Zeiss LSM 510 Manual by 6rlEDhL

VIEWS: 0 PAGES: 25

									ZEISS LSM 510
 CONFOCAL
MICROSCOPE


  OPERATING
INSTRUCTIONS
   (Last Revision: 5/21/2009)
ZEISS LSM 510 POWER ON/OFF PROCEDURES
POWER ON:
1. Ensure that all of the equipment is turned off
2. Start the system in the order given:
   A. the mercury lamp is never switched off; make sure that it is on
   B. switch on the Remote Control switch




  C. press the On/Off button on the computer tower if the computer did not
      start
  D. wait for the Windows login window to appear on the monitor
  E. simultaneously press the ctrl-alt-delete keys and log in as follows:
     1) User name: your unique name
     2) Password: assigned password
  F. if the UV laser is to be used, wait for the Desktop to load and open the
      cold water valve handle by rotating it to the vertical position in the
      alcove next to the entry door to the confocal room then flip the
      Coherent Enterprise UV laser Main Power black toggle switch (first
      confirm that the key switch is at the 12 o’clock position) to the up
      position (ON) and then rotate the key switch to the 3 o’clock position
      (ON)
POWER OFF:
1. Before switching off the laser(s) check the microscope schedule to see if
   another user is scheduled to use the microscope later. If this is the case
   contact that next user and inform him or her that the microscope is open
   for use and that it will be left on and running; otherwise, proceed with step
   2. below, also, see page 25 step 7.
2. Click the Load button in the Stage and Focus Control window and remove
   the sample, blow off the water on the 40X or 63X objective lens (if used),
   and click the 5X objective lens button in the Microscope Control window
3. In the LSM 510 Expert Mode window in the Acquire mode, click on
   the Lasers button and switch off all of the lasers; if either or both the UV
   and/or the Argon laser(s) is/are on, first move the % output button fully
   left, click the Standby button and then the Off button for each laser
4. Click File and Exit in the Expert Mode window
5. Click Exit in the LSM Switchboard window
6. Transfer the images made during this session to the Photon server
7. Click on the Start button > Shutdown > OK
   A. wait for the computer to shut down and for the Argon laser cooling fan
       to stop (if it is on) and then switch off the Remote Control switch
8. Switch off the Coherent Enterprise UV laser power supply key switch (if
   it is on) by rotating it to the 12 o’clock position (Off) and then push the
   black Main Power switch down (Off)
   A. wait 5 minutes and close the cold water valve in the alcove
OBJECTIVE LENSES

   2.5X (Plan-Neo) NA=0.075 WD=9.3mm
     10X (Plan-Neo) NA=0.3 WD=5.6mm
     20X (Pan-Neo) NA=0.5 WD=2.0mm
 40X (C-Apochr) NA=1.2 WD=n/a (with collar)
 63X (C-Apochr) NA=1.2 WD=n/a (with collar)




                     4
Zeiss LSM 510 Operating Procedures
1.   Upon completion of the Power On steps given above, one should
     ensure the following:
     A.    all unnecessary filters are removed from the pathway of the
           tungsten, or transmitted, light
     B.    the fluorescence exciter shutter is closed (pulled fully right)
     C.    all fluorescence filter modules are removed from beneath the
           objective lens (fully right)
     D.    the lower polarizing filter is removed from beneath the
           objective lens (fully right)
     E.    the Side Port LSM rod is pushed fully left and the VIS rod is
           pulled fully right
     F.    each ocular is adjusted to its midrange position
     G.    the condenser lens accessories turret is set to DIC .5-1.4 and the
           condenser aperture is open (fully left)
     H.    the ocular shutter rod is pulled fully right

Confocal Microscopy

2.   Confocal microscopy mode operation
     A.   ensure that the whole system is powered up as indicated above
     B.   double click on the LSM 510 desktop icon




     C.    in the resulting LSM 510 Switchboard window, click on the
           Scan New Images and Start Expert Mode buttons
           1) wait for the Expert Mode SP2 window to open




