Quick Start Guide Zeiss LSM 510

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Quick Start Guide Zeiss LSM 510 Powered By Docstoc
					Quick Start Guide
 Zeiss LSM 510
Table of Contents                             (Continued on next Page….)



1. SYSTEM OVERVIEW
      1.1 System Components……………………………………………………………. ……………………..1
      1.2 Basic Microscope Components………………………………………………………………….2
2. STARTING UP THE SYSTEM
      2.1 Start the Hardware……………………………………………………………………………………3
      2.2 Start the Software………………………………………………………………………………….. 5
      2.3 Turn on the Lasers……………………………………………………………………………………..6
3. SETTING UP THE MICROSCOPE
      3.1 Finding the Specimen………………………………………………………………………………….7
      3.2 Focus and Align the Condenser………………………………………………………………..9
      3.3 Using DIC Optics………………………………………………………………..……………………...11
      3.4 Check Specimen for Fluorescence………………………………………………………….12
4. CONFOCAL CONFIGURATIONS
      4.1 Configurations ……………………………………………………………………………………………..13
      4.2 Single Track………………………………………………………………………………………………….15
      4.3 Multi Track……………………………………………………………………………………………………16
      4.4 Configurations Explained……………………………………………………………………………17
5. SCAN PARAMETERS
      5.1 Frame Size…………………………………………………………………………………………………….19
      5.2 Data Depth…………………………………………………………………………………………………… 20
      5.3 Frame Size and Bit Depth Explained………..………………………………………….. .21
      5.4 Scan Speed…………………………………………………………………………………………………….22
6. PINHOLE SETTTINGS
      6.1 Confocal Principle Explained………………………………………………………………………23
      6.2 Set the Pinhole- Single Channel……………………………………………………………….24
      6.3 Set the Pinhole- Multi Channel………………………………………………………………..25
Table of Contents                           (Continued)



7. IMAGE ACQUISITION
      7.1 Setting Gain and Offset……………………………………………………………………………26
      7.2 Scanning Options………………………………………………………………………………………..27
      7.3 Optimization- Single Channel…….…………………………………………………………….28
      7.4 Optimization- Multi Channel……………………..……………………………………………..30
      7.5 Gain and Offset Explained……………………………………………………………………….32
      7.6 Optical Zoom………………………………………………………………………………………………..33
      7.7 Optical Zoom Explained………………………………………………………………………………34
      7.8 Line Averaging……………………………………………………………………………………………..38
      7.9 Frame Averaging…………………………………………………………………………………………39
8. Z-SERIES
      8.1 Collecting a Z-Series…………………………………………………………. ……………………..40
      8.2 Optical Thickness Explained..…………………………………………………………………. 43
      8.3 Viewing a Z-Series……………………………………………………………………………………….44
9. REGION OF INTEREST
      9.1 Scanning an ROI…………..………………………………………………………………………………47
      9.2 Scanning Multiple ROI’s………...…………………………………………………………………. 49
10. TIME SERIES AND BLEACHING
      10.1 Setting up a Time Series………………………………………………. ……………………..50
      10.2 Setting up a Basic Bleach Experiment…….…………………………………………. 51
11. WORKING WITH IMAGES
      11.1 Adding Overlays..………..………………………………………………………………………………54
      11.2 Extracting a Region…….………...…………………………………………………………………. 55
12. SAVING AND EXPORTING
      12.1 Saving an Image..………..………………………………………………………………………………56
      12.2 Exporting Data….………...………………………………………………………………………….. 58
13. SHUTDOWN PROCEDURE..……………………………………………………………………………………59
Note
•   Much of the information contained within this
    booklet is derived from the LSM510 Guided Tour
    by Carl Zeiss Inc. available on the Zeiss ftp site:
    ftp://lsm.zeiss.com/User_Area/HowTo_Guides/Co
    nfigGuides/Literature%20&%20Guided%20Tour/
  System Components
                                            Lasers
 Axiovert 100M Inverted Microscope




  Halogen Lamp            Control Panel
  Power Supply



Mercury Lamp
Power Supply                              Computer




                                                     1
  Basic Microscope Components

  Eyepieces               Field
                          Diaphragm


                           Condenser
                           Focus


                           Condenser

                           Mercury
                           Bulb
                           Housing


                            Remote
                            Control



                                Stage
                                Control



Control
Panel                  Tube Slider
              Focus
              Knob
                                          2
Start the Hardware

                1)   Open the valve on
                     the Nitrogen tank
                     located just
                     outside the
                     confocal room.




