Instruction for the MRC-600 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 for unusual comments and sign out time of the previous user
• Mercury Bulb (Optional, applicable only if you use conventional epifluorescence to locate your specimen)
  1. 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 h 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 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.

• Laser
  1. check to make sure that the fan is not running (which indicates the laser has just been turn off), wait until it is off before switching on the laser
  2. under the AV Table, turn the key on the power supply clockwise to the horizontal position to switch on the laser
  3. the switch on the laser housing should be on "norm"—pointing to the back

     Note: The laser should not be switched on and off unnecessarily. You can leave it running if it is going to be used within the next 2 hr. Please call the next user at the end of your session to see if the laser can be left on.

• Excitation wavelength

  Tip: Most users will proceed to optimize the spatial filters, to use K1+K2 one should select position # 3 (568) for excitation filter, and # 3 (1 %) for neutral density filter now.

  • rotate the Excitation Filter wheel in front of the laser housing to select the desirable wavelength

    Table 1 Excitation filters

    Position #	Wavelength (nm)
    0	488 + 568 + 647, no filter
    1	488
    2	488 + 568
    3	568
    4	647
    5	488 + 568 + 647, no filter

• Laser intensity
  • rotate the adjacent Neutral Density Filter wheel to select the desirable laser intensity
    • the neutral density filters are the primary way to regulate the amount of laser intensity impinging on your sample: start with #3 for fixed and #0 for live samples
    • always use the lowest possible laser intensity (the least transmission), increased laser intensity results in faster bleaching of your sample

    Table 2 ND filters

    Position #	% Transmission
    1	10.0
    2	3.0
    3	1.0
    0	0.1

• Scan Head
  1. turn the scan head on with the toggle switch on the back of the unit, at the top and under the power cable

     Front panel of the scan head

     

  2. GAIN should be mid-range (around 6) and AUTO is off (push-button out)
  3. BLACK LEVEL should be on 5 and AUTO is off (push-button out)
  4. ENHANCE should be on LOW SIG. and External is off (push-button out)
  5. select the Emission Filter

     Table 3. Filter block selection

     Mode	Channel 1	Channel 2	for Excitation (nm)
     single	BHS	Blank	488, position 1
     	YHS	Blank	568, position 2
     	RHS	Blank	647, position 3
     	Reflectance	Blank	488, position 1
     dual	K1	K2	488 + 568, position 3
     	Reflectance	BHS, YHS, or RHS	use an excitation filter appropriate for channel 2

     Note: see appendix for filter specifications and the procedure for removal and replacement of filter blocks.

Computer

1. turn on both monitors
2. turn on the computer if it is off, hit OK when prompted for windows network login, no password needed
3. double-click the desktop icon of CoMOS to launch the software
4. if there is any initialization error, STOP and seek help from Facility staff

Confirm the presence of the laser

1. swing the perspex prism into the light path with the flat face outward so the black target ring should be visible
2. clear the light path by GENTLY swing the binocular head to the left, and disengaging (pulling out) both epifluorescent filters
3. start the laser scanning by pushing the spacebar on the keyboard, or clicking the laser scan button in CoMOS

   Caution: If there is no laser, check every component in the light path; still nothing, STOP and notify Facility staff.

4. check to see if the laser is centered on the target, If not, try gently rotate the filter block in Channel 1 to make sure it is properly seated; if still not centered, STOP and notify Facility staff

   Note: We do not adjust M1 anymore, this section in the appendix explains why.

5. if the laser is flickering or throbbing (vs. gentle pulsating for a scanning laser), it is off focus, please notify Facility staff for adjustment.

Nikon Optiphot Microscope

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

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

  • 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. lower the stage using the coarse focus knob (counterclockwise)

     Caution: 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 objective assembly to bring the desirable lens into position; use the turret, avoid grabbing the objectives
  5. raise the stage until the objective lens contacts the oil
  6. use the fine focus knob to bring your object into focus
  7. when you are done, carefully lower the stage
  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)
  • Bright field
    1. use the thumb wheel in the front to turn on the transmitted light
    2. select A on the condenser turret
    3. locate your specimen first using low power (e.g., < 10×)
    4. 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
         • appropriate position for phase contrast
       • close down the Field Diaphragm with the knurled plastic ring on the base
       • focus on the leaves of the iris diaphragm with the Condenser Focus Knob
       • 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.