                                     5
D.   click on the Acquire button and then the Laser button in the
     Expert Mode SP2 window




                              6
     1)     in the Laser Control window switch on each laser which
            one intends to use
            a)     always choose the Standby button to switch on the
                   UV or the Argon laser
                   (1) wait for the laser status to change from
                         warming up to Ready and click the On
                         button (about 60 seconds)
                   (2) if the Argon laser is to be used, set the laser
                         tube current by sliding the Output [%]
                         button to the right to 50
                   (3) if the Enterprise, or UV, laser is to be used
                         leave the Output [%] button fully left
                   (4) click on Close
E.   click on the Micro button in the Expert Mode SP2 window and
     move the resulting Microscope Control window to a convenient
     and accessible location and select the objective lens to be used
     by clicking of the Objective button and clicking on the desired
     objective lens




                              7
F.   click on the Stage button in the Expert Mode SP2 window and
     move the resulting Stage and Focus Control window directly
     below the Microscope Control window and click on the Load
     button
G.   click on the Config button in the Expert Mode SP2 window
     1)     if one intends to image more than one fluorophore
            simultaneously, click on the Multi Track button in the
            Configuration Control window; otherwise, make sure
            that the Single Track button is pressed in (the default
            position)




                              8
     2)     click on the Config button in the Configuration Control
            window (this is a separate Config button from the one
            above and is located on the right side of this window)
     3)     in the resulting Track Configuration window, click on the
            Configuration subordinate dropdown menu and select the
            combination of fluorophores from the dropdown menu
            which apply to you by clicking on it
            a)     click Apply
                   (1) never click the Store or Delete buttons
H.   click on the Scan button in the Expert Mode SP2 window
     1)     in the resulting Scan Control window Mode page
            a)     make sure that the Frame button is active
            b)     choose the desired image Frame Size [usually 512
                   (for large z-stacks) or 1024 (for single images or
                   small z-stacks)]
            c)     in the Pixel Depth, Scan Direction & Scan
                   Average selection area, ensure that the Data Depth
                   is set to 12 Bit, the Scan Direction is set for
                   unidirectional scan, Mode is set to Line, Method is
                   set to Mean, and Number is set to 1
            d)     in the Zoom, Rotation & Offset selection area, set
                   Zoom to 1, Rotation to 0, and Offset to 0 (these are
                   defaults)




                              9
I.   click the Channels button in the Scan Control window
     1)     in the Channel Settings page, one will notice one or more
            small colored rectangles, i.e., Channels Buttons (usually
            in a vertical stack which indicates that each laser will
            separately scan the sample in the order of the indicator
            colors)
     2)     configure each channel separately prior to scanning the
            sample with the laser(s) by performing the actions listed
            below
            a)     set pinhole diameter to the desired optical section
                   thickness (usually 1.0 micron with the 40X and
                   63X objective lenses)
                   (1) one may set the pinhole diameter to any
                          desired size within its limits if it suits one's
                          purpose, e.g., in multitrack mode, set
                          pinhole diameters to produce optical
                          sections of equal thickness for each channel



                               10
     (2)    one will have to reset the pinhole diameter
            each time after switching objective lenses
b)   move the Detector Gain button to the midrange
     position
c)   set the Ampl Offset button fully right
d)   set the Ampl Gain button fully left
e)   in the Excitation of Track submenu window, set
     those which apply to your setup
     (1) set the 364 nm laser % Transmittance to 10
     (2) set the 488 nm laser % Transmittance to 10
     (3) set the 543 nm laser % Transmittance to 100
     (4) set the 633 nm laser % Transmittance to 50
            Note: the above laser settings may need to
            be changed later if necessary to increase
            fluorescence emission from the sample




                11
J.   before placing a sample onto the microscope stage, click on the
     Load button in the Stage and Focus Control window
     1)     if one has selected either the 40X or 63X objective lens,
            the lens correction collar must be set to the thickness of
            the coverslip, e.g,. if one is using a No. 1 coverslip, set
            the correction collar to 0.14; if one is using a No. 1.5
            coverslip, set the correction collar to 0.17
            a)     the correction collar may require additional small
                   adjustments later in order to produce the sharpest
                   image, i.e., reduce spherical aberration
            b)     one must carefully place a drop of double-distilled
                   water on the top of the objective lens using the
                   syringe provided for this purpose
                   (1) caution: this applies only to the 40X and
                           63X objective lenses
     2)     place the slide on the stage with the coverslip facing
            down and click on the Work button in the Stage and
            Focus Control window
K.   with the microscope set up in either Nomarski (DIC) mode (see
     section 3.A. below) or epifluorescence mode (see section 4.
     below), locate and bring to center an area of interest on the
     specimen
     1)     if the microscope is in DIC mode, pull the lower
            polarizer fully to the right, switch off the transmitted
            light in the Microscope Control window, pull the LSM
            selector rod fully to the right, push the VIS selector rod
            fully to the left




