 574 604 590 617 599 590 596 605 601 >= 600 595 617 600 590 599 617 610 590 602 603 596
Red emitting fluorescent probes Bodipy TR ceramide DASPMI DiA DiD DiQ FM1-43 LysoTRacker Red DND-99 SNARF (Base) Sulforhodamine 101 Sulforhodamine B Sulforhodamine G Sulfonerhodamine Bis-(PEG 2000) Far red emitting fluorochromes and stains Acridine orange (RNA) Allophycocyanin 7-aminoactinomycin D Bodipy 630/650 CY5 LDS 751 Napthofluorescein TOPRO-3 TOTO-3 Far red emitting fluorescent probes Fura red Napthofluorescein RH-414
589 475 491 644 562 510 577 490 586 565 529 581
617 605 590 665 681 626 590 630 605 586 548
487 650 555 630 650 543 598 642 642 436/500 605 532
650 661 655 650 670 712 668 661 660 640 675 716
4.9.2 FLUOROPHORES EXCITED BY 2-PHOTON EXCITATION
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4.10 Dynamic range

In order to correctly set the dynamic range it is helpful to understand a little about signal digitisation in the Radiance systems. Light detection is achieved when one or more photons strike the photocathode of the photomultiplier giving rise to photoelectrons. The resulting current is converted to a voltage, amplified and fed to an analog-to-digital converter (A/D), where it is sampled and then converted to a digital number (see figure below). In order to realise the full 12 bit resolution of the ADC it is important to set the gain and offset of the detectors correctly. To achieve this reliably a false colour look up table (LUT) called setcol.lut is supplied and can be quickly loaded from the pop-up menu. ThisLUT sets the lowest few grey levels to green and highest few grey levels to red. In direct scanning mode the gain and offset (black level) should be set so that a few green and few red pixels are visible in the image. When a frame averaging filter is invoked (e.g. Kalman) you will probably notice that the green and red pixels disappear or reduce in number (due to the improvement in S/N) - this is to be expected and will result in an image with a perfect dynamic range.
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4.11 Monitor adjustment

Clearly, the adjustment of the monitor doesn t have a direct effect upon the quality of the images one collects. However, one should be aware that the monitor settings together with the ambient lighting can influence your judgement of the dynamic range in the image (if one is not using the setcol LUT). If you are working in a darkened room then it is very easy to misjudge the dynamic range in the image and to consequently collect images in which the brightest pixels fall far below a grey level of 255.
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4.12 Sampling resolution

One must consider sampling resolution both in the XY plane and the Z axis. XY Plane Ideally (according to Nyquist) one should strive to set the pixel size to be half of the optical lateral resolution. This resolution is given by:
Rlat =
0.61 N . A.
as discussed in section 4.1. However, one should be aware that this may well result in the collection of very large files so the sampling resolution could be reduced so that the pixel size is equivalent to the optical resolution. Z axis Again, the ideal sampling resolution in the Z axis (the Z step) should be half the axial resolution which is given by:
Rax =
2 2 = n(sin 2) n sin 2 sin 1 ( N . A. n)
as discussed in section 4.1.
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4.13 Signal strength and noise

It is well understood that that in confocal fluorescence imaging the range of light levels likely to be encountered will result in a photon flux of between 10 and 1,000 photons per pixel at a pixel dwell time of 2 microseconds. (Reference: The Pixelated Image, R. H. Webb & C. K. Dorey - Chapter 4, Handbook of Biological Confocal Microscopy second edition - Ed. J Pawley) This range extends from extremely low signal levels in live cell applications through to the brightest possible fluorescence. For the purposes of the following discussion a flux of 200 photons per second, relating to bright immunofluorescence, is used. Poisson statistics tells us that the signal to noise ratio is equal to the square root of the number of detected photons. So, for a signal of 200 photons the noise will be 14 photons or 7% of the total signal. An 8 bit ADC has a digitisation resolution of 1/255 or < 0.4% so it is immediately obvious that it has much more than sufficient resolution to faithfully sample signals from typical fluorescently labelled specimens. Indeed, it is worth noting that one would need to detect more than 65,000 photons per pixel to fully utilise the sampling resolution of an 8 bit ADC. It is clear from the figures above that most confocal fluorescence images will require a degree of averaging to improve the S/N level to an acceptable level. This can be achieved either by scanning more slowly (intra-pixel averaging) or by frame averaging (e.g. kalman filter). The slow scan speed on the Radiance2000 increases the pixel dwell time by a factor of three and will increase the S/N by approximately 1.7 (square root of 3). The frame averaging filters offer a very flexible method of increasing the S/N until it is judged to be satisfactory. Again, the S/N will improve proportionally to the number of photons detected (or frames scanned). Caution should be taken when averaging for a large number of frames (> 10) that the sample is not photo-bleaching as this will result in the S/N rising to a peak value and then falling off with further scanning.
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5. SAMPLE PREPARATION SUGGESTIONS FOR CONFOCAL AND MUTI-PHOTON