  • Phase contrast (not covered here)
• Epifluorescence
  1. gently push the appropriate lever in to engage one of the 2 filter blocks
     • B-2A: EX 450–490, DM 510, BA 520
     • G-1B: EX 541–551, DM 565, BA 590
  2. open the epifluorescence Shutter only when you are examining your specimen

Optimize the spatial filter

The spatial filters: M4 and M5 are mirrors directing signal to each of the 2 detectors. Each filter has 2 Allen socket adjustment screws. Failing to properly align the spatial filters will lead to degraded images.

Adjusting M4, the spatial filter for PMT1

Note:

• This procedure is for single channel or Channel 1 of dual channel imaging.
• For single channel, use the green plastic slide for 488 nm, red one for 568 nm, and the tetraspeck preparation for 647 nm.
• For dual channel imaging with K1+K2, use 568 nm and the red plastic slide.

1. with transmitted light at 4×, focus on an edge of the cross-mark on the surface of the plastic slide or the tetraspeck beads
2. increase magnification to 20× and focus
3. turn off the transmitted light and prepare for laser scanning (see step 2 in Confirm the presence of the laser)
4. select the appropriate excitation filter (see note above)
5. a good starting point is: ND 3, GAIN 6, BLACK LEVEL 5 for Channel 1, aperture at half open
6. select PMT1 and start scanning
7. focus on an edge of the cross-mark
8. If you can't find the cross-mark, check the light path; still not, STOP and get help from Facility staff
9. move over to an unmarked area
10. apply SETCOL.LUT (see Selecting LUTs)
11. adjust the BLACK LEVEL properly e.g., the black corners on the right should just turn solid green
12. close aperture down to the minimum, adjust the GAIN to obtain some red pixels such that any slight increase or decrease in signal can be detected easily
13. use the fine focus to get the brightest possible image which is the surface of the slide, you should see a "hot spot" somewhere on the monitor
14. adjust the 2 screws for M4 in turns to center the "hot spot", adjust the GAIN as needed

    Note: M4 is very sensitive, so only make very slight turns (< 1/16 of a turn)

Adjusting M5, the spatial filter for PMT2

Note:

• This procedure is for Channel 2 of dual channel imaging and you MUST adjust M4 first.
• For K1+K2, use 488 nm and the green plastic slide.
• For reflectance use the green plastic slide for 488 nm, red one for 568 nm, and the tetraspeck preparation for 647 nm.

1. do the same as in Adjusting M4, except use Channel 2 and select PMT2
2. with PMT2, you can expect to get an evenly illuminated field when M5 is properly adjusted
3. M5 is not as sensitive as M4 so larger displacement (1/4 turn) is needed to produce any noticible signal change

Using CoMOS

Scanning

1. select the correct detector (PMT1, PMT2, BOTH) on the left
2. click on the top left large laser icon or press the space bar to toggle on or off laser scanning

Selecting Lookup table (LUT)

1. go to Display, select Output LUTs ...
2. click Open at the bottom of the Edit Output LUTs dialog box
3. in the Load Output LUT File dialog box, click on the desirable file e.g., SETCOL.LUT, and then OK to load the LUT
4. go to Display and toggle on "Hold LUTs" (a check mark will appear before it)
5. to return to the default greyscale LUT, either toggle off "Hold LUTs" and scan, or select Monochrome in the Edit Output LUTs dialog box

Collecting a Z-series

1. locate your object of interest, focus, and adjust Zoom, GAIN, BLACK LEVEL, and aperture for an optimal image
2. set Objective to the same as used on the microscope
3. select ON for the focus motor

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

4. select an appropriate Z-Step size
5. while scanning, focus to the top of the region you wish to image by clicking the left arrow for Position which moves the stage down

   Note: While scanning through the whole volume, you should also watch out for over- or under-exposed pixels in the area of interest, adjust gain and black level as needed.

