Instruction for the Radiance2000 MP laser scanning confocal microscope

Note: A typical workflow can be found at the end of this manual.

Starting the system

• Sign in
  • Check the log and extended log for unusual comments
• Mercury Bulb (Optional, applicable only if you use conventional epifluorescence)
  1. check the log, you should only turn the power on if it is turned off at least 20 min. ago
  2. check the counter of the power supply, if it is over 300 hr. please notify Facility staff first before turning it on
  3. turn on the mercury lamp power supply: hit the power switch, press and hold the IGNITE button for 1 s to 2 s

     Note:The READY light will come on very briefly, go out, and then come back on again after the lamp is stable. The mercury lamp should burn for at least 15 min. after ignition. It should not be switched on and off unnecessarily. Please call the next user at the end of your session to see if the mercury lamp can be left on.

     Warning: If the Mercury Lamp bursts, leave the room immediately and notify Facility staff.

• Dell PowerEdge 1300 Computer
  1. if the computer is not already on, make sure the ICU is off before turning on the computer

     Caution: Do not turn on the ICU (lasers + PMTs) until the computer has finished booting up, or you will damage the ICU. Do not launch Lasersharp 2000 until the ICU is on or it will not find the hardware components. If the computer needs to be restarted, the ICU must be turned off first.

  2. if the computer is left on by the previous user, it is a good idea to reboot the computer for your session
  3. make sure the ICU is off, then turn on the monitor and boot up the computer
  4. press CTRL-ALT-DEL and log in as _____ (passwd: _____)

     Note: If you wish to burn a CD, you may have to log in as a different user. Please see burn a CD in Moving files.

• Instrument Control Unit (ICU) and lasers
  1. when the computer has finished booting, go behind the AV Table, turn on the ICU with the rocker switch near the top (the key is always in the ON position, horizontal)
  2. you should hear a series of snaps indicating mirrors and filters are being moved into position

     Note: If you hear a dull noise instead, the initialization has probably failed and LaserSharp will not launch properly.

  3. launch LaserSharp and log in

     Note: If hardware check fails, quit LaserSharp, turn off ICU, wait 1–2 min., and then restart ICU. Note this event in the error log. If it fails again, please stop and notify Facility staff.

  4. turn on only the laser you will need by pushing the round buttons on the front panel
     • Kr/Ar: 488 nm and 568 nm

       Caution: The Kr/Ar laser will beep for about 30 s during startup. The cooling fan for the Kr/Ar laser is automatic and will continue running for a few minutes after the laser is powered off; even if the ICU is switched off. Do not turn on the laser while the fan is still running, doing so will decrease the life span of the laser.

     • Red diode: 637 nm

Nikon Eclipse E600 FN Microscope

• Objective lenses
  • for optimal images, objective lenses should be clean

    Note: If you see oil on the dry objectives 20× or lower, please notify Facility staff for cleaning. The mixed pollen slide is good for checking the dry objectives

  • read the section below on using oil immersion objectives if you will use them
  • only use the supplied Ross lens paper; under no circumstance should you need to rub the lens dry, not even with lens paper
  • do not sweep dry lenses through immersion oil
  • never use slides with wet nail polish or other media that may damage the lenses
• Using oil immersion objective lens
  1. locate the object at a lower magnification, if needed
  2. raise the objective assembly using the coarse focus knob (clockwise)

     Caution: This is a fix stage microscope. Turn the coarse focus knob SLOWLY and make sure the objective is moving away from your slide. This help to protect the objectives and your specimen.

  3. apply a drop of immersion oil on the coverglass
  4. rotate the turret of the objective assembly to bring the desirable lens into position; avoid grabbing the objectives
  5. lower the objective assembly SLOWLY onto the slide until the objective contacts the oil
  6. use the fine focus knob to bring your object into focus
  7. when you are done, carefully raise the objective assembly
  8. swing the high power objective out and then remove your slide
  9. at the end of the session, take a piece of Ross lens paper and gently draw it flat across the front lens surface in ONE direction only

     Caution: do not rub the surface in a circular or back and forth manner

  10. 3 to 4 passes should remove most of the oil on the objective lens
• Transmitted light (bright field and DIC)
  • Bright field
    1. push the Light Path Selector to BINO
    2. gently slide the Cube Selector lever to INT