                2)   Make sure the
                     front valve reads
                     40. If it doesn’t,
                     adjust the
                     pressure by
                     turning the black
                     knob to the right
                     of the gauge until
                     the pressure
                     reads 40.




                                          3
     Start the hardware
3)   Turn on the mercury
     arc lamp (located
     underneath the
     microscope table).
     Note the start time
     on the sign-up sheet.

     Note- The mercury
     lamp should be left
     on for at least 30
     minutes. After
     turning it off, it
     should not be turned
     on again for 30
     minutes.

4)   Turn on the Remote
     Control switch
     (located to the right
     of the microscope).

5)   Turn on the PC if it
     does not boot up
     automatically.

6)   Login with your lab’s
     username and
     password.
                             4
     Start the software

1)    Double click the LSM 510 icon.

2)    The LSM 510 Switchboard window will appear.

3)    Select Scan New Images and then Start Expert
      Mode.

      Note! If Scan New Images is not selected, the
      software will still initialize but you will not be able to
      control the lasers or the microscope.




                                                                   5
          Turn on the lasers




1)         Select the Acquire
           button on the toolbar.
           The lower toolbar will
           change to display the
           acquisition controls.

2)         Click on the Laser
           button.

3)         Turn on the lasers that
           you will need to acquire
           your images.


     a)    To turn on the Argon laser, click the Standby button
           and wait approximately 30 seconds. When the status
           shows Ready, click the ON button and run the Tube
           Current up to 6 amps by dragging the Output slider bar.

     b)    To turn on the HeNe1 and/or HeNe2 lasers simply click
           the ON button (they do not have a standby mode).

                                                                     6
     Find the specimen
1)     Select the Micro button on the
       toolbar.

2)     Turn ON the transmitted light
       source (halogen lamp):
                                    a)   Click here.


                                    b)   Transmitted
                                         Light window
                                         appears; click
                                         ON.




3)   Select an objective lens. This can be done
     through the software or by selecting a button
     on the control box to the left of the
     microscope.
                                                          7
     Find the specimen

4)   Place your slide
     coverslip-side down on
     the microscope stage.
     Note- If you are using
     an oil immersion lens,
     place a small drop of oil
     on the coverslip first.

5)   Pull out the bottom
     tube slider on the
     microscope to direct
     light to the eyepieces
     (the top slider should
     always be pushed in).
                              Focus Knob      Stage control


6)   Focus and position your specimen. You can
     adjust the illumination intensity by dragging
     the slider bar in the Transmitted Light
     window.




                                                              8
     Focus and Align Condenser

1)   While looking at your
     specimen, stop down
     (close) the field
     diaphragm most of
     the way by turning
     the diaphragm
     clockwise.

2)   Focus the condenser
     until you see a small    Condenser focus knob
     circle/octagon of        (raises and lowers condenser)
     illumination (this is
     the field iris
     diaphragm). Focus
     until the edges of the      Alignment screws
     octagon are clear and
     in sharp focus.

3)   Center the octagon by
     turning the alignment
     screws.

4)   Open the field
     diaphragm back up so
     that you can see the
     entire field of view
     again.

                                                              9
     Focus and Align Condenser

5)     Adjust the numerical
       aperture (NA) of the
       condenser to
       increase/decrease
       contrast and
       resolution.



     Note- When taking DIC images using the laser, the
     laser illuminates the specimen and some of this light
     is transmitted through the specimen to form an
     image. That light is collected by the condenser lens
     and transferred to a detector (Channel D). So, if you
     want to take a nice looking DIC image with the laser,
     you’ll need to focus,align and adjust the NA of the
     condenser lens.




                                                             10
     Using DIC Optics

1)     To find your specimen using DIC (differential
       interference contrast) microscopy, push the
       Analyzer in.

      Analyzer Out                   Analyzer In




2)    Rotate the polarizer or adjust the DIC slider
      (underneath the objective lens- not shown)
      until you achieve the contrast you desire.



                                      Rotate polarizer




     Note- Before taking a confocal image with the
     laser, pull the analyzer back out of the light
     path. Because laser light is inherently polarized,
     it will interfere with confocal imaging.
                                                          11
     Check Specimen for Fluorescence

1)    Under the Micro
      button on the toolbar,
      turn off the
      transmitted light
      source by clicking the
      ON button again.

