Instructions for the Leica SP5 II laser scanning confocal microscope

Content: Check-in and Start up • Set up acquistion parameters • Optimize acquistion parameters • Acquire a z-stack • Sequential scan • Check out • Appendix • a printable version of this manualof an older version

Check-in and Start up

1. enter the In time on the log sheet and check previous entries for unusual remarks
2. if needed, record counter of FLUO (the metal halide epifluorescence light source) and turn it on

   Note: Please report to facility staff if counter is over 2000.

3. on the Main Switch Board: switch on PC Microscope, log on when Windows XP is fully loaded
4. on the Main Switch Board: switch on Scanner Power
5. launch LAS AF (desktop shortcut), check Configuration and scanner mode, then click ok

   Note: Usually, Configuration is Machine and Activate Resonant Scanner is unchecked. However, if the previous user has selected simulated SP5 or used resonant scanner, these will become the default when start up, therefore please check carefully before clicking OK.

6. click OK to initialize the stage when prompted
   • this enables Mark and Find, and Tile scan
   • stage is configured to return to the position before initialization
7. on the Main Switch Board: when the software finish loading, switch on Laser Power and turn the key clockwise to On-1 to enable Laser Emission
8. in LAS AF, click on Configuration tab on top left to bring up Hardware Configuration
   • Laser: check to activate lasers that will be used for the session

     Note: If you need the Ar laser, set the power to 20 %. You can use higher power which will shorten the life span of the laser; but one should never go above 80 % into the red region.

   • (optional) Settings > Resolution > Bit Depth: select 12 Bit for 4096 grey levels, the maximum this system can offer

Set up acquistion parameters

1. 3 ways to configure excitation and emission parameters (Beam Path Settings in the central Work area)
   1. use a preset
      1. click on Load/Save single settings
      2. select a Leica or User Settings (default is simultaneous scan, see below for sequential scan)
   2. reuse a setting from a previous scan
      1. open a previous experiment
      2. in the Experiments tab, click to select the desirable scan
      3. right-click to choose Apply Setting or hit Apply button at the bottom
   3. assemble from scratch
      1. set laser power
         • activate the shutter
         • set power level of the desirable laser line, 5 % is a good starting point
      2. select the beam splitter (dichroic)

         selections for different laser lines

         beam splitter \ laser line (nm)	RT 30/70 for backscatter or reflection	Substrate	TD 488/561/633	DD 458/514	RSP 500	DD 488/561
         spectra or details for the beam splitters are in the Appendix
         405	✓	✓
         458	✓			✓	✓
         476	✓		✓		✓	✓
         488	✓		✓		✓	✓
         514	✓			✓
         561	✓		✓			✓
         633	✓		✓

      3. setup the detector
         • select the emission band: drag the lower and upper bounds or double click the slider to enter the begin and end wavelengths

           Caution: Drag the sliders slowly or else they will jam!

         • click on PMT n to adjust PMT Gain* and Offset*; good starting values are 800 and 0, respectively
         • select a LUT as default
         • activate the PMT
2. select the Acquire Operation step on top left, click the Acquisition tab to set up the data format
   1. Acquisition mode panel
      • defaults to xyz simultaneous scan
      • seq. button enables sequential scan
      • Tile scan, and Mark and Find
      • Best Focus
   2. XY panel (click ▶ on upper right corner to expand)
      • Format: frame resolution defaults to 512x512 pixels, max 8192x8192 pixels
      • Speed: line frequency defaults to 400 Hz, scan area will be reduced above 700 Hz, max 2800 Hz
      • Zoom* and panning
      • Average and Accumulate
      • scanfield Rotation*
      • Pinhole*: default is 1 AU and displayed in µm

*these parameters are also adjustable via Smart Panel

Optimize acquistion parameters

1. center your prep in the field of view with either BF or FLUO (see Modes of operation in the Appendix)
2. to toggle on scanning, click the Live button (lower left pane of LAS AF) or use the button on the left side of Smart Panel

   Caution: if there is no signal in the viewer display Window on the right, DO NOT move the stage controls (XYZ) but try the following

   • with BF or FLUO, make sure object of interest is still in focus
   • open up pinhole to increase the slice thickness
   • raise PMT gain
   • lastly, increase laser power