IMAGING
5.1 Fixatives for biological tissue

There are basically two types of fixative: The cross-linking fixatives such as paraformaldehyde and Glutaraldehyde are normally used to preserve morphological structure and localisation of antigenic sites. However, they render the cells/tissues fairly Impermeable to fluorescent antibody molecules, and detergents such as Triton X or DMSO are required to make the membranes more permeable. The second type of fixative is known as 'precipitating', and includes the methanol/acetic acid and acetone groups of compounds. Whilst these render the tissue permeable to antibodies, they will precipitate the proteins, thus endangering the retention of antigentic site localisation. Sometimes a crosslinking fixative is applied first, in order to preserve the structure, and a precipitating fixative used afterwards to render the membranes permeable prior to staining. Note that Glutaraldehyde is highly fluorescent and may obscure fluorochrome staining. The use of NaBH4 can quench this fluorescence if required.
5.2 Bleaching and anti-fade agents

Light-induced damage to fluorochromes can occur largely (though not entirely) due to the presence of molecular oxygen. The total extent of bleaching of the fluorochrome is in general less than in conventional microscopy. However the addition of an anti-fade agent (antioxidant) to the mounting medium appears to be even more important for laser scanning fluoroscence than for conventional fluorescence. Various anti-fade reagents are available (e.g. p-phenylene-diamine, DABCO [Diazabicy clooctane], Propylgallate Hydroquinone and Citifluor), but the most recently introduced reagent is called FluoroguardTM. FluoroguardTM is the first of many fluorescence-based reagents from Bio-Rad. Quantitative results show that Fluoroguard reduces photo-bleaching in an aqueous sodium fluorescein solution by greater than 95% i.e. it prolongs fluorescence by >20 fold and reduces photo-bleaching in a solution of DAPI stained DNA by greater than 85% i.e. prolongs fluorescence by greater than 6 fold compared to identical solutions substituting buffer for anti-fade. Rhodamine derivatives Texas Red, AMCA, propidium iodide and other dyes appear to be protected from photo-bleaching. Further information can be received by requesting Bulletin 2047 from your local Bio-Rad representative.
5.3 Mounting media

Canada Balsam and certain epoxy resins fluoresce strongly. This defectmay be put to advantage in confocal imaging of non-fluorescent specimens such as silicaceous shells and mineral grains, which appear in negative contrast. The same technique can be used in measurement of the thickness of live cells. Surounding the cells in a weakly fluorescent medium permits x-z sectioning through the cell thickness with the cell section appearing dark
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against a bright background. Fluorochromes attached to large molcules like dextran are very useful for this purpose. In the vast majority of situations, a non-fluorescing mounting medium is desirable. These may range from glycerol based media to PVA based media. The type of medium used will largely depend on whether the sample is aqueous or solvent based following fixation/clearing etc. Fluid mounts which are made with anti-fade solutions should be sealed with, for example, nail varnish. Media which solidify seem to have poor anti-fade properties, but are convenient to use with specimens resistant to photo-bleaching, e.g. those stained with acridine orange or Lucifer Yellow. Non-fluorescent media are available, e.g. Fluoromount (British Drug Houses) or Citifluor (Agar Scientific Ltd.) Using anti-fade with living specimens is much more of a problem because of the concommitant removal of oxygen. However, attempts are being made to use vitamin derivatives as live cell anti-fade agents. The success of these remains to be seen.
5.4 Autofluorescence
Autofluorescence can be useful for some forms of imaging e.g. the 488 nm line excites skin, feathers, chitinous structures, crustacean muscle and plant cell walls. The green 514 nm line of the Argon ion laser gives strong stimulation of autofluorescence in chloroplasts. However, in the vast majority of cases, autofluorescence is a gremlin which reduces image clarity hence, rendering information extraction more diffficult. Sometimes, autofluorescence can be removed by bleaching the specimen with potassium permanganate or other recommended agent, or reduced, by clearing the tissue with an agent such as methyl salicylate or methyl benzoate. The latter method renders the tissue more transparent, thus permitting optical sectioning at far greater depths than would otherwise be possible. Autofluorescence can also be circumvented by using a fluorochrome which emits at a totally different wavelength from the autofluorescent colour.
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6. SOFTWARE REFERENCE
This chapter provides a reference to the LaserSharp2000 software.
The various components and modes of image acquisition are explained in the following subsections.
6.1 Menu bar
6.1.1 FILE MENU
The File menu contains entries to:
6.1.1.1 Create a New Experiment

Select this item from the menu or use the button on the application toolbar to create a new experiment. You will be prompted for an experiment name and then an image display window to suit the currently selected Method.
6.1.1.2 Open an existing Experiment
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Opens an existing experiment from disk. The experiment will appear in the experiment browser and the desired data can be opened from there.
6.1.1.3 Close a currently Open Experiment