                              12
     2)      if the microscope is in epifluorescence mode, pull the
             fluorescence exciter shutter fully to the right, pull the
             LSM selector rod fully to the right, push the VIS selector
             rod fully to the left
L.   click on the Fast xy button in the Scan Control window
     1)      the laser beam(s) will simultaneoulsy scan at a rapid rate
             and allow for quick changes in the sample focal plane
             with the focus knob and sample placement with the stage
             translators and Detector Gain settings
             a)     caution: do not operate the laser(s) in the Fast xy
                    mode any longer than necessary as it may damage
                    the scanning head device
     2)      click the Stop button
M.   click the Mode button
     1)      select the scan time which will produce the best signal-
             to-noise ratio in the image (often 3.93 secs works well)
     2)      click on the Cont (i.e., continuous) button in the Scan
             Control window in the Channels page of the Scan
             Control window
N.   if the sample image contrast and/or brightness is not properly
     set, the Detector Gain and Ampl Offset must be changed:
     1)      if in multichannel mode (i.e., more than one Channel
             button showing in the Channels page of the Scan Control
             window), click on the Split xy button in the sample image
             window




                              13
2)   decide which channel requires adjustment and click on its
     corresponding Channels button
3)   if necessary change the Detector Gain by sliding the
     button to either increase or decrease image brightness
     (i.e., white level) [with the laser(s) running] and/or
     change the Ampl Offset (i.e., black level) button to reduce
     background signal levels; repeat this for any other
     channels which require adjustment
     a)      be careful not to produce large areas of excessive
             brightness or the result will be saturated pixels
     b)      make sure that all channels are adjusted at the scan
             speed selected earlier
4)   if the image exhibits a low signal-to-noise ratio, one may
     want to use mean averaging of multiple scans to improve
     image quality prior to saving it
     a)      click on the Mode button in the Scan Control
             window
     b)      in the Pixel Depth, Scan Direction & Scan
             Average selection area, click on the arrowhead in
             the Number drop-down menu and choose a scan
             iteration number (e.g., 4, 8 or 16)
             (1) one should see an improvement in image
                    quality as the laser(s) scans the sample


                       14
           e) one may require further Detector Gain and
               Ampl Offset adjustments
5)   a more systematic and reproducible method for adjusting
     the image quality than is outlined in step 3) above is as
     follows:
     a)    with the laser(s) scanning in Cont mode with the
           scan iteration number set to 1 [see N. 4) b) above]
           and, if in multichannel configuration, click on Split
           xy in the image window; otherwise, click on the
           Palette button in the image window
     b)    in the resulting Color Palette window, click on
           Range Indicator
           (1) the color image(s) become greyscale
                  image(s)




     c)    adjust the Detector Gain for each channel while
           observing its corresponding image, if there are no
           red areas in the sample image increase the
           Detector Gain until one notices the appearance of
           a few small red areas (i.e., gain is slightly too high)



                       15
           d)      adjust the Ampl Offset to the left for the same
                   channel to the point at which blue areas first
                   appear in areas of the image which have little or no
                   fluorescence emission (the amount, or intensity, of
                   blue determines the "blackness" of that area)
            e)     one may wish to make a further minor adjustment
                   to the Detector Gain and click the Stop button
            f)     click No Palette in the Color Palette window to
                   restore color to the image
O.   set the scan iteration (or Kalman) number to 4, 8 or 16 and click
     the Single button in the Scan Control window to scan in the
     image to be saved
     1)     sample images should be saved into a database folder
            (.mdb) in .lsm format by the following procedure:
            a)     click on the Save As button in the image window




           b)     in the resulting Save Image and Parameter As
                  window, click on the New MDB button to create a
                  database folder in which to store images in lsm
                  format and corresponding microscope imaging
                  parameters
           c)     in the resulting Create New Database window,
                  navigate to your user folder (if present) in the
                  Shortcut to Users folder on the Desktop or create
                  your folder
           d)     enter the database folder name in the File name
                  blank and click on the Create button




                              16
     e)    in the Save Image and Parameter As window,
           enter the image name and other information as
           desired and click on the OK button
     f)    never place current images into an older database
           folder; always create a new database folder each
           separate occasion one uses the instrument