2)    Select a fluorescence
      filter from the
      software window
      (Click on Reflector) or
      by using the control
      panel.

3)    Then Select….
     a. Rhodamine
     b. FITC (fluorescein)
     c. FSet16wf (Long
         Pass 515nm)

     Note: When you are finished
     finding and screening your sample,
     Select Reflector: None. This will
     block the light from the mercury
     lamp so that you do not
     photobleach your sample!
                                          12
 Configurations
Now that you’ve found
your specimen, it’s time
to set up the microscope
to take a confocal image.

The first step is to set
up a configuration based
on the fluorescent dye(s)
you are using.

Select Config and the
configuration control
window will appear.

You may choose Single
Track or Multi Track
depending on your
application.




                            13
     Configurations

            Single Track                   Multi Track

     Used for single, double          Used for double and
       and triple labeling               triple labeling

      Simultaneous scanning               Sequential
              only                         scanning

           Advantage:                       Advantage:
     Faster Image Acquisition          When one track is active,
                                       only one detector and one
                                     laser is active. This reduces
                                           emission crosstalk


            Disadvantage:                 Disadvantage:
       Cross talk between channels   Slower Image Acquisition


*After Zeiss LSM510 Guided Tour



    Recommendation- Unless you need to acquire images
    quickly, only use a single track configuration when
    you have a sample that is labeled with one
    fluorescent dye. Choose multi tracking when your
    sample is dual or triple labeled. This is particularly
    important if you are doing colocalization studies.
                                                                     14
     Single Track

1)   Select Config
     from the main
     toolbar.

2)   The
     configuration
     control window
     will appear.
     Select Single
     Track.

3)   Click on the
     Config button to
     load a standard
     configuration.

4)   Open the drop-
     down menu to
     choose a
     configuration.
     Click Apply.

5)   If you want to
     take a
     transmitted
     light image,
     choose ChD.

                        15
      Multi Track
1)   Select Config.

2)   The Configuration
     Control window will
     appear. Select Multi
     Track.

3)   Click on the Config
     button to load a
     standard
     configuration.

4)   Open the drop-down
     menu to choose a
     configuration. Multi
     tracks are indicated
     by a * in front of the
     track. Click Apply.

5)   You can switch tracks
     after each Line or
     Frame.

6)   To take a
     transmitted
     light image,
     choose ChD.
                              16
       Configurations explained
     Note- For more information, see the Beampath Configuration Guide by Zeiss




                                                                        Confocal
                                                                        Detectors




Secondary
dichroic
beamsplitter



Primary
dichroic
beamsplitter



  Single Track Example- The HFT 488 (laser dichroic) will direct 488nm
      light to the specimen and allows the longer wavelength emitted light
      emitted by the sample to pass through. This light is directed to one
      of the three detectors (in this case, Channel 1) by the use of plates
      (transmit 100% light) and mirrors (deflect 100% light). In this case we
      want the light to pass through to Channel 1, so we can leave the
      additional beamsplitters blank (None). The emission filter located in
      front of the detector (in this case, a long pass 505nm filter) selects
      the wavelengths that are allowed to pass through to the detector. The
      confocal pinhole is located in front of the detector.
                                                                             17
     Configurations explained
 Note- For more information, see the Beampath Configuration Guide by Zeiss




Multi Track Example (Rhodamine/FITC line-switching) - The HFT 488/543
    will direct both 488nm and 543 light to the specimen and allows the
    longer wavelength light emitted by the sample to pass through. In the
    case of Rhodamine, 543nm laser light is directed to the specimen and
    light longer than 545nm passes through the secondary dichroic
    beamsplitter (NFT 545) to Ch1. In the case of FITC, 488nm light is
    directed to the specimen and the light emitted by the specimen that is
    shorter than 543nm is directed to Ch3. Scanning sequentially reduces
    the possibility of emission crosstalk that occurs when using
    simultaneous excitation and detection.
                                                                         18
     Scan Parameters – Frame Size
1)   Select Scan.

2)   Select Mode




3)   Select the Frame Size. You may choose a
     predefined value (for example 512 x 512) or enter
     your own values. A larger frame size means you will
     collect more pixels per unit area scanned,
     effectively increasing the resolution of the image.
     However, it will also mean a larger file size. A good
     starting point is 512 x 512.