Closes the experiment - shuts all windows in the experiment.
6.1.1.4 Save an Experiment

This item is unused (greyed) as the experiment is automatically saved on closure.
6.1.1.5 Print an image

Prints the selected pane from the currently highlighted image. LaserSharp2000 prints to the default printer as determined by Windows NT. For details on setting the default printer see Windows NT Help.
6.1.1.6 Log Out

Logs out the current user and displays the Login dialog ready for the next user.
6.1.1.7 A list of recently opened Experiments

The eight most recently used experiments are shown for rapid access.
6.1.1.8 Exit the application

Exits LaserSharp2000.
6.1.2 EDIT MENU
A single entry, copies the currently selected image to the clipboard for transfer to other applications.
6.1.3 METHODS MENU
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This menu contains entries to Save a newly created or modified method and to edit an existing method or to create a new method. The Radiance2000 system will be supplied with a set of standard methods as below: Method name Single green fluorophore Single red fluorphore Double label green/red fluorophores Double label green/far red fluorophores Triple label green, red & far red fluorophores Suitable fluorophores FITC, Cy2, BODIPY, Fluo-3, Calcium Green, GFP Texas Red, Propidium Iodide, Cy3, TRITC Combinations of fluorophores shown above Combinations of green fluorophores shown above with Cy5 Combinations of fluorophores shown above
6.1.3.1 Save Method

Saves the currently loaded method with modifications to the optics or mixer settings.
6.1.3.2 Edit... (Method)
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The Edit Methods dialog shows a graphical representaion of the methods and their settings on a per user basis. To edit an existing method select the method name in the list and press the Method Editor Wizard button . To create a new method press the Method Creator
Wizard button . The Method Wizard will take you through the required steps to create a new Method or to edit an existing one.
6.1.4 METHOD WIZARD

Step One
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Enter your chosen Method name and description if desired.
Step Two
Set up the multi-pane layout by choosing the number of panes and the number of rows. Assign one pane only as the merge pane by dragging and dropping the Merge icon into that pane. Step Three
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The setting manager allows you to create your Sequential settings (many) and one Simulataneous setting. On pressing the New Sequential setting button you will be prompted to assign that setting to one of the panes (normally you will simply need to accept the default shown).
Step Four
Use the Setting Wizard to define the setting name and the colour of the icon which will appear in the control panel. Step Five
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Set up the instrument appropriately for the setting you desire in the following sequence: 1. Turn on the laser(s) to excite the fluorophore. 2. Set an appropriate power. This can be adjusted later so don t worry too much about the level set - use less rather than more. 3. Reflect the emission signal into the chosen PMT and turn on that PMT. Edit the name of the PMT if desired. 4. Select a suitable emission filter fom the drop down. 5. Select the laser line which is causing the fluorescence from the drop down.
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Step Six
To define the contents of the Merge pane drag and drop the chosen panes into the Merge pane and then apply the look up tables (LUTs) to the individual panes and to the merged pane.
Step Seven
Finish. Your new method will now appear in the Methods menu and is ready for use. To edit the method use the Wizard and step through to the appropriate stage to make the change.
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6.1.5 ACQUIRE MENU
The functionality of the Acquire menu is replicated in the control panel with the collection buttons except to set the pixel depth at 8 or 16 bits.
6.1.5.1 Live Scan

On pressing the live scan button the system will start scanning the galvos and acquiring an image into the active image display window. Whilst the system is scanning almost all system parameters can be changed. For example, detector gain and iris, image zoom, Setting etc.
6.1.5.2 Sequential live scan

The sequential live scan button automatically cycles through the sequential settings and updates the merge pane (if you have configured one) frame by frame. This mode of scanning is useful when imaging samples which can only be imaged in sequential mode due to bleedthrough.
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6.1.5.3 XY (Z-Series) collection

On pressing this button the XY dialog box is shown; Z-Start, Z-Stop and the Z Step can be reset.
The Channels page is used to select Simultaneous or Sequential acquisition and the settings to be included in the sequence.
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The Time Series page allows either an XY-T series or an XYZ-T series to be acquired.
6.1.5.4 X-Z (vertical section) collection
Pressing the XZ button causes the XZ dialog to be shown. The scan line (Y position) is selected using the spin buttons.
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The Channels and Time Series pages function in the same way as they do for XY images.
6.1.5.5 X-T (line scan) collection

XT data collection runs in two distinct modes: 1. As fast as possible - limited to a maximum of 1024 lines. 2. Slower mode - an unlimited number of lines can be collected. If the image is already rotated then the XT line will be rotated as well.
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6.1.5.6 Pixel Size (depth)
This setting determines whether you save data with 16 or 8 bit resolution. Remember that 16 bit images will take up twice as much space as the equivalent 8 bit image.
6.1.6 IMAGE MENU
The image menu currently only has one entry - Adjust contrast.
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6.1.6.1 Adjust contrast
The adjust contrast dialog allows you to change the brightness, contrast and gamma settings for the current image. You can also load different look up tables from here.
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6.1.7 TOOLS MENU
The tools menu contains options for setting up the system configuration, user login details and access rights.
6.1.7.1 System Setup