       Caution: do not pull the lever out or it will jam

    3. turn on the transmitted light with the controller next to the monitor
    4. make sure the DIC Analyzer located in the center of the Epifluorescence Module is out
    5. locate your specimen first using low power (e.g., < 10×)
    6. adjust for Kohler illumination (only needed if you will acquire images with the transmitted light detector or you need optimal conditions to locate your sample)
       • select the objective that you will use for the experiment
       • focus on the specimen
       • fully open the Aperture Diaphragm and rotate the condenser to
         • A for brightfield
         • DIC L for DIC at 10×
         • DIC M for DIC at 20×
         • DIC H for DIC at 40× and 60×
       • close down the Field Diaphragm with the knurled plastic ring
       • focus on the leaves of the iris diaphragm with the Condenser Focus Knob

         Caution: the condenser can travel through the stage and pop out your slide if you raise it up too far.

       • open the Field Diaphragm until the leaves are just within the field of view
       • center the iris diaphragm with the 2 Condenser Centering Screws
       • close down the Aperture Diaphragm until the intensity just start to drop

         Note: This reduces glare and corresponds to about 70 % of the field.

  • Differential Interference Contrast (DIC)
    1. do 1 to 3 as in Bright field above
    2. insert the DIC Analyzer located in the center of the Epifluorescence Module
    3. do 5 as in bright field but leave the Aperture Diaphragm fully open
    4. rotate the Substage Polarizer until the fine structures of your specimen gain sufficient contrast
• Epifluorescence
  1. gently slide the Cube Selector lever to engage one of the 3 filter blocks
     • UV-2E/C: EX 330–380, DM 400, BA 435–485
     • B-2E/C: EX 465–495, DM 505, BA 515–555
     • G-1B: EX 541–551, DM 565, BA 590

       Caution: do not pull the lever out or it will jam

       Note: If a specific block is not in the microscope, please see Facility staff. There is additional information about the filter blocks in the appendix.

  2. disengage the DIC Analyzer

     Caution: the Analyzer can be pulled out completely and fell off the microscope!

  3. open the epifluorescence Shutter only when you are examining your specimen

Main features of LaserSharp2000

(see the appendix for Basics in LaserSharp2000)

• Acquire an image (assuming you have located an object of interest at the desirable magnification)
  1. slide the Cube Selector lever to the appropriate position (do not pull the lever out!)
     • INT for the conventional PMT with all visible wavelength lasers and Mai Tai
     • DDS for the Direct Detector with the Mai Tai alone
  2. pull the Light path selector out to PHOTO
  3. pull out the DIC Analyzer (don't drop this!)
  4. make sure transmitted light is off, Epifluorescence Shutter is close, and ICU is on
  5. double-click the LaserSharp2000 icon (not the No Hardware version) and login

     Note: The software will load the previously used method and open a new experiment.

  6. in the upper most pane of the Control Panel: Microscope
     • set a speed and zoom
     • select the objective to match the one you are using
     • select Direct
     • set the resolution (Pixels × Lines) of your image
  7. in the middle pane of the Control Panel: Channels
     • select either a Simultaneous or Sequential setting
     • move the sliders on the Gain, Iris, and laser power to a desirable value

       Note: The Lock buttons will lock the parameters across the Simultaneous and Sequential setting.

  8. start scanning by clicking the Exploration icon (topmost, far left button)
  9. optimize the image by picking an appropriate setting for the Iris, Gain, laser power, and Offset with respect to the nature of your sample

     Note: There are compromises in any parameter changes.

     • increase gain enhances both signal and noise; this can be partially compensated for using Kalman filter or slower scan speed, but with increase exposure to laser
     • increase the confocal aperture size (Iris) allows more out of focus signal to come through to yield a brighter but less sharp image; if the aperture is wide open, you are essentially using the equipment as a glorified epi-fluorescence microscope.
     • higher laser power increases signal but will bleach your preparation faster

• Some tips on obtaining an image
  • rough in the settings with the slider, and then fine tune with the small arrow keys; adjust the settings slowly while scanning your sample so you can see the changes
  • If you do not know the response of your sample
    1. pick a Sequential setting to start with
    2. select a low laser power e.g., 1.0
    3. open the Iris completely
    4. slide the Gain to about 30
    5. leave the Offset at 0.0
    6. start scanning by clicking the Exploration button (topmost, far left button)
    7. if there is nothing, stop scanning (click Exploration) and check the light path
    8. if everything is set correctly, start scanning and then pull up the Gain slowly (as appropriate for the scan speed), adjust the focus as needed
    9. if still nothing with Gain at 100, pull up the laser power SLOWLY
    10. unless your sample is really weak or completely out of focus, you should start to see something
    11. as your object comes into view, you can start to optimize it by checking the focus, and then picking an appropriate setting for the Iris, Gain, laser power, and Offset
• Image Optimization
  • to maximize the dynamic range, apply SETCOL.LUT to help set the correct Gain and Offset first and then fine tune them for your image (see the appendix for an explanation about using SETCOL)
  • use Kalman: select the number of passes in the N box (reselect Direct when you are done taking your image)
  • start with an aperture size optimized for the objective selected—Target Iris, click on the target icon beneath the slider, and open it as wide as you can tolerate so you can use a lower gain