3. bring the object of interest into focus, adjust PMT gain, offset, and laser power to obtain an image with good dynamic range

   Note: the "glow-over, glow-under", Glow (OU) LUT which displays 0 in green and saturation in blue (255 for 8-bit or 4095 for 12-bit), facilitates this setup

   1. activate the Glow (OU) LUT via the Quick LUT button on the top left side of the viewer display Window; this button cycles through 3 LUT: default , Glow (OU) , and grey 
   2. adjust offset so the pixels in the area you think should be darkest just turn green, usually around -0.2 %; increase the sensitivity of the control panel offset knob may help
   3. vary the PMT gain or laser power so a few pixels in the brightest object of interest just turn blue

4. with multichannel simultaneous scan, always check for cross-talk between channels by dropping the laser power of the suspected channel to zero; if cross-talk does present, use sequential scanning

Acquire a z-stack

1. Z-Stack panel
   • z is selectable: z-Galvo (default) or z-Wide
   • current z-Position can be adjusted with the Smart Panel or via software (see z-drives in the Appendix)

     z-drive controls

     controls \ z-drives	microscope focus knob*	Smart Move*	Smart Panel	range
     the objective turret i.e., focus is always adjustable with the microscope focus knob and Smart Move, indepent of the z-drive selection of the software
     z-Galvo			✓	± 250 µm
     z-Wide	✓	✓	✓	> 4 mm

   • in Live mode, move deeper (z more positive) into the prep to where the stack should start, click Begin to register this z-Position (black: unset, red: set)
   • move towards the outside of the prep to where the stack should stop, click End to register this z-Position
   • stop scanning, click the z-step size button and then enter the appropriate slice thickness (check setting slice thickness in Appendix for recommendations)
2. select the number of passes for average in the XY panel
3. click Start (lower middle) to collect the stack

   Note: Capture Image is for taking a single plane at the current z-position with averages or accumulate applied

Sequential scanning

1. set up a simultaneous scan for the fluorophores needed with regard to laser power, emission band, and PMT settings
2. click the button on Acquistion Mode on the upper left to open the Sequential Scan panel
3. click the + button to add as many scans as needed
4. select between lines, -frames, or -stacks (see useful tips below)
5. for each scan
   • drop the unwanted laser power to 0 %
   • select the appropriate beam splitter
   • deactivate the unwanted PMTs
6. (optional) save the setting

useful tips:

• if you use different beam splitters, have overlapping emission bands, apply different number of passes in average or accumulate, or need different size pinhole for different scans, you will need to use the slower "between frames" or "between stacks"
• keep hardware changes to a minimum between scans will ensure fast and smooth operation e.g., use the same emission band for the same PMT in each of the scans
• each scan can have multiple simultaneous channels e.g., the most common usage is adding the transmission detector, or have 2 simultaneous channels with no cross-talk

Stage automation

Caution: whenever the Tile Scan or Mark and Find window is displayed, all tiles or points marked will be processed when you click Start.

• Tile Scan
  1. click to toggle displaying the Tile Scan window
  2. go Live
  3. move the prep/stage to the lower left of the region of interest, focus
  4. click to mark the current position
  5. move the prep/stage to the upper right of the region of interest
  6. click to mark the current position, the software will calculate the matrix needed

     Note: Marking any points outside the established coverage area will expand the matrix but marking any points inside will not change anything. Unfortunately, there is no provision to save the points here or reuse the points from Mark and Find.

  7. move about to make sure the coverage is sufficent
  8. setup Z-stack, configure Average etc., as needed
  9. click Start to acquire data
• Mark and Find
  1. click to toggle displaying the Mark and Find window
  2. if there are existing coordinates, you can save or clear them first
  3. go Live
  4. move the prep/stage to the desirable area and focus on the object of interest
  5. click to mark the current position, which will be listed as positionN
  6. repeat the above 2 steps to mark more positions as needed, i.e., positionN+1, N+2... etc.
  7. you can go back to any position by selecting it from the drop down list
• relevant details
  • for tile scan, current rotation calibration for stitching is -0.19 ° with a 10% overlap
  • tile scan order is row-wise in the viewer display window, starting from the lower left, which corresponds to column-wise starting from the lower right as displayed in the Tile Scan window

    Note: The orientation of the specimen as displayed in the Tile Scan or Mark and Find window is rotated when compared to that as observed through the ocular of the microscope or the viewer display window. Confusing? Indeed!