4)   If you select Optimal, the software will calculate
     the appropriate number of pixels depending on the
     NA of the objective and the wavelength of light you
     are using.

     Note- The number of pixels influences the image
     resolution!
                                                             19
     Scan Parameters – Bit Depth

1)   Still in the Scan Control Window, select your
     Data Depth (Dynamic Range).




     8 bit data depth will give 256 gray levels - each
     pixel in your image will be scaled between 0
     (black) and 255 (white). 12 bit will give 4096
     gray levels. Photoshop 5 will import 12 bit
     images.

     Publication quality images should be acquired
     using 12 bit.




                                                         20
    Frame Size & Bit Depth Explained
    When your sample is scanned, the emitted fluorescence
    signal is detected by a PMT (photomultiplier) and
    converted into digital picture elements (pixels).

    Pixels are represented by x and y values. The x value
    specifies the horizontal position (column) and the y value
    indicates the vertical position (row). A larger frame size
    means you will collect more pixels per unit area scanned,
    effectively increasing the resolution of the image.
      x


y




    Fewer Pixels          More Pixels
    Smaller Frame Size    Larger Frame Size
                          (same field of view)

    Each of these pixels has a specific intensity value. The
    range of intensity values you collect depends on the bit
    depth you select. 8 bit data depth will give 256 gray levels
    - each pixel in your image will be scaled between 0 (black)
    and 255 (white). 12 bit will give 4096 gray levels.

    Thus, the quality of the digital image is determined
    by the number of pixels (frame size) and the bit
    depth (range of intensity values) for each pixel.
                                                                   21
     Scan Parameters- Scan Speed

1)   Still in the Scan Control Window, select
     your Scan Speed.




     Note- A higher scan speed will reduce the
     possibility of photobleaching your sample
     but will result in a noisier image.

     Note- A slower scan speed will increase the
     time the laser illuminates the specimen
     (increasing the possibility of
     photobleaching) but will result in a less noisy
     image.

     **For superior image acquisition, I
     recommend a scan speed of 7-9 coupled with
     Line or Frame Averaging to produce an
     image with the best signal to noise ratio.
                                                       22
        Confocal Principle Explained
                                           Detector
                                       (Photomultiplier)

     Source pinhole                          Detector Pinhole
(Illuminating Aperture)                    (Confocal Aperture)


                                     Dichroic
                                     Beamsplitter


                                          Features above and
     Light Source                         below the plane of
        (Laser)                           focus fall outside the
                                          pinhole and are not
                                          collected by the
                                          detector- producing a
                                          true optical section




                                      Specimen
 Focal Plane                                               Focus




   *After Zeiss LSM510 Guided Tour
                                                                   23
     Set the Pinhole- Single Channel
1)    Select the
      Channels
      button.

2)    For single
      channel
      acquisition,
      highlight the
      channel and
      select a
      pinhole size of
      1.0 Airy Units.
                          y

3)    0.8-1.0 Airy
      units produces
      the best                                          x

      signal : noise
      ratio.                                       Diffraction
                                                   pattern
      Note-
      Adjusting the
      pinhole                 *After Zeiss LSM510 Guided Tour
      changes the
      Optical Slice.


     Note- opening up the pinhole will allow more out
     of focus light to reach the detector but will also
     decrease resolution in the z-axis.
                                                                 24
     Set the Pinhole- Multi Channel

1)    Select the
      Channels
      button.

2)    When collecting
      multi channel
      images,
      highlight the
      longer
      wavelength
      channel first (in
      this case red)      y

      and select a
      pinhole size of
      1.0 Airy Units.                                  x


3)    Then, highlight                             Diffraction
      the shorter                                 pattern
      wavelength
      channel and
      adjust the
      Optical Slice to         *After Zeiss LSM510 Guided Tour
      match.

     Note- When taking multi channel images, make
     sure the pinholes are adjusted so that each
     channel has the same Optical Slice. This is
     very important, particularly for colocalization
     studies.                                                    25
      Setting Gain and Offset

1)      Select each Channel
        and set the
        Detector Gain
        relatively high- 850
        to 1000volts is a
        good starting point.

2)      Leave the Amplifier
        Offset at 0 for now.

3)      Make sure the
        correct laser is
        checked in the
        Excitation window
        and adjust the laser
        attenuation by
        dragging the slider
        bar.


     Suggestion- Keep the 488nm laser power at 10%
     or below. The 543nm and 633nm lasers can be set
     at 50-100%.