The system setup dialog consits of three pages. The first is the scan system setup:
Check the Close shutter between sectionsto ensure that the beam is turned off between sections. Leave this option unchecked to achieve the fastest possible series acquisition. The Delay to start of scanningoption allows the user to determine the period between the opening of the shutter (or operation of the AOTF) and commencement of scanning. The Direction controls allow you to imvert the direction of scan in both axes to orient the image on the screen to be the same as that seen down the eyepieces. The scale factor determines the XY calibration of the system and will be set up at the time of installation. Changing this will render measurements incorrect.
The second page is for set up of the focus motor:

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Again, all of these settings will be made at installation and will only need changing if you change the microscope to which your system is attached. The last page determines how the Z-Focus icon operates dependent upon the type of microscope you are using:
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6.1.7.2 User set up
The User set up dialog allows authorised users to add or delete other users or to edit their access rights. There are three access levels; System, Read/Write and Read Only. It is recommended that the appointed system manager has System rights, all authorised users should have Read/Write access (so that key system configurations cannot be accidentally modified) and that unauthorised users are give Read Only access. Only users with System access can create, delete or edit other users or edit their access level. A default working directory can be assigned for each user so that their data is automatically saved in a known folder on the system.
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6.1.7.3 Objective lens set up
The Objective lens page of this dialog allows you to add, edit or delete lens descriptions from the list which appears in the control panel. The Magnification value is used for calibration of measurements. The default focus motor step size should be set to a convenient value for each lens. The Depth Correction page activates a correction algorithm to correct for axial geometric distortion caused by mismatching of refractive indices above and below the cover slip.
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6.1.8 SCRIPT MENU
The script menu contains entries for editing and running scripts. These scripts are standard Microsoft Visual Basic Scripts (VB Script) and their documentation should be consulted for details of this programming language. Scriptable functionality within LaserSharp2000 which is available at release of V1.0 is: Instrument Control Start a scan series (currently XY scan only) Detector gain, iris, offset Laser attenuation (power) ZMotor position, move, switch on/off Setting selection (within given a method) Application Access Current scanned 2D images Windowless image processing operation (yet to be fully implemented) Confocal data manipulation and access Creation of multi-dimensional confocal image matrix Initialise and allocate image buffers for image matrix Access/manipulation of dimension information Access/manipulation of Calibration information Access/manipulation of individual 2D images and their individual pixels Fast memory copy between 2D images Scriptable Status Bar Write text to the status bar from within a script
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6.2 Image display window
Each image display window houses its own set of controls:
Apply LUT
Set zoom to 1
Increase zoom
Decrease zoom
Toggle on/off Fit toview mode
Toggle between single and multi-pane view. can also be achieved by double clicking the image panes
Show montage display of data
Rocking animation of multi-image data set
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Looping animation of multi-image data set
Stop animation
Increase/decrease animation speed
The slider at the bottom of multi-image files can be used to manually animate the images. The Small black pointers at the top and bottom are used to set the extent of the animation.
Note that the iamge can be displayed in full screenmode by pressing the return to windowedmode press escape.
button. To
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6.3 System control panels