    

    1. microscope
    viewer display rotated 90 ° right

    

    2. Tile Scan or Mark and Find window
    viewer display rotated 90 ° left

    

    3. viewer display window
  • coordinate system
    • values (mm) displayed in the window denotes the upper right corner of the image in the viewer display window
    • upper left of the stage is 0, 0
    • lower right of the stage is 126, 82

Check out

• in LAS AF, go to Configuration tab > Hardware Configuration > Laser to uncheck all lasers
• lower the turret, remove your prep, remove oil/immersion fluid on immersion objectives, if you have used them; switch to the 10 × objective
• save your experiments and quit LAS AF
• copy your data to external storage media or network file servers, as needed
• turn off FLUO, if applicable
• if no one signs up for the next 2 sessions, please proceed to shutdown everything; otherwise, log out of Windows and complete the log
  • shutdown Windows XP
  • on the Main Switch Board, turn the key counterclockwise to Off-0 to disable Laser Emission and switch off Scanner Power
  • after computer has shutdown, switch off PC Microscope
  • when the Argon laser cooling fan stops, switch off Laser Power
  • complete the log
  • put the cover back on the microscope
• clean up the work area

Appendix

Content

• DMI 6000 inverted microscope: basic controls • modes of operation • using immersion objectives
• beam splitter spectra • working with lif files • scanner

software overview and layout

LAS AF user interface

DMI 6000 inverted microscope

Controls

• focus knob on either side of microscope (z-Wide)
• commonly used functions are controlled by 5 sets of buttons
  1. left rear variable function buttons
     • CHG TL ◑: switch to transmitted light, TL and rotate through different modes TL_BF (bright field), _DIC, _POL (polarization)
     • CHANGE CS: toggle between confocal SCAN and the last selected TL mode
     • COMBI ◑: epifluorescent + DIC
     • Z COARSE Z FINE: toggle z control
  2. left front: Illumination Manager
     • TL/IL: toggle between TL and IL (incident-light i.e., epifluorescence)
     • AP: aperture diaphragm for TL
     • INT : light intensity, press both together to toggle between Coarse and Fine mode
     • FD: field diaphragm for IL
  3. front
     • top row switches should not be activated, or else it may block viewing through the binocular, to rectify
       • press the leftmost button to select binocular
       • press the rightmost button to deselect the supplemental magnification changer
     • SHUTTER: for epifluorescent illumination
     • bottom 6 buttons: for selecting specific cube (DAPI, GFP, DsRed) for epifluorescence; press the middle 2 buttons together to display the programs assigned to the variable function buttons
  4. right rear variable function buttons
     • CHGOBJ ↻: rotates the objective turret one position clockwise in the current mode (dry or immersion)
     • CHGOBJ ↺: same as above but counterclockwise
     • DRY/IMM: toggles between dry and immersion mode
  5. right front: Focus buttons
     • Z ↑: moves the Z drive up when a upper focus stop has been set
     • Z ↓: moves the Z drive down
     • SET + Z ↑: toggles set and unset upper focus stop
     • SET + Z ↓: toggles set and unset lower focus stop
• SmartMove remote control for stage movements, as observed in the viewer display window of the software
  1. X
  2. Y
  3. Z (focus, z-wide)
  4. left button set selects Precise or Fast XY movement
  5. right button set selects FINE or COARSE focus control
• front LCD panel displays the current status
  1. Contrasting method e.g., TL, FLUO, or SCAN; and IL SHUTTER status
  2. Objective lens and Magnification, see front top buttons above
  3. Illumination and Apertures
  4. Camera Ports/Eyepieces, see front top buttons above
  5. Focus control

     Note:

     • display on the right shows z at 0 µm with upper focus stop set and lower focus stop unset
     • if upper focus stop is unset, display will indicate --.-- mm and
     • indicates lower focus stop is set