     Note- it is best to keep laser power as low as
     possible to avoid damaging or photobleaching your
     sample.
                                                         26
Scanning Options
You have several options to scan your image….

                  •   Selecting Fast XY will provide
                      a continuous fast scan of the
                      specimen. This is useful for
                      finding and changing the focus.

                  •   Single will take a single scan of
                      the image at the Scan Speed
                      previously selected.

                  •   Stop blanks the laser beam and
                      stops the scanning mirrors.

                  •   Select Continuous to
                      continuously scan your
                      specimen at the Scan Speed
                      previously selected.




Note- before scanning your
image, push the microscope
tube slider back in. If you
forget, the software will
remind you.
                                                          27
     Optimization- Single Channel
                         1)   Push the tube
                              slider on the
                              microscope back
                              in and scan your
                              image using one of
                              the scan options
                              previously
                              outlined.

                         2)   Stop the laser
                              and click on the
                              Palette button.




3)   Choose Range
     Indicator.
     This puts your
     image in gray-
     scale and
     highlights the
     saturated
     pixels (red)
     and highlights
     where the
     signal is at a
     minimum
     (blue).

                                                 28
      Optimization- Single Channel

4)    Click on Cont. and start
      scanning your image.

5)    If you have a lot of
      saturated pixels, reduce
      the gain to ˜750-800V.
      If you still have red
      pixels, decrease laser
      intensity in the
      Excitation panel to
      reduce the intensity of
      the laser light.

6)    Increase the Amplifier
      Offset until all the blue
      pixels disappear, and
      then make it slightly
      positive.
 7)    Make final adjustments to the image by
       increasing/decreasing the Detector Gain. Your
       image should now have very few red and blue pixels.

     Note- if you do not see any saturated pixels, even with a
     high gain, try increasing laser power. Keep in mind that
     higher laser power means increased risk of photobleaching.

Note- If you have decreased your detector gain below 750V
   and are still getting plenty of signal, try decreasing laser
   power instead of lowering the gain any further.
                                                                  29
     Optimization- Multi Channel

1)   Scan your image using one of the scan options
     previously outlined.

2)   Stop the laser and click on Split xy so that you
     can view each channel separately.

     Channel 1 (red)     Channel 3 (green)




        Mixed Image

3)   Select Palette, Range Indicator.

                                                        30
      Optimization- Multi Channel

4)    In the Scan
      Control menu,
      click on each
      Channel
      separately to
      adjust the Gain
      and Amplifier
      Offset (See
      page 29 for
      details).

      Note- It is
      easier to make
      these
      adjustments if
      you are
      scanning line
      by line. Also,
      you may also
      choose to scan
      one channel at
      a time by
      turning off
      each channel in
      the track
      menu.
     Click the box
     to unselect a channel
                                    31
     Gain and Offset Explained
•   Detector Gain determines the sensitivity of the
    photomultiplier by setting the maximum limit.

•   Amplifier Offset determines the minimum intensity
    limit.

•   Amplifier Gain determines signal amplification.



                            Saturation at the maximum;
                            Reduce Detector Gain
                            Saturation at the minimum;
                            Increase Amplifier Offset



                             Gain and Offset set correctly-
                             You are now taking advantage of the
                             entire dynamic range.



                                   Amplifier Gain increases the
                                   whole signal, and the
                                   Amplifier Offset will need to
                                   be decreased.



*After Zeiss LSM510 Guided Tour                                    32
     Optical Zoom

1)   The level of zoom
     can be changed by:

     a)   Using the
          Zoom, Rotation
          and Offset
          control in the
          mode menu.

     b)   Selecting Crop
          in the image
          menu (xy view)




                           33
         Optical Zoom Explained*
        When you use the zoom feature on a confocal
        microscope, the laser is scanned over a smaller area
        of your sample.

        Because the decreased scan area is displayed
        within the same frame size on the computer screen,
        the image is magnified.

        For example, if you scan an image at 512x512 pixels
        and then zoom in on a region of interest, those
        same 512x512 pixels are now obtained from a
        smaller area of your sample- effectively increasing
        the resolution of the microscope.

        Keep in mind, though, that you are still limited by
        the theoretical resolution of a light microscope
        (100-200nm).

        Zooming beyond ~6x will result in “empty
        magnification”, that is, magnification without any
        actual increase in resolution.