This section describes the control panels on the right-hand side of the screen which are used to control the microscope. The panel is composed of three sections.
All of these panels can be shown or hidden using the
button.
6.3.1 MICROSCOPE CONTROL
Speed The scan speed has a range of possible settings. For Radiance2000 systems the line frequency spans from 750Hz to 25 Hz, and for MicroRadiance systems the range spans from 500Hz to 25Hz. In addition to controlling the line scan frequency one can separately modify the frame rate: Normal At the Normal speed if the line (or X) galvanometer is set at 500Hz it takes about 1 second to acquire a 512x512 image. X2 The X2 speed setting increases the Frame (or Y) galvo frequency by a factor of two (compared to Normal), so that a 512x512 frame is scanned in half a second. It is important to remember that the Line frequency is not increased in this mode so there will be half the number of 'real' lines in the image. For example, in a 512x512 frame only 256 lines will be scanned on the sample, with every line being duplicated in the image.
X4
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The Fastest speed setting increases the Frame (or Y) galvo frequency by a factor of four (compared to Normal), so that a 512x512 frame is scanned in a quarter of a second. In a 512x512 frame only 128 lines will be scanned on the sample, with every line being replicated four times in the image.
Collection filters In the following sections: Pn is the new pixel value Pn-1 is the previous pixel value I is the input value n is the current frame number f is the factor
Direct This is the default setting. The contents of the image window(s) is overwritten with the new data. The factorvalue is ignored, and the entry boxes for this value are disabled in this filter mode. Pn = I Kalman With this filter, the pixel values are calculated: Pn = I/n + Pn-1 (1 - 1/n) This filter enables a display of the average of all the frames since filtering was started. Full intensity is always maintained, but the signal to noise ratio increases. If collection is stopped and then started, the image is cleared before restarting. The factorvalue is ignored, and the entry box for this value is disabled in this filter mode. When the user stops scanning in this mode, the filter selection returns to Directand N is set to zero.
Accumulate In accumulate filter mode 'N' frames are accumulated (added). The resultant values are scaled by 1/Factor. If 'Factor' and 'N' are set to the same value, then the final result will be the same as if Kalman filtering had been used. The image is not cleared automatically before scanning in the accumulate mode, so it is necessary for the user to do this using the eraser button. Accumulate to 'N' can be used to produce brighter images, and can be set prior to the collection of a multi-image file like a z-series. Before doing this, it is worth checking that image saturation will not occur and that N is not set too high. Saturation occurs when so many pixels within a structure attain a value of 255 that the greyscale information is lost. To combine Accumulation of signal with averaging, set a ratio of N:F where the greater N is, the better the averaging, and where the N:F ratio determines how many times brighter the eventual image will be compared to a single 'Direct' scan. For instance, if the original image needs to be 5 times brighter and averaged, N can be set to 30 and F to 6 so the image is averaged over 30 frames. Because the pixel values in
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each frame are divided by a factor of 6, the final image is 30:6 or 5 times the original intensity.
Factor (F) This value is only used by the Accumulate filter. It is described above.
Number of scans (N) This is the number of scans to perform. If it is set to zero, then the instrument will continue scanning until the laser button is pressed again.
Objective This is the magnification of the objective lens on the microscope. This value is used to calculate distances on an image. Users must remember to change this value to match the lens being used in order to collect calibrated data.
Zoom Changing this value controls the amplitude of the angle through which the galvanometer mirrors scan, and hence the area of the sample that is scanned. The value in the edit box is a magnification value. This is a true optical zoom. An optimum value associated with each lens is shown in the table on the next page, which can be used as a guide. Exceeding these values will make the image bigger but with no further improvement in resolution (empty magnification). Moreover, any fluorochrome bleaching will be accelerated because a constant flux of laser light will be concentrated into a much smaller area. Of course, zooming can be used to deliberately apply a high irradiation dose to a small area for applications such as FRAP or caged compound release.
Table showing maximum useful zoom for various lenses:
Objective lens
Pixel size at Zoom 1
Theroretical lateral (XY) resolution (m)
Maximum useful Zoom factor
Nikon Fluor 10x/0.5 Fluor 20x/0.75 Fluor 40x/0.85 Fluor 40x/1.3 oil Fluor 100x/1.3 oil Plan Apo 10x/0.45 Plan Apo 20x/0.75 Plan Apo 40x/0.95 Plan Apo 60x/0.95 Plan Apo 40x/1.0 oil Plan Apo 60x/1.4 oil Leica 25x/0.6 water
1.38 0.690 0.345 0.345 0.138 1.38 0.690 0.345 0.230 0.345 0.230
0.49 0.33 0.287 0.188 0.188 0.54 0.33 0.26 0.26 0.24 0.17
5.6 4.2 2.4 3.6 1.5 5.0 4.2 2.6 1.8 2.8 2.6
0.552
0.41
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2.2
50x/1.0 water
0.276
0.24
2.2
Pan Press the appropriate arrow to move the scanned area in the required direction. The central button returns the scanned to region to the centre or homeposition. The bitmap at the bottom right of this window indicates how much of the image is being shown. The outer ring is constant, and represents the full field of view of the objective lens. The rectangular border represents the scanned region. The shape of this will depend on the box size chosen. As the zoom factor is increased above 1.0 this rectangle will become smaller. The solid rectangle represents the region that can be seen on the screen. When the box size is, for example, 256 by 256, the solid rectangle will fill up the border because all of the image can be seen. When, for example, the box size is 1024 by 1024, and the system is in quad mode, then not all of the acquired image can be seen. The solid rectangle will indicate this.
Scan rotate The scan rotate buttons cause the sacnned region on the sample to be rotated. The scan can be freely rotated to any angle.
Box size The box size determines the resolution of the acquired image. A user defined box can be set by checking the Definebox.
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6.3.2 CHANNELS CONTROL
The appearance of this panel is determined by the currently selected Method. All the controls for a particular channel are grouped on a single page within the panel. The Laser slider controls the intensity of laser light delivered to the sample. If an AOTF is fitted then this is a continuous control, but it moves in steps for a system with ND filters. The Iris control allows the user to select an iris diameter from 0.8mm to 12.0mm in 0.1mm steps. The iris is a variable iris diaphragm, which acts as the confocal aperture in front of each PMT. The theoretical optima for different lenses are given in section 4.2. Opening the iris beyond the value given in the table will reduce the confocal effect, but will allow more light to enter the PMT from the sample. The Gain control allows the user to select a value from 0 to 100 in steps of 1. The gain is (non-linearly) proportional to the voltage applied across the PMT's dynode chain. Increasing the gain will produce a brighter image but will also increase noise in the image. This noise can be reduced by Kalman averaging as described previously. If the gain is set too high, the image may be saturated, that is, too many pixels are at peak intensity. The Black level control allows the user to select a value from -199 to 200 in steps of 1. The default value is 0. The black level sets a DC offset on the electronic signal. The offset should normally be set to a value close to zero. Precise setting of this value is best achieved using the SETCOL look- up table (LUT) as decribed in Tutorial 1. In some circumstances the offset can be used together with the gain control to enhance an image with low contrast. An example of such an instance is in transmitted light imaging using phase contrast optics. The contrast mechanism of phase contrast imaging generates an image with positive and negative intensity deviations from a mid-grey background. Caution should used when setting the black level - too high a setting will compress the effective dynamic range of the detctors and a too low a setting will exclude low intensity information from the image (clipping).
The Photon Counting button switches the confocal detectors between:

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Analog detection mode and
Photon counting mode
6.3.3 FOCUS MOTOR CONTROL

This portion of the microscope control panel only appears if the z-drive system is connected and active when the software is initialised. The commands in this panel control the stepper motor which is attached to either the microscope fine focus knob or in the case of Zeiss microscopes to the coarse focus knob. The z-position is specified in microns. The motor has a resolution of 2000 increments per revolution. This translates into a minimum stage or lens movement (resolution) of 0.05 microns. On/Off This control determines whether the stepper motor coils are powered. None of the other commands in this panel have any effect upon z-position unless the motor coils are powered. You may notice that the fine focus control offers considerable resistance and becomes diffficult to turn when the coils are powered. It is inadvisable to make a habit of turning the fine focus by hand when the motor is on, as this could lead to wear in the linkage between the motor and the focus knob. Whenever the motor is switched to from On to Off, the Position value is reset to zero. However, if a value of zero is entered while the focus motor is ON, the stage will move to the zero position relative to its current position. The focusing drive motor has 2000 increments per revolution. On Nikon, Olympus and Leica microscopes, this corresponds to 20 increments per micron of travel, and thus a minimum vertical movement of 0.05 microns. In the case of the Zeiss microscopes an 18:1 gear box is used to drive the coarse focus and a minimum movement of 0.05 is achieved. For all microscopes, motor operation will be calibrated by the Bio-Rad installation engineer, using the Tools drop-down menu. Position This value changes by an increment determined by the Z-step each time one of the arrows is pressed, either with the mouse or by pressing the function keys F7 and F8 on the keyboard. An arbitrary z-position can also be entered directly. Whenever the motor is switched to from ON to OFF, the Position value is reset to zero. Changing the Position affects the z position icon if it is displayed. Step This value sets the distance from one Z-level to the next. A recommendation is provided, based on the optical sectioning capability of the currently specified microscope objective, determined by its numerical aperture. The recommendation is for a lower limit. As a rule of thumb, it is best to use a Z-step which is one-half the vertical spacing between the features to be resolved. Start
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This determines the starting level for Z-series and Vertical sections. Clicking on the button loads the current position into the Z-Start value. Stop This determines the stopping level for Z-series and Vertical sections. Clicking on the button loads the current position into the Z-Stop value. Middle Pressing this button moves the focus to the mid point between the Start and Stop positions.
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6.3.4 OPTICS PANEL
The optics panel gives a graphical representation of the system s current configuration and allows this to be modified as desired. Filters and laser power settings can be freely changed whilst the system is scanning.
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6.3.5 MIXER CONTROL PANEL
There are three digital Mixers available in the system. The mixers are particularly useful for on-line bleed through correction. The schematic below shows an example of how the mixers would be set to correct for optical bleedthrough of FITC into PMT2. In Mixer A, a fraction of the FITC (green) signal is subtracted from the red signal.
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6.4 Image operators

Image operators (or analysis and processing functions) are accessed via the pop up menu simply right click on an image and select New .
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6.4.1 ARITHMETIC
The Arithmetic operator caters for arithmetic operations on images. These include: Add Subtract Multiply Divide Average And Or XOr Operations can be made between images (e.g. divide one image by another) or upon images by a numerical value (e.g. subtract 20 from all pixel values in an image).
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6.4.2 HISTOGRAM
The Histogram operator shows a histogram of all the pixels in the image. The option to show a graph, a table or both is given. The data from the histogram is saved in a file called data.hst in the histogram folder.
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6.4.3 LINE PROFILE