6. click the Z-Stop button to set the upper limit of the scan, or you can type in the number directly
7. using the right arrow for Position, focus through your sample (the stage moves up) to find the lower limit of the scan
8. click the Z-Start button to set this limit
9. select a Collection Filter and set the number of passes (N scans) and Factor, as needed
10. from Collect, select Z-Series\Time Series… to bring up the Parameters dialog box
11. make sure that "Save to File" and "Enable Z-Step" are checked and click OK
12. in the Save Z-Series to File dialog box, enter a file name (8 characters max), make sure Byte (8-bit) is selected, and click OK to begin collecting your data

    Note:Files are saved in C:\COMOS by default, if you want to save your file elsewhere, enter the relative path for the first file and all subsequent file in the session will be saved in that subdirectory. E.g., you want the files to go into C:\COMOS\IMAGES, create the subdirectory in windows first, then for your first save, type in IMAGES\filename

    Note: Your files can be stored temporarily on the harddrive 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.

Some tips on obtaining an image

• at a Scan Speed of F2 each scan takes 1 s, you should adjust the settings slowly while scanning so you can see the changes
• if you do not know the response of your sample
  1. select a low laser power e.g., ND position 3 for fixed and 0 for live samples
  2. open the aperture completely
  3. set the GAIN to about 8
  4. leave the BLACK LEVEL at 5
  5. start scanning
  6. if there is nothing, stop scanning and check the light path
  7. if everything is set correctly, start scanning and then increase the GAIN slowly, adjust the focus as needed
  8. if still nothing with GAIN at 10.0, select the next higher ND filter
  9. unless your sample is really weak or completely out of focus, you should start to see something
  10. as your object comes into view, you can start to optimize it by checking the focus, and then picking an appropriate setting for the aperture, GAIN, ND filter, and BLACK LEVEL

Image Optimization

• to maximize the dynamic range, apply SETCOL.LUT to help set the correct gain and black level first and then fine tune them for your image (see the appendix for an explanation about using SETCOL)
• use as large an aperture size as you can tolerate so you can use less laser intensity and lower gain
• use Kalman: select the number of passes in N scans
• use a slower scan speed—more laser exposure!
• for really low signal, try the accumulate filters

Routine Shutdown (as listed in the log sheet)

1. select OFF for the focus motor, exit CoMOS
2. turn off the laser and scan head
3. start transferring your files using the zip, 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 60× objective
8. rotate the objective turret to put the prism in the light path
9. turn off the image monitor
10. when you are done transferring your files, turn off the menu monitor
11. put the cover on the microscope
12. complete the log and record Time Out
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.

The specification of the filter blocks

*DF:

discriminating filter with very steep-sided passbands

DRLP:

dichroic longpass filter

EF:

edge filter

EFLP:

longpass edge filter

LP:

longpass filter

OG:

orange glass (absorbs blue light)

Removal and Replacement of the Filter Blocks

Caution: ALWAYS stop scanning first before changing filter blocks and do NOT touch the optical surfaces.

Why you should not adjust the mirror, M1

It is critical that M1 be centered or you will experience a severe drop in intensity and uneven illumination. It should only be adjusted by Facility Staff with the REFL block installed. All other blocks should have the beam centered by a slight and gentle rotation of the block.

Here is the reason. The filter block is held in place by 3 pins against the mounting ring. Wear and tear on the pins and the seats on the mounting ring has created some play which allow the block to rotate a few degrees upon its longitudinal axis. Therefore, it is not always possible to place the block in the appropriate position. The REFL block is the least worn and still maintains the factory alignment. Once the beam is centered with the REFL block, the correct placement of the other blocks on the mounting ring can simply be found by keeping the beam in the center of the target.

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. powerup laser
4. powerup scan head
5. launch CoMOS
6. optimize M4 (and M5, if applicable)
7. locate specimen at low magnification
8. move to your area of interest
9. begin scanning and get data!
10. quit CoMOS
11. poweroff laser
12. poweroff scan head
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