Modes of operation

• TL
  1. press CHG TL ◑ to rotate through BF, DIC, and POL
  2. adjust intensity (INT) and aperture (AP) as needed
  3. optional, press CHG CS to scan mode thus shutoff transmitted light
  4. a knurled wheel (arrowhead) on the left side below the objective turret adjusts the DIC shear; it's a rather tight space!
• IL/epifluorescence
  1. turn on FLUO (fluorescent light source)
  2. press the button for the desirable cube, see the front buttons in Controls
  3. press Shutter to illuminate specimen
  4. adjust the intensity (INT) and field diaphragm (FD) as needed
  5. press Shutter to block illumination when done viewing

Using immersion objective lenses

• 2 ways to switch from DRY to IMM objectives
  • semi-auto with Mark and Find (recommended)
    1. locate the area of interest with a dry objective and focus
    2. click to bring up the Mark and Find interface
    3. click to record the current position: positionN
    4. press and hold Z ↓ to lower the turret completely
    5. switch immersion objective into light path
       • via the objective link in LAS AF (recommended)
       • or at the microscope: press DRY/IMM to switch mode, press either button CHGOBJ ↻ or ↺ to select the desired objective
    6. move the stage to expose the objective so you can apply immersion fluid
    7. apply an appropriate amount of immersion fluid
    8. select positionN from the drop down list and the stage should move back and bring the objective to focus
    9. click to dismiss the Mark and Find interface
  • manual (not recommended)
    1. locate the area of interest with a dry objective and focus
    2. make sure upper focus stop is set
    3. press and hold Z ↓ to lower the turret completely
    4. switch immersion objective into light path (see manual procedures above)
    5. offset the turret ~15 ° counterclockwise manually and apply a small drop of immersion fluid on the objective
    6. swing the turret back into position
    7. press and hold Z ↑ to put the prep back to the previously set focal plane
• when finish
  1. press and hold Z ↓ to lower the turret completely
  2. remove slide
  3. at the end of the session, use the provided lens paper to remove the excess immersion fluid from the front lens
  4. switch in the 10 × objective
• Best practice
  • always set the upper focus stop whenever you put on a prep, press SET + Z ↑
  • switch objective lenses via LAS AF software or the right rear buttons on the microscope
  • if you want to manually switch the objective, DO NOT grab the objective lens barrel, use the turret instead

    Caution: All our immersion objective lenses have aperture or correction collar, grabbing the barrel can easily move the collar and also strain the lenses.

  • select the 10 × objective at the end of the session

objective lenses (coming soon)

Smart Panel (coming soon)

beam splitter spectra

• RT 30/70 is 30% reflection and 70% transmission
• Substrate is just plain glass (50/50)
• RSP 500 is a 500 nm long pass filter
• TD and DD are triple and double dichroics (transmission bands in blue)
  
  

  TD 488/561/633

  

  DD 458/514 is similar to the TD 458/514/594 shown here

  

  DD 488/561

working with lif file

• use ImageJ and loci_tools, see details on using ImageJ
• use the free Leica LAS AF Lite application from Leica's ftp depository, Windows only

scanner

Z

software control of the z-drive

This provides alternate means to control the z-Position, especially when the Smart Panel is unavailable

• enter a number directly into the text boxes of z-Position, Begin, or End
• mouse: place cursor on z-plane (cursor change into double arrow), either click and drag, or click and then use thumb wheel

  Caution: Use extreme caution with mouse control! It is very easy to drag the z-position too far and ram the objectives onto the slide damaging the objectives.

setting z-step size or slice thickness

• the general guideline is to set the z-step size to ½ to ⅓ of the section thickness (clickable link) shown in the XY panel
  • section thickness (dz) is dependent on the emission wavelength (λ), refractive index of the immersion medium (n) and NA of the objective, and the size of the pinhole (AU)
    dz = √((λ×n÷NA²)²+(AU×n×√2×1.22×λ÷NA²)²)
  • section thickness can be adjusted to the mean of the emission wavelength by following the link and then Apply to the step size
  • usually the shortest wavelength emission band is used to set the section thickness in a multichannel scan
• use System optimized in the Z-Stack panel if you plan to do deconvolution

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You can contact us for comments and inquiries.

Updated on 2012 May 08. Copyright© 2012 University of Washington, unless indicated otherwise.

Department of Biology • UW