The line profile tool allows measurement of intensity versus XY position. The line thickness can be set to values gretaer than one to effect smoothing of noisy traces by averaging across the thickness of the line. The data from the line profile measurement is aved in a file called data.lpf in the lineprofile folder.
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6.4.4 MERGE
The merge operator can be used to merge upto three images. At present, only images which have been acquired together or have been produced by the Projection operator can be merged.
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6.4.5 PROJECTION
The projection operator allows either single or multiple views to be generated using a wide range of projection algorithms.
6.4.5.1 Single view
There are six preset views available: Front Back Top Bottom Left Right Additionally, a Custom option is given where any angle of tilt or rotation can be selected.
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6.4.5.2 Multiple Views
On selecting Multiple Views the extent of the tilt and rotation angles and the steps between the views can set.
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6.4.5.3 Projection method (type)
In all of these projection modes imaginary lines (of sight) are traced through the data set and the intensity values of the voxels that lay on each of these lines are used to calculate the resultant single value for that projected line. There are five projection methods available: Maximum - Chooses the highest voxel value and ignores the rest. This is very well suited to the majority of Confocal fluorescence data sets, particularly those that have very open structures e.g. Neurones, cytoskeletal labelling or surface labelling of cells. Minimum - Chooses the lowest voxel value and ignores the rest. This can be used to good effect with negative labelling techniques such as imaging living cells in a medium containing FlTC-Dextran. Average - Calculates the mean of all voxel values along the line. Useful where a maximum brightness projection discards important information from the less bright regions of the data set. An average brightness projection will almost always produce a dimmer image than the equivalent Maximum brightness projection. This is because many zero or low brightness values will be included in the averaged values along the line. This can be compensated for by adjusting the Contrast in the image. Above threshold - Chooses the first voxel value to exceed the selected threshold. This can be useful for imaging an anterior (front) surface in a data set which may not be the brightest feature along that line of sight. A more powerful method of imaging anterior surfaces or features is to use a Two Pass projection as described in the next section. Below threshold - Chooses the first voxel value to fall below the selected threshold. Again, useful for negative staining techniques where one wishes to image the anterior surface of a dark object in a bright surrounding medium.
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6.4.5.4 Source data
This page is used to set the z-axis to xy-axes aspect ratio. If the aspect ratio is set to a value greater than 1.0 then a Z-fill value and method are required. The Z-fill value determines the number of additional points which will be added between sections and the method offers the choice of either replicating the preceeding value or using linear interpolation between adjacent values.
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6.4.5.5 Two Pass Projections
These compound projections can be used to great effect when reconstructing Confocal fluorescence data sets. In the most commonly used single pass projection (maximum brightness) it is clear that two views of a data set from opposite directions (180 degrees opposed) will be exactly the same in everything but orientation. In fact one is a mirror image of the other. If one could 'see' a fluorescence image, confocally, by eye then this would indeed be what we would see. In other words, the sample would be totally transparent. In every day life the vast majority of things that we see and interact with in three dimensions are totally opaque. It is this very 'Opacity' that plays an enormous role in our perception of depth or 3-Dimensionality. In other words, we need a projection algorithm that shows us different views of the sample from front and back. A two pass projection allows us to achieve this by using the first pass to 'locate' a surface by a selectable criterion (e.g. first voxel above a given threshold) and then to make a local projection (e.g. maximum or average) a limited distance away from that point, towards the viewer or a combination of the two. The first pass projection is referred to as the 1st pass reference rule and the second pass as the projection method. The projection methods are exactly the same as those used in single pass projections and are accessed from the same page in the notebook. The Single View projection option is designed to allow the user to quickly ascertain the optimal projection algorithm for a data set prior to generating a set of projections in the batch mode menu.
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6.4.6 SEED FILL
Seed Filling uses a simple algorithm to segment pixels (or voxels in 3-D) by the dual criteria of intensity and connectivity. A starting point (the seed voxel) is selected using the mouse pointer and the algorithm checks either all orthoganally adjacent (face connecting) voxels (as shown below), or all surrounding (edge connecting) voxels, to see whether or not they meet the intensity criterion set with the parameters in the dialog box.
If the voxel does meet this criterion then it is selected and written into a new data volume in a new window. Having been selected the same check is made on the new neighbouring voxels, and so on. In this way continuous structures can be separated from complex 3-D volumes.
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This process works particularly well for neuronal and vascular structures. A particularly useful 'by-product' of this operation is that the segmented structure's volume is directly measured (within the limits set by the contrast and signal/noise in the original image data). The starting point is selected simply by clicking in the image and this is reported on the left hand side of the dialog box. The intensity range can be set either by setting the upper and lower valuse manually, or by using the start voxels value and setting a range around that value.
6.4.7 IMPORTING AND EXPORTING FILES

To export a file select Export from the pop-up menu for the image.
The file formats currently supported are shown above. To import a file right click on an experiment folder in the Experiment Browser
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7. TECHNICAL DETAILS 7.1 Filter specifications