Reference-
*Confocal Microscopy for Biologists by Alan R. Hibbs.
                                                               34
         Optical Zoom Explained*
       Zooming is also used to match the objective lens
       resolving power to the image resolution (number of
       pixels collected) in order to satisfy the Nyquist
       sampling criteria.

       The Nyquist Sampling Theorem states that, when an
       analog signal is digitized, in order to retain all of the
       information contained within the signal the diameter
       of each pixel collected must be at least 2.3 smaller
       than the optical resolution of the microscope.

                         No Zoom                            2x Zoom



                                                                                  3-dot specimen
                                                                                  and sampling
                                                                                  intervals




                                                                                   Pixel display
                                                                                   on the monitor




               **Graphic after Douglas B. Murphy




References-
* Confocal Microscopy for Biologists by Alan R. Hibbs.
**Fundamentals of Light Microscopy and Electronic Imaging by Douglas B. Murphy.
                                                                                                    35
         Optical Zoom Explained*

       The optimal zoom factor is determined by the
       magnification and NA of the objective lens, as well
       as the wavelength of the excitation light.

       For the Zeiss LSM 510 microscope, the minimum
       zoom factor (z) is calculated by the following
       formula:



                  z ≥ 3.92 * NA * system constant (8.94mm)
                                # pixels * magnificationobj * λExc



         The following page contains a table with minimum
         zoom factors and corresponding pixels sizes
         (calculated using the previous equation) required for
         each of the three major objective lenses on our
         microscope.




References-
*Confocal Laser Scanning Microscopy – Principles, by Carl Zeiss Inc.

                                                                       36
      Optical Zoom Explained
512x512 Frame Size
      Objective   Excitation Wavelength         Minimum Zoom   Corresponding
        Lens               (nm)                     Factor       Pixel Size
                                                                    (nm)
40x, 1.3NA        458                     4.9                  89.9
40x, 1.3NA        488                     4.6                  95.8
40x, 1.3NA        543                     4.1                  106.6
40x, 1.3NA        633                     3.5                  124.2
63x, 1.4NA        458                     3.3                  83.5
63x, 1.4NA        488                     3.1                  89.9
63x, 1.4NA        543                     2.8                  98.9
63x, 1.4NA        633                     2.4                  115.3
100x, 1.4NA       458                     2.1                  83.5
100x, 1.4NA       488                     2                    88.9
100x, 1.4NA       543                     1.8                  98.9
100x, 1.4NA       633                     1.5                  115.3



1024x1024 Frame Size
      Objective   Excitation Wavelength         Minimum Zoom   Corresponding
        Lens               (nm)                     Factor       Pixel Size
                                                                    (nm)
40x, 1.3NA        458                     2.4                  89.9
40x, 1.3NA        488                     2.3                  95.8
40x, 1.3NA        543                     2.0                  106.6
40x, 1.3NA        633                     1.8                  124.2
63x, 1.4NA        458                     1.7                  83.5
63x, 1.4NA        488                     1.6                  88.9
63x, 1.4NA        543                     1.4                  98.9
63x, 1.4NA        633                     1.2                  115.3
100x, 1.4NA       458                     1.0                  83.5
100x, 1.4NA       488                     1.0                  88.9
100x, 1.4NA       543                     0.9                  98.9
100x, 1.4NA       633                     0.8                  115.3
                                                                               37
     Line Averaging
1)   Averaging
     improves the
     image by
     increasing
     the signal to
     noise ratio.

2)   Under Mode
     in the Scan
     Control
     window,
     select Mode:
     Line.



                             3)   Select Method: Mean.

4)   Choose a Number for averaging. For instance, if you
     select 4, each line will be scanned four times and
     the average pixel intensity will be calculated and
     displayed.

5)   When you are ready to scan, select Single scan.

     Note: The more you average, the better the signal
     to noise ratio (particularly if you have a low signal
     and are using high gain), BUT averaging increasing
     the exposure time of the sample.                        38
     Frame Averaging
1)   Under Mode in
     the Scan
     Control
     window, select
     Mode: Frame.

2)   Select
     Method: Mean.




3)   Choose a Number for averaging. For instance,
     if you select 4, each frame will be scanned four
     times and the average pixel intensity will be
     calculated and displayed.

4)   When you are ready to scan, select Single scan.