2)   sample images can be saved in uncompressed tif format
     by the following procedure:
     a)    click on the xy button in the image window
           (1) if the Split xy button is active, the split
                  channel image will be saved as seen in the
                  image window
     b)    click on File in the Expert Mode SP1 window and
           choose Export Images
     c)    in the resulting Export Images and Data window,
           click into the drop-down menu in the Image type
           selection area and choose Full Resolution Image
           Window Single
           (1) if one is saving a z-stack, choose Full
                  Resolution Image Window Series




                      17
           d)      select the destination folder in the Save in selection
                   area (one should create a separate folder in one’s
                   user folder for tif images)
                   1)     never attempt to store tif images in your
                          database folder
            e)     enter an image file name
                   (1) do not use commas, periods, hyphens, or
                          backslashes
            f)     select tagged image file format (tif) in the Save as
                   type selection area
            g)     click the Save button
P.   to scan a sequential series of optical, or "z", sections at a
     particular xy-coordinate, one must first locate the z-plane of
     brightest fluorescence emission (for each channel if using
     multitrack) and fully maximize the image quality as indicated
     above for a single image, then
     1)     click the Stop and the Z Stack buttons in the Scan Control
            window




     2)    click the Fast xy button
     3)    rotate the right-hand focussing knob clockwise to move
           the objective lens focus point to the upper limit of the z-
           stack
     4)    in the Z Settings submenu, click on the Mark First/Last
           subordinate menu tab
     5)    click on the Mark First button
     6)    move the objective lens down (focus knob
           counterclockwise) to the lowest z-plane in the z-stack


                               18
            and click on the Mark Last button and click on the Stop
            button
     7)     click on the Z Slice button in the Z Settings window
     8)     click on the Optimal Interval button in the resulting
            Optical Slice window
            a)      close the Optical Slice window
            b)      this satisfies the Nyquist sampling rate
            c)      one can ignore the Z Slice button and set the z-
                    interval to equal the optical slice thickness if one
                    wants to do so, while realizing that there would be
                    no oversampling
     9)     set the scan iteration number to the desired value and
            click on the Start button to begin the z-stack collection
   10)      save the z-stack as described above, except one should
            create a separate folder for the z-stack images within the
            tif folder
Q.   to create a projection of the z-stack, one may want to first delete
     images from either the beginning or ending points or both of the
     z-stack images
     1)     click on the Gallery button in the image window




             a)    in the resulting montage image window of the z-
                   stack images, click on the Subset button and



                               19
                       choose new beginning and ending points for the
                       modified z-stack and click OK
                       (1) this creates a new image window with the
                              truncated z-stack in it; the original z-stack is
                              left unchanged
          2)    click on the 3-D View button and the Projection button in
                the Expert Mode window
          3)    in the Projection window make the necessary settings
                which will produce the various projected views desired
                or do nothing to produce the "classic" single projection
                image
                a)     click the Apply button
          4)    the resulting projection image window contains all of the
                projections in the z-stack, which may be viewed using
                the Slice, Anim or Gallery buttons as needed
          5)    save the projection(s) as described above

3.   Nomarski (DIC) Mode Microscopy (visible and LSM methods)

     A.   Visible Method
          1)    configure the microscope as indicated above (see Step 1)
          2)    ensure that the objective lens correction collar is set
                correctly (applies only to the 40X and 63X lenses, see
                above)
          3)    switch on the transmitted light in the Microscope Control
                window
                a)    click on the Transmitted Light button




                b)    click the ON button to switch on the transmitted
                      light source, i.e., 12V 100W tungsten/halogen bulb