In all graphs in this section the horizontal axis the wavelength in nm and the vertical axis is the transmission value as a fraction of 1.0, where 1.0 = 100% transmission.
7.1.1 RADIANCE2000 MP DICHROIC FILTERS
440DCLPXR
1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 100 200 300 400 500 600 700 800 900
500DCLPXR
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 430 480 530 580 630 680 730
560DCLPXR
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 430 480 530 580 630 680 730
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650DCLPXR
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 379 429 479 529 579 629 679 729 779 829 892 992 1092
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7.1.2 RADIANCE2000 MP EMISSION FILTERS

HQ390/70
1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 100 200 300 400 500 600 700 800
HQ450/80
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1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 100 200 300 400 500 600 700 800
E460LP
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HQ500LP
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 405 430 455 480 505 530 555 580 605 630 655 680 705 730
HQ515/30
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 450 470 490 510 530 550 570
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HQ530/60
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 430 480 530 580 630 680 730
E570LP
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 405 430 455 480 505 530 555 580 605 630 655 680 705 730
E600LP
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1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 405 430 455 480 505 530 555 580 605 630 655 680 705 730
HQ600/50
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 405 430 455 480 505 530 555 580 605 630 655 680 705 730
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HQ660LP
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 430 480 530 580 630 680 730
HQ590/70
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00 380 430 480 530 580 630 680 730
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7.1.3 RADIANCE2000 MP BLOCKING FILTERS

HQ575/150
1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 100 200 300 400 500 600 700 800
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7.2 System calibration

The system will be calibrated at installation and should not require alteration. If you are concerned about system calibration contact your local service representative or email [email protected].
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7.3 Switching the scanhead between microscopes

For the purposes of laser safety this operation is considered to be a maintainance procedure and should only be carried out by users who have been properly trained and are fully aware of the laser safety issues. A mechanical laser safety interlock device is built into the scan head. For this to operate (to open the beam path for the scanning laser) the appropriate Microscope Adaptermust be fitted to the host microscope. These microscope adapters are manufactured solely by BioRad and users should never attempt to install the scan head on a micrscope for which an adapter has not been supplied by Bio-Rad. The microscope adapter comprises two parts - the Optical Adapterwhich screws into the scan head and the Safety Adapterwhich connects directly to the host microscope. Only when these two parts are connected will the system function correctly.
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What safety precautions are mentioned regarding the laser?

What safety precautions are mentioned regarding the laser?

What does the manual say about switching the scanhead between microscopes?

What does the manual say about switching the scanhead between microscopes?

Radiance2000 MP Operating Manual

Radiance Operating Manual

Radiance Operating Manual

Radiance Operating Manual

.................................................. 69 Close a currently Open Experiment Save an Experiment................................................................................... 69

Sequential live scan XY (Z-Series) collection

X-Z (vertical section) collection

X-T (line scan) collection .................................................................. 79 Pixel Size (depth)....................................................................................... 80

Radiance Operating Manual

Radiance Operating Manual

1. PREFACE AND WARNINGS

1.1 LaserSharp2000 Software - limited use licence conditions

1.2 Software copyright

1.3 Software use

1.4 Unauthorized use of software

1.5 Software support limitations

1.6 Other products referred to in this manual

1.7 Manual part number

Radiance Operating Manual

1.8 Laser Safety

1.8.1 LASER WARNINGS

The white BCU LED is illuminated when laser radiation is present.

WARNING: EYE PROTECTION MUST BE WORN WHEN THE SYSTEM IS SCANNING.

1.8.2 LASER CAUTIONS

Radiance Operating Manual

1.8.3 LASER SAFETY LABELS 1.8.3.1 Identification and Certification Labels

Identification label for Ion Secondary Laser

Radiance Operating Manual

FOR EXPORT OR OEM USE ONLY

1.8.3.2 Warning Logotype Label

Warning Logotype Label (fitted to the BCU)

Radiance Operating Manual

1.8.3.3 Aperture Label

Aperture Label (fitted to the scan head focus ring)

1.8.3.4 Labels for Non-interlocked Housing

Radiance Operating Manual

1.8.4 MICROSCOPE INTERFACE SPECIFICATION

Radiance Operating Manual

1.9 Electrical safety

1.9.1 ELECTRICAL WARNINGS

1.9.2 ELECTRICAL CAUTIONS

1.10 System Cooling

1.10.1 COOLING WARNINGS

1.11 System Management

1.13 Symbols and conventions

Radiance Operating Manual

Radiance Operating Manual

2. THE SYSTEM AT A GLANCE

Radiance Operating Manual

2.1.1 SCAN HEAD

Radiance Operating Manual

Figure 3 Schematic view of collector module

2.1.2 INSTRUMENT CONTROL UNIT

Figure 4 Internal view of Instrument Control Unit

Radiance Operating Manual