     Note: Frame averaging helps reduce
     photobleaching, but does not give as smooth an
     image as line scanning. There is also a longer
     delay between each track when using Multi
     Track.
                                                        39
     Collecting a Z-Series

1)   Focus up and down through your specimen to
     find the brightest focal plane.

2)   Note- Focusing can be achieved manually or by
     selecting Stage from the main menu.




                                        Focus up
                                        and down




                 Set step size


3)   At the brightest focal plane in the sample, use
     the range indicator and adjust the gain and
     amplifier offset until you see very few red and
     blue pixels.
                                                       40
     Collecting a Z-Series
4)   In the Scan
     Control
     window, select
     Z-Stack.

5)   Select Mark
     First/Last.

6)   Select Keep
     Interval.

7)   Start scanning
     using Fast XY
     or Continuous.

8)   Keeping your
     eye on the
     image, focus up
     through the
     sample and
     then select
     Mark First.

9)   Move the focus back in the opposite direction
     to the end of the Z-Series and select Mark
     Last.

10) Click on Z-Slice.
                                                     41
   Collecting a Z-Series
11) The Optimal Slice
    window will appear.
    Click on Optimal
    Interval. The
    computer will
    calculate the
    optimal number of
    slices and ensure    Note- Check here to make sure each
    correct Nyquist     Channel has the same optical slice.
    sampling (slices
    overlap by half
    their thickness).

12) Close the Optical
    Slice window and
    select Z
    Sectioning.

    Note- For more or
    less sections,
    adjust Num Slices.

13) Select Start.

    Note- The Z-
    Series can be
    stopped at any
    time by selecting
    the Stop button.
                                                         42
      Optical Thickness Explained

 1)    The optical thickness
       depends on:
       a) Wavelength λ
       b) Objective lens NA         z                   y
       c) Refractive index n
       d) Pinhole diameter P                 x
       e) D=Pnλ / (NA)2
           (~0.5μm @ 63x1.4)
2)    To ensure optimal sampling (so that you are not
      missing any information), slices should overlap by half
      their thickness. This is called Nyquist sampling.

      Incorrect sampling          Correct (Nyquist) Sampling




*After Zeiss LSM510 Guided Tour                                43
     Viewing a Z-Series

                          1)   In the image
                               window…
                               a) Select
                                   Slice
                               b) Use the
                                   scroll bar
                                   to view
                                   individual
                                   sections.




2)   To animate
     the series,
     select
     Animate. You
     can adjust
     the speed of
     the animation
     by dragging
     the slider
     bar.



                                                44
     Viewing a Z-Series- Gallery

1)   In the image window, select Gallery




2)   Select Data to
     show the scale
     on the image.

3)   Use subset if
     you want to
     extract
     sections.
                                           45
     Viewing a Z-Series- Orthogonal

1)   In the image window, select Ortho




2)   Select mouse (Select).

3)   Position the cut lines using the mouse or by
     adjusting the slider bars.


                                                    46
     Scan a Region of Interest (ROI)
     Note- For faster image acquisition and to save disk
     space, you may choose to scan a region of interest
     (ROI), rather than scanning the entire field of
     view. When you define an ROI, the laser scans
     only that particular region - image resolution and
     magnification remain unchanged (Note- this is
     UNLIKE the zoom feature).

1)   Select Edit ROI from the menu bar.


2)   Select Fit
     Frame Size to
     bounding
     Rectangle.

3)   Choose the
     shape of the
     ROI.

4)   Position the
     mouse pointer
     over your image
     and draw the
     shape around
     the region you
     want to scan.


                                                           47
     Scan a Region of Interest (ROI)
5)   Scan your image.




6)   To remove the
     ROI, you must
     deactivate the
     ROI (unselect it)
     or select the
     overlay and click
     on the blue trash
     bin. Closing the
     ROI window only
     removes the
     overlay- the ROI
     will still be
     active.


                                       48
     Scanning Multiple ROIs

1)   Select Edit ROI from the menu bar.


1)   Unselect Fit
     Frame Size
     to bounding
     Rectangle.

2)   Choose the
     shapes of the
     ROIs.

3)   Position the
     mouse pointer
     over your
     image and
     draw/position
     the shapes
     around the
     regions you
     want to scan.

4)   Scan your
     image.




                                          49
     Time Series

1)   Set up scanning
     parameters as
     previously
     explained.

2)   Select Time
     Series from the
     Main Menu.

3)   Enter the Number
     of scans you would
     like to perform.