                                   20
           c)      adjust the light intensity by sliding the button to
                   the right
     4)    place the sample onto the microscope stage and bring the
           objective to focus on a chosen area using the focus knob
     5)    focus and center the condenser lens by Koehler's method
           as follows:
           a)      fully close the field diaphragm
           b)      using the condenser lens focussing knob, bring the
                   inner edge of the field diaphragm into sharp focus
           c)      open the field diaphragm as fully as possible
                   without allowing any part of the inner edge to go
                   outside of the field of view
           d)      if necessary center the field diaphragm image
                   using the condenser lens centering knobs
           e)      once centered, open the field diaphragm only to
                   the point at which the inner edge goes just outside
                   the field of view
           f)      if the 40X or 63X objective lens is used, set the
                   condenser aperture lever so that it is aligned with
                   the D in the DIC .5-1.4 logo on the accessory turret
                   immediately below the condenser aperture lever
     6)    insert the polarizer at the top of the condenser lens and
           make sure the polarizer handle is set to the 6:00 o'clock
           position
     7)    slide the lower polarizer (located below the objective lens
           turret but just above the reflector turret filter modules and
           has a white dot on the handle) fully to the left
     8)    the sample can now be viewed in the DIC mode
B.   Digital Image Acquisition Method
     1)    before proceeding take note of the fact that the Nomarski
           (DIC) digital image is not a confocal image, i.e., not an
           "optical section"; it is a transmission light microscope
           image and must be focussed as one would do for any
           light microscope
     2)    one's sample should first be located and focussed as
           described in the above visible DIC mode section
     3)    if the transmitted light is on, switch it off by moving the
           Intensity % slider button to 0 and clicking on the ON
           switch button, which turns it off, and close the window



                              21
4)    rotate the condenser lens polarizer to the 3 o'clock
      position, i.e., 90º counterclockwise
5)    fully open both the field diaphragm and the condenser
      aperture
6)    pull the objective lens polarizer rod fully to the right
      to remove it from the light path
7)    pull the LSM selector rod fully right
8)    push the VIS selector rod fully left
9)    one should use the argon laser as the illumination source
      as described in the following steps
10)   for example, if the FITC/Rhod multi-track configuration
      is in use, click once on the Rhod track configuration in
      the Configuration Control window checkmark in the List
      of Tracks area to disable it (checkmark removed from
      box)
11)   click once on the FITC track in the above selection area
      to highlight it (do not remove the checkmark)
12)   below click on the checkmark in the Beam Path and
      Channel Assignment area (to remove it) next to the green
      channel (i.e., Ch 3) symbol [this switches off the
      photomultiplier tube (PMT), or detector]
13)   click on the empty check box next to the Transmission
      button in the Beam Path and Channel Assignment
      selection area of the Configuration Control window (to
      activate the transmitted light detector)
14)   click on the Transmission button
      a) in the resulting Channel Color Selection window,
          click on the white button and close the window
15)   make sure that the Detector Gain button is fully left
      and click on the Fast x,y button, adjust the detector gain
      and amplifier offset and focus the sample image and click
      the Stop button
16)   set the scan time to 3.93 secs in the Mode page, click on
      the Cont button, adjust the detector gain and ampl offset
      for best image quality; click the Stop button
17)   in the Mode page choose Number 8 or 16 in the Scan
      Average selection area and click the Single button
18)   save the image as described above




                        22
4.   Epifluorescence Mode Microscopy
     A.    ensure the the following are set correctly:
           1)    the transmitted light is off
           2)    the objective lens polarizing filter is pulled fully right




           3)     the LSM rod is pushed fully left
           4)     the VIS rod is pulled fully right




                                     23
     5)    the ocular shutter rod is pulled fully right




B.   click on the Reflector button in the Microscope Control window
     and click on one of the three available fluorescence modes, i.e.,
     blue, green or red
     1)     the filter module selected will move into position beneath
            the objective lens
C.   push the fluorescence exciter shutter fully left and view the
     sample




                              24
           1)     to return to confocal mode, pull the exciter shutter fully
                  right, reverse steps 4.A.3) and 4) above, and begin
                  scanning the sample
5.   Photon Server File Transfer and Temporary Storage
     A.    never separate lsm-formatted images from their respective
           database folders
     B.    close all image folders and image windows
     C.    double-click on the Shortcut to Photon folder on the Desktop
     D.    connect to the server as milguest and use milguest as the
           password
     E.    if you have an existing folder in the server, open it or create
           a folder and name it with your uniquename
     F.    open the Shortcut to Users folder on the Desktop
     G.    highlight the image folders for transfer and drag and drop them
           into your folder in the Photon server
     H.    when completed close both Photon and Shortcut to Users
           folders
6.   be sure to remove all image files from the confocal computer and
     Photon server as soon as possible
     A.     all image folders and images 30 days or older will be deleted
            from the Shortcut to Users folder without notification
     B.     all user folders will be deleted from the Photon server after
            seven days without notification
7.   See page 2, Power Off procedures
     A.    if another user will operate the microscope later, be sure to
           leave on any laser(s) to be used by that user
           1)     leave the Enterprise and Argon lasers in the standby
                  mode if they are on




                                    25

								
To top