    Note: Target Iris is the Airy disk diameter as calculated with respect to the NA of the objective and the refractive index of a common mountant.

  • use a slower scan speed
  • the Optics Diagram page (topmost, second right button) allows you to change the following even as you are scanning
    • Signal Enhancing Lens
    • Dichroic Mirror
    • Emission Filter
    • Polarization Filter
    • Blocking Filter
    • Power Meter setting for the Mai Tai

    Note: Occasionally, the software fails to perform a mirror/filter change that you have selected. If you do not hear the clicking, it is likely that it does not occur. Try again, and if the problem persists, please notify Facility staff.

• Collecting Z-series data (XY Series; XZ Series, or Line Scan are possible)
  1. the lowest pane of the Control Panel: Focus motor
     • turn on the Focus motor, the current Position will be defined as 0

       Note: Whenever you turn the focus motor on, the current position of the objective assembly is defined as 0.

     • set a step size appropriate for the object of interest and the objective used
     • step through your object with the arrow keys in the Position box and define the Start and Stop position of the series, and also adjust for the optimal exposure of the image

       Note: click on the Start button will set the current position as the start of the series, or simply enter the value in the box

     • before collecting the Z-series, confirm that all of the software settings are correct, especially if you want to do Kalman averaging
  2. click on the XY Series icon to open the XY Images window
     • click the Enabled box under the Focus Motor tab
     • verify the values in the Position boxes and the details given in Collection Information
     • click Start to collect the images
     • save the series when it's done, your files should only be in F:\Radiance files

       Caution: Files saved elsewhere will be deleted without further notice.

Mai Tai infrared laser: 780 nm to 920 nm (effectively 390 nm to 460 nm)

Start up

1. if the water chiller and T40 Power Supply is on, skip to step 4 below

   Note: To maintain temperature equilibrium, both the water chiller and the T40 power supply should be left on at all times. In emergencies, please turn off the T40 first and then the water chiller. Follow the directions below to re-start the system.

2. if the water chiller is off, switch it on

   Caution: The chiller should be set to 21 °C; wait until the temperature settles before switching on the Mai Tai’s power source. THE WATER CHLLER MUST ALWAYS BE ON WHEN THE MAI TAI POWER SUPPLY (T40) IS ON. LASER COMPONENTS WILL BE FATALLY DAMAGED OTHERWISE.

3. under the AV table, switch on the T40 power supply using the orange rocker switch
4. boot up the Dell Optiplex computer if it is off
5. press CTRL-ALT-DEL and log in as _____ (passwd: ____ )
6. double click the Mai Tai software icon
7. turn on the Pockel’s cell power source

   Note: The Pockel's cell modulates the laser power. If it is not on, you will be imaging with full laser power!

Mai Tai Controller software basics

1. once launched, the software will monitor the operation of the pumping lasers as indicated by the flashing RS-232 icon

   Note: if the T40 is just powered up, a “% warmed-up” will be displayed. The laser should only be turned on when the System Status reads “Ready to turn on”.

2. select a wavelength around 800 nm for start (you can select any wavelength later after the laser is stabilized)
3. click and hold the ON/OFF button until “EMISSION” displays; the Power Meter will rise, stabilize, and begin pulsing (box is green) in a few minutes; if not, STOP
4. select the desirable wavelength

   Note: Move the gray slider to the desired wavelength with the mouse and release or type the desired wavelength in the Set Wavelength box (or use the arrow keys). You can rely on published literature or vendor specification in selecting the wavelength, or sometimes the easiest way to find the best wavelength to excite your fluorophore is by observation. Tune across all the wavelengths while scanning, and watch until the brightness of the fluorophore is the strongest.

5. if the Shutter button is white (close), click and hold the button until it turns red (open)
6. to power off the laser, click once on the ON/OFF button

Using the Direct Detector

Note: It is not possible to use the DDS and the other PMT in the same method.