4)   Enter a Cycle
     Delay between
     scans if needed
     (select 0 for no
     delay).

5)   Select StartT to
     start time series
     acquisition.

     Note- Start and
     Stop the series
     using the StartT
     button.
                          50
     Basic Bleach Experiment

1)   Set up scanning parameters as previously explained.



2)   Select
     EditBleach
     from the Main
     Menu.

3)   Select Bleach
     after number
     scans and
     enter the
     number of
     pre-bleach
     scans you
     would like to
     take.

4)   Trigger in and
     Trigger out
     should read
     ‘none’.

5)   Select the number of Iterations. This number is
     sample-dependent and depends on how easy it is to
     bleach your region of interest. Iterations: 20
     means the laser will scan the ROI 20 times in
     order to bleach the area.
                                                           51
     Basic Bleach Experiment

6)   Define the ROI
     that you’d like to
     bleach.




7)   Select the
     bleach laser
     and use the
     slider bar to
     run the laser
     up to %100.




                               52
     Basic Bleach Experiment

8)   Open the Time
     Series Control
     Window.

9)   Set up a time
     series as
     previously
     explained (enter
     number of scans
     and time delay
     between scans).

     Note- The
     number of scans
     includes any pre-
     bleach scans you
     entered in the
     Bleach Control
     window.

10) Select StartB to
    start the bleach
    acquisition series.




                               53
     Adding Overlays
1)   In the Image Window select Overlay.




2)   To add a micron bar, click on 1μm, place the cursor
     on the image and drag the bar to elongate it.

3)   You may also add shapes, text, arrows etc.

4)   Click the ruler button to measure the length of a
     line or the area of a shape.

                                                           54
     Extracting a Region
1)   In the Image Window select Overlay.




2)   Using one of the
     overlay shapes, draw a
     shape around the
     region you’d like to
     extract.

3)   Click on Extract
     Region.

4)   Save the new image.

                                           55
      Maximum Intensity Projection
1)   Open your image and select 3D View from the
     Main menu. Select Projection




1)   Open your image
     and select 3D
     View from the
     Main menu.
     Select Projection
     from the sub-
     menu bar.

2)   In the Projection
     menu, select
     Turning Axis=Y,
     First Angle=0
     and Number of
     Projections=1.

3)   Note- the
     difference angle
     does not matter
     because this is a
     single projection.


                                                   55
     Saving an Image
1)   First, ensure that you will be saving your images in
     the correct format. From the Main Menu, select
     Options and the Settings.




2)   Select the Save tab and make sure that the third
     option, (At “Create Database”…) is selected. If it
     isn’t, select it. Click OK.




                                                            56
     Saving an Image
3)   From the Main Menu bar, Select
     File, Save As or select Save As
     from the image window.




4)   Select New MDB to create a new database. File up
     to NewVolume F: (never save to drive C or D).

5)   Type in a file name for your database and hit
     Create. (It is best to create a new database each
     session- that way if a file becomes corrupted, you
     won’t lose all your data).

6)   Back in the Save Image and Parameter As window,
     type in the name of the current image and click OK.
                                                           57
     Exporting an Image
1)   Select File, Export from the Main Menu bar.




2)   Select Image Type-
     a)   To save the raw image data, select Raw data single (for
          a single image) or Raw data series (for a z-series).
     b)   To save your image with any overlays added to it, select
          Full resolution image window single (single image) or
          Full resolution image window series ( z-series).
     c)   Contents of image window saves the image as shown on
          the screen (at screen resolution).
3)   Select Save as type.
     a)   TIF-Tagged Image File is recommended for 8-bit
          images.
     b)   Choose Tiff-12 bit for 12 bit images.
                                                                58
     Shut down procedure
1)   If another user is signed up within 30
     minutes of the end of your session, simply
     log out of the software and leave everything
     on.

2)   If the microscope will not be used again for
     30 minutes, shut OFF all the lasers in the
     software.




3)   Exit the software.




                                                    59
      Shut down procedure

 4)   Shut down the
      computer.

 5)   Turn off the Remote
      Control button.

 6)   Note the time of the
      mercury lamp and write
      it down on the sign-in
      sheet. Turn off the
      mercury lamp.

 7)   Fill in the rest of the
      information on the sign-
      in sheet.

 8)   Shut the valve on the
      nitrogen tank.


9)    Put the cover on the microscope!!!




                                           60