Leica SP5 II Manuel utilisateur
8/31/2020 Instruction for Leica SP5 confocal microscope
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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
Check-in and Start up
1. Main Switch Board: switch on PC Microscope, log on and wait until Windows XP is fully loaded
2. Main Switch Board: switch on Scanner Power, wait 5 S
3. launch LAS AF (desktop shortcut),
select the desirable 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.
4. make sure the condenser is in the "up" position, click Yes to initialize the stage when prompted
this enables Mark and Find, and Tile scan
stage is configured to return to the position before initialization
5. Main Switch Board: when the software finish loading, switch on Laser Power and turn the key clockwise to
On-1 to enable Laser Emission
6. in LAS AF, click on the Configuration Operating step 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
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Set up acquistion parameters and acquire an image
1. Beam Path Settings in the central Work area: there are 3 ways to
configure excitation and emission parameters
A. use a preset
i. click on Load/Save single setting
ii. select a Leica or User Settings (for multichannels, the default
is simultaneous scan, see below for sequential scan)
B. reuse a setting from a previous scan
i. open a previous experiment
ii. in the Experiments tab, click to select the desirable scan
iii. right-click to choose Apply Setting or hit Apply button at the bottom
C. assemble from scratch
i. set laser power
activate the shutter
set power level of the desirable laser
line, 5% is a good starting point
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ii. select the beam splitter (dichroic)
selections for different laser lines
beamsplitter
\
laserline(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 ✓ ✓
iii. 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
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2. select the Acquire Operating step on top left, click the Acquisition tab to set up the data format
A. Acquisition mode panel
defaults to xyz simultaneous scan
seq. button enables sequential scan
Tile scan, and Mark and Find
Best Focus
B. XY panel (click ▶ on upper right corner to expand)
Format: frame resolution defaults to 512x512pixels, max 8192x8192pixels
Speed: line frequency defaults to 400Hz, scan area will be reduced above 700Hz, max 2800Hz
Zoom* and panning
Average and Accumulate
Scan Field Rotation*
Pinhole*: default is 1AU and displays in µm
3. acquire an image (buttons in the lower left pane of LAS AF)
Live: scan, apply accumulate if set
Capture Image: acquire an image at the current z position, apply both average and accumulate if set
Start: acquire all configured scans, e.g., z-stack, stage positions, tile scan, time points, spectral series etc.
*these parameters are also adjustable via the Smart Panel with the default configuration
Optimize acquisition 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 Live 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) or increase the laser power; try the following
i. with BF or FLUO, make sure object of interest is still in focus
ii. open up pinhole to increase the slice thickness
iii. raise PMT gain
iv. 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 intensity in green and
saturation in blue (255 for 8-bit or 4095 for 12-bit), facilitates this setup
i. top left side of the viewer display Window: activate the Glow (OU) LUT via the Quick
LUT button which cycles through the default , Glow(OU) , and grey LUTs
ii. 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
iii. 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
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Z-stack
Z-Stack panel
focus control is selectable: z-Galvo (default) or z-
Wide
focus (z-Position) can be adjusted with the
microscope controls, Smart Panel and also via
software (see z-drives in the Appendix)
z-drive controls
controls
\
z-drives
microscope
focus knob* Smart Move* Smart Panel range min.
step size
*the objective turret i.e., focus is always adjustable with the microscope focus knob and Smart Move, regardless of the z-drive
selection in the software
z-Galvo ✓±250µm 42 nm
z-Wide ✓ ✓ ✓ >4mm 49 nm
Acquire a z-stack
1. in Live mode, move deeper into the prep (z is more positive) to where the stack should start, click
Begin to register this z-Position (black: unset, red: set)
2. move towards the outside of the prep to where the stack should stop, click End to register this z-
Position
3. stop scanning, click the z-step size button and then enter the appropriate slice thickness (check setting
slice thickness in Appendix for recommendations)
4. select the number of passes to average in the XY panel
5. click Start (lower middle) to collect the stack
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Sequential scanning
1. set up a simultaneous scan for the fluorophores needed with regard to laser power, beam splitter, emission
band, and PMT settings; activate one laser line and detector combination at a time to find out the optimal
acquisition parameters
2. click the button on Acquistion Mode on the upper left to open the Sequential Scan panel
3. select between lines, -frames, or -stacks (see useful tips below)
4. click the + button to add as many scans as needed
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"
with "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., adding the transmission detector, or have 2
simultaneous channels that do not show any cross-talk
the function buttons act differently in different sequential scan mode
display behavior of the function buttons in sequential scan
mode
buttons
\
scan mode
Live Capture Image Start
*to obtain all scans at the current focus (z-position), switch to xyt mode, set frame to 1, and then click Start
between lines all scans all scans acquire all scans
between frames currently selected scan currently selected scan* acquire all scans
between stacks currently selected scan currently selected scan* acquire all scans
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Stage automation
Caution: whenever the Tile Scan or Mark and Find window is launched
i. any tiles or points marked will be processed when you click Start
ii. the grey area represents the stage, a left mouse click will cause the stage to move to that specific
location and can be dangerous for any objective lenses except the 10×
Tile Scan
1. click to toggle displaying the Tile Scan window
2. check that Scan Field Rotation is set correctly (see
r elevant details
below)
3. go Live
4. move the prep/stage to the left boundary of the region of interest, focus
5. click to mark the current position
6. repeat the last 2 steps for the top, right, and bottom boundries, 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 apply any points already saved from Mark and Find.
7. move about to make sure the coverage is sufficent, especially with multichannel acquistion
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
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1. viewer display window 2. at the microscope
viewer display windows rotated 90 ° right
3. Tile Scan or Mark and Find window
viewer display windows rotated 90 ° left
relevant details
for tile scan, current rotation calibration for stitching is -0.19 ° (-0.18 ° is also OK) 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 shown in the viewer display window (1) is rotated
when compared to that as observed through the ocular of the microscope (2) or represented
in the Tile Scan or Mark and Find window (3). Confusing? Indeed!
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
8/31/2020 Instruction for Leica SP5 confocal microscope
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Check out
1. lower the turret, remove your prep; if applicable, remove oil/immersion fluid on immersion objectives
2. switch to the 10× objective
3. if there are users signed up within the next 6 hours do the following, otherwise go to the next step
i. save your experiments and quit LAS AF
ii. copy your data to external storage media or network file servers, as needed
iii. submit your usage using the google form and log out of Windows
4. if no one signed up within the next 6 hours, please proceed to shutdown everything
i. save your experiments and quit LAS AF
ii. Main Switch Board: turn the key counterclockwise to Off-0 to disable Laser Emissions
iii. Main Switch Board: wait 10 s, switch off Scanner Power
iv. copy your data to external storage media or network file servers, as needed
v. submit your usage using the google form
vi. shutdown Windows XP
vii. Main Switch Board: after computer has shutdown, switch off PC Microscope
viii. Main Switch Board: when the Argon laser cooling fan stops, switch off Laser Power
ix. turn off FLUO, if applicable
x. put the cover back on the microscope
5. clean up the work area
8/31/2020 Instruction for Leica SP5 confocal microscope
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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
CHGTL◑: switch to transmitted light, TL
and rotate through different modes TL_BF
(bright field), _DIC, _POL (polarization)
CHANGECS: 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)
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