1. select a method that uses DDS or create a new one, DD1 is the green and DD2 is the blue channel
2. open the Filter Carousel Wheel Cover and check the Filter Carousel Wheel to make sure the desired cube is selected

   Filter Carousel Wheel Elements

   position	name	emissionA (DD1)	dichroic	emissionB (DD2)
   1	empty	empty	empty	empty
   2	B/G	HQ 515/30	DC 500 LP	empty
   3	empty	empty	empty	empty
   4	495 DCXR	HQ 528/50	495 DCXR	HQ 450/80

3. make sure the Filter Carousel Cover is properly installed (window is closed)
4. gently slide the Cube Selector lever to the DDS position (far left)

Moving Files

• via the network using SSH secure file transfer
  1. double click the SSH icon on the desktop
  2. select the profile "Imaging Facility" or other server you prefer
  3. enter the password when prompted
  4. navigate to your folder or create a new one as needed
  5. open up Windows NT Explorer, navigate to your files, drag and drop them into the right pane of the SSH window
• burn a CD
  • simple
    1. double click the CD icon on the desktop
    2. either use the wizard or click return to RecordNow to make a new data CD
    3. open up Windows NT Explorer, navigate to your files, drag and drop them into the RecordNow window
    4. burn away!
  • more options
    1. you need to log in as _____ (passwd: _____ )
    2. double click the create CD icon on the desktop and click data CD
    3. on the upper panes, navigate to your files, drag and drop them into the lower right pane
    4. 2 options available in layout (File | CD Layout properties) are worth notice
       • You can limit the filetypes to write to the CD by going to the File type tab, select "do not add ..."
       • If you are going to write an ISO 9660 CD, make sure the filenames are under 30 characters long. The Joliet file system does not have this limitation but Mac OS 9 or earlier will need extra software to read the CD. The file system for the CD is specified in the General tab.
    5. burn away!
    6. this is a privilaged user accout, please log out promptly when you are done

One way to save time and storage space: You can select just the raw PIC (or other files) to transfer or burn to CD. However, this will prevent you from opening your experiments ever again in LaserSharp. If you will never use LaserSharp to view these files again, this should not be a problem.

1. it will make life a lot easier if you put all the files in the current session in a single folder
2. do a file search in windows, enter "*.pic" in the box labelled, add ", *.tif" if you have any TIFF to move or add other file types separated by coma as needed
3. browse to your folder and hit search
4. select the files in the result window, drag and drop them into the SSH or RecordNow window

Routine Shutdown (as listed in the log sheet)

• for Mai Tai only
  1. power off the Mai Tai, exit the software, and turn off the monitor
  2. turn off the Pockel's cell
  3. check and make sure that the water chiller and T40 are still on

     Note: If you need to shutdown the Mai Tai system, turn off the T40 first and then the water chiller.

  4. complete the Mai Tai log and record the Time Out
• all lasers
  1. exit LaserSharp
  2. turn off the laser and ICU
  3. start transferring your files using the zip, CD or network
  4. if the Hg bulb is still on, call the next user to ask if the Hg bulb can be left on
  5. if you finish early, notify the next user
  6. make sure the transmitted light is off
  7. remove any excess oil from the 40× or 60× objectives
  8. rotate the objective turret to put the prism in the light path
  9. when you are done transferring your files, shutdown the computer and turn off the monitor
  10. put the cover on the microscope
  11. complete the Radiance 2000 log and record Time Out
  12. if applicable, provide details for any problems encountered in the Radiance 2000 Extended log and notify Facility staff
  13. cleanup the work area before you leave

Appendix

Setting the gain and black level/offset

At each pixel of the specimen, the fluorescent photons are collected and amplified by the photomultiplier tube. The analog output signal (voltage) is then passed to an analog to digital converter (ADC). The 8-bit ADC assigns 256 levels to represent the image. The value 0 is assigned as the minimum intensity whereas 255 is the maximum intensity. By setting the black level, you tell the ADC that below a certain voltage everything should be assigned as 0; this controls the brightness. The gain, on the other hand, defines the range and thus sets the highest voltage beyond which the ADC will assign as 255; it determines the contrast.

In order to display the image on the monitor, you instruct the computer to map the 256 levels to some display characteristics according to a LookUp table (LUT), e.g., to display a grey scale image, 0 is choose as black, 255 white, and shades of grey in between. To make full use of the ADC’s capability, you will need to make sure that the black level and gain are selected properly for the parts of the image you are interested in. Since human eyes are less sensitive to variations at the 2 extreme ends of the range, mapping these display levels to a different color can help ascertain these settings, e.g., SETCOL.LUT maps 0–5 to green and 250–255 to red.

