Zeiss LSM 800 User manual

Imaging and Flow Cytometry Core CPOS LSM 800 SOP A-1
ver. 2.1 (Updated: 11/13/2020)
LSM 800 Confocal Microscope
Standard Operation Protocol

Imaging and Flow Cytometry Core CPOS LSM 800 SOP A-2
ver. 2.1 (Updated: 11/13/2020)
Contents
A. Overview ............................................................................................................................................................... A-3
B. Microscope Lens list...............................................................................................................................................B-3
C. System start up ......................................................................................................................................................C-4
D. Graphical User Interface Description.................................................................................................................... D-6
E. Locate specimen, focus, find cells / region of interest ..........................................................................................E-7
F. Confocal imaging set up.........................................................................................................................................F-8
G. Saving an Image file ............................................................................................................................................ G-11
H. Removing sample from microscope stage.......................................................................................................... H-12
I. Z-stack set up ........................................................................................................................................................I-13
J. Airyscan imaging set up ........................................................................................................................................J-15
K. Tile / Position set up (Simple) ..............................................................................................................................K-17
L. Tile / Position Advanced Setup ............................................................................................................................ L-19
M. Time series set up .......................................................................................................................................... M-24
N. Oil immersion Lens clean procedure .................................................................................................................. N-25
O. System Power down ........................................................................................................................................... O-26

Imaging and Flow Cytometry Core CPOS LSM 800 SOP B-3
ver. 2.1 (Updated: 11/13/2020)
A. Overview
This guide will cover operation of the Carl Zeiss GmbH LSM 800 scanner paired with an inverted microscope
setup and ZEN Blue edition ver. 2.3 driving software. For specific operations of the ZEN Blue software please
read http://med.hku.hk/corefac/downloads/ZEN_BLUE2.3SOP_20200415.pdf . It is recommended that user study
both guides side-by-side.
B. Microscope Lens list
Position
1
2
3
4
5
6
Magnification
10x
20x
40
63
EMPTY
5x
Numerical Aperture
0.3
0.8
1.4
1.4
0.16
Free Working Distance
[mm]
2 mm
0.55 mm
0.13 mm
0.19 mm
18.5 mm
Coverglass Thickness
[mm]
0.17 mm
0.17 mm
0.17 mm
0.17 mm
0.17 mm
Immersion
Air
Air
Oil
Oil
Air
Diagram showing arrangement of objective positions. Position 5 is reserved for special application lens thus will not
be populated at all times.

Imaging and Flow Cytometry Core CPOS LSM 800 SOP C-4
ver. 2.1 (Updated: 11/13/2020)
C. System start up
1. Toggle main power supply on wall (labelled
○
1…..
wait 5 seconds….and ○ 2)
2. Turn on PSU # ○ 3and ○ 4.
3. Turn on Laser module “LM” turn-key ○ 5.
4. Turn on power for Metal halide lamp for finding
your specimen under fluorescent light ○ 6.
5. Turn ON computer ○ 