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Center for Cell Death, Injury and Regeneration

Leica

Before using these instructions make sure you have had at least one training sessions on using the system.

Leica TCS SP2 AOBS confocal imaging operating instructions:

 Turn on the red button switches to start the scanner and the PC if necessary.

  1. Scanner should be on for about 1 min before starting the LCS program.
  2. Turn on the mercury lamp
  3. Turn on the microscope control box.
  4. To start lasers:
    1. Turn on only the lasers you will use.  They do not need more than a minute or so to warm up and stabilize, so don’t bother turning them on until you know that you will need them.
    2. Argon (458, 476, 488, 496):

                    i.      turn on the red button switch

                    ii.      turn the power level knob down to ‘min’

                   iii.      turn the left key to ‘on’, then push to ‘start’ and release

                   iv.      adjust power upwards as necessary

  1. Green and orange HeNe’s (543 and 594):

                    i.      turn on the middle key

  1. Red HeNe (633)

                    i.      turn on the right key

  1. Start the LCS program (personal as default, click the start button or “enter”).
  1. If you get an error in the LCS program when it tries to initialize the microscope, you may have to lower the objective until it reaches the bottom of its travel and then try reinitializing the microscope.
  2. It will ask you if you want to initialize the stage.  When you say yes, the objectives will lower and the stage will move to its corners to calibrate itself.  The stage is currently configured to avoid bumping the condenser, so just click OK unless you have some unusual equipment on the stage that might bump when the stage moves long distances. 
  3. The objectives rise again to the same plane after the stage is done initiazing.

    Other things to consider before imaging
  1. Is your sample clean?  Coverslips on slides or in live experiments should be wiped clean; salt and mounting medium should not get on the objectives.
  2. Is the air table floating?  (Vibrations easily and noticeably degrade the image.)
  3. Is the objective clean?
  4. Which objective are you using? 
    1. The 20x objective is an air objective, and should NEVER have oil on it.  There are 63x and 100x objectives that are oil immersion, and there is a 63x glycerol immersion objective.  Oil and glycerol are NOT interchangeable. 
    2. The 100x objective is not UV-corrected, meaning that it is not suitable for use with the 405 nm laser.  However, it will give better Z colocalization for long-wavelength fluorophores like mRFP and Alexa 633 than will the 63x.
  5. The 63x and 100x objectives have aperture collar adjustment rings, which should for almost all experiments be set wide open. The effect of these apertures is dramatic, so if your samples look dim or the whole field is not illuminated evenly, check these collars.  The stop position is sometimes hard to feel, especially on the 63x, but turn till there’s a gentle stop, and the light will be the brightest and the circle of illumination will be the largest.
  6. Is the DIC analyzer pulled out?
  7. The field diaphragm and aperture diaphragm in the excitation path of the mercury lamp are controlled by round, black dials on the left side toward the back of the microscope, and control the fluorescence illumination. For almost all experiments, these should be set wide open, turned all the way counter-clockwise.  These settings don’t matter for confocal imaging.

If you need more help than what’s written up here, check out the program’s help file.

  1. Find your sample visually at the microscope (switch “MicCtrl” to visual if necessary)
  2. Switch “MicCtrl” to Scan mode
  3. Press “Beam” button in the “Acquisition” section
    1. Choose a previously stored setting for your fluorophore.
    2. Or, select the laser line you want from the “Beam Path setting” dialog window, activate one of the PMTs, choose the fluorophore from the dropdown list, and adjust the PMTs detection window to overlap your emission.
  4. Select the scan mode (I always start with xyz, which is the default).
  5. Select the scan format, speed, bit (I always start with 512*512, 400Hz, zoom 1, 8 bit, which are the defaults).
    1. It’s rarely useful to acquire in 12-bit mode.  Clicking back and forth between 8 and 12 bit images scanned with all other settings the same will show you that the dynamic range doesn’t come primarily from the bit depth.  Dynamic range depends not just digitizing small changes in the signal, but also on the noise in the image.  Thus, a 12-bit image is useful only if you have both large and small signals that you want to measure and if you also have low enough noise.  Amazingly enough, most fluorescence images of the type we acquire have many fewer than 255 photons per pixel, in fact more like 10 or 20 photons per pixel.  So in principle, a 5-bit detector would be good enough!  As a rule of thumb, consider using 12-bit acquisition only if your background noise is extremely low (with a very low PMT voltage or large amounts of averaging) and your signal contains areas of both very high and very low intensities that you will have to measure. 
  6. Press “Continuous” button to start scanning
  7. Optimize parameters including:
    1. The z-position within the specimen
    2. The zoom factor (keep an eye on the voxel size)
    3. Pinhole (usually set at 1, but often useful at larger values)
    4. PMT offset
    5. PMT gain (rarely used over 800, better under 700, least noise below 600)
    6. Laser intensity
    7. See the section Optimizing image quality for more thoughts on this process.
  8. Stop scanning, change format, speed, bit to optimize your image and press “Continuous” button to check.
  9. SEQUENTIAL SCANNING
    1. Up to three fluorophores can be detected simultaneously, but excitation of one may cause emission in the acquisition range of another. To avoid this, scan the fluorophores sequentially by having only one laser and one PMT active at any given time.  The confocal can switch between settings at the end of each scanned line, so the acquisition is for most purposes simultaneous. 
    2. To do this, you must set up and save separate Beam settings for each fluorophore.  These two (or three or four) settings must be identical in every respect EXCEPT the laser that is active and the PMT that is active.  In particular, the PMT windows must be the same for each setting, and the line averaging, beam expander, scan speed, zoom, etc. will be the same for all settings. 
    3. Here is one way to set up sequential scanning for GFP and Cy3 together.

                    i.      Frame up the image, determine scan speed, XY resolution etc. that you’ll use for both channels.

                    ii.      Determine the PMT acquisition window settings and lasers that will work for all fluorophores (e.g. PMT1
                            491-540 with the 488 for GFP, PMT2 550-650 with the 543 for Cy3). 

                    iii.      Activate only the first PMT and turn on only the 488 laser, scan continuously, and adjust the voltage, offset, 
                             and laser power for the GFP image.

                    iv.      Save this Beam setting as a distinctive name, e.g. “GFP-Cy3 g”

                    v.      Unclick PMT1, click on PMT2, turn the 488 line to 0%, turn up  the 543 line, and adjust the voltage, offset, 
                             and laser power for the Cy3 image.

                    vi.      Save this Beam setting as a DIFFERENT name, e.g. GFP-Cy3 c”

  1. To scan sequentially

                     i.      Click the “Seq” button in the Beam window

                    ii.      Add the settings you just made by selecting them in the beam window and then clicking Add in the 
                            sequential setup window.

                   iii.      Keep the setting at “by line”

                   iv.      Make sure to leave the sequential window open.

                    v.     Start the acquisition.

    1. NOTE: to change the PMT voltage for one channel, you need to re-save the beam setting for that channel. 
  1.  Additional basic acquisition notes
    1. The overhead light doesn’t interfere with your images as much as you might suspect.  The condenser blocks most light from reaching the objective, and of course the confocal is simply designed to reject out-of-focus light.  Light from the open door to the hallway certainly has no effect on images, and even with the pinhole wide open and the PMT gain up in the normal range (600), there is essentially no increase in the background signal when the overhead lights are on.
 
 
 

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