Basics in LaserSharp2000

• The online help file and the Radiance 2000 MP manual (c:\Radiance_pdfs\9M60UM04.PDF) provide some useful information even though they are based on an older version of the software.
• The Experiment is a basic unit of operation in LaserSharp. It is a collection of files in a folder containing the raw images from each channel, the Merge Pane, and other derivatives. After collecting a XY or XZ Series, or Line Scan, you must either save or discard the current result before you can launch a new experiment to collect more images.
• The Experiment can only be opened in LaserSharp whereas the raw pics can be opened in any software that can read BioRad PICT files e.g., ImageJ.
• If you change the name of any folders and files in the Experiment, LaserSharp will fail to open the files; except in the case with a Merge Pane, the merged images (xxx_MergeData.pmf) can be deleted to save disk space without affecting the integrity of the Experiment. LaserSharp can regenerate the merged image. However, you can import any BioRad PICT files by right clicking an Experiment listed in the Experiment Browser.
• Images can be exported at anytime when an experiment is opened by right clicking on the image, select export, and choose an appropriate file type.
• Right clicking on the image will also allow for various operations such as projections, merge, orthogonal view, etc.
• The Method contains details for the Simultaneous and Sequential settings. These include the optical configuration and most of the acquisition parameters displayed in the Microscope and Channels Panes of the Control Panel. Any changes made in the Optics Configuration and the acquisition parameters can be saved by going to Methods | Save Settings. The file Creating Methods (c:\Radiance_pdfs\9M60UM31.PDF) provides examples in dealing with methods.

  Note: when you create a new method, put your username in parenthesis at the end of the name to make it unique. All methods on this computer must have an unique name.

• Depending on the amount of RAM available, you can open any previously acquired experiments. The file will be added to the Experiment Browser window where you can simply click to open the experiment again.
• Your files can be stored temporarily in D:\Radiance Files for up to a month. However, files will be erased sooner if disk space is low. Please back up your files and make sure they are readable as soon as possible.

  Caution: Files saved elsewhere on this computer will be deleted without further notice.

The Epifluorescense Attachment Slider

Caution: Do not try this unless you have been trained on this procedure!

• There are labels in the front of the Attachment which show the configuration of the Slider. The default is (from left to right): DDS, INT, G-1B, B-2E
• Filter cubes can be accessed from either side of the Attachment by opening or slide out the protective cover.
• The physical position of the cubes on the slider is (from left to right): B-2E, G-1B, INT, DDS
• Filter cube on the far left or right can be removed to accommodate the UV-2E cube: if you do not use DDS, take the far right one out; otherwise, take the far left (B-2E) out.
• The necessary tools (2 mm Allen wrench) and filter cubes are in the drawer opposite the microscope
• Procedure to replace the cube

  Caution: make sure you do not touch any of the optical surfaces!

  1. open or slide out the protective cover
  2. move the slider to one end to expose the appropriate filter cube
  3. loosen up the set screw securing the cubes 1 to 2 turns
  4. grab the filter cube and slide it off the rail

     Note: The big round lens is on the right side of the DDS cube and there should be a filter on the top (barrier or emission) and the back (excitation) of the other filter cubes.

  5. slide on the replacement cube and tighten the set screw to finger-tight

     Caution: Do not overtighten!

  6. replace the cover
  7. put or replace the label on the front in the appropriate slot to indicate the change e.g., if you take out the DDS, put the label in the far left slot
  8. put the replaced cube in one of the protective plastic box together with its label
  9. restore the default configuration at the end of the session unless the next user requested the same configuration

A Typical Workflow

Caution: This is NOT a step by step instruction but an example of a typical workflow. You should follow the specific instructions in the respective sections in this manual.

1. check log for unusual activities or errors
2. sign in
3. reboot or startup computer
4. start up ICU
5. launch Lasersharp
6. powerup laser
7. locate specimen at low magnification
8. move to your area of interest
9. begin scanning and get data!
10. quit lasersharp
11. poweroff laser
12. poweroff ICU
13. transfer data out
14. sign out
15. clean up

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You can contact us for comments and inquiries.

Updated on 2009 April 24. Copyright© 2009 University of Washington, unless indicated otherwise.

Department of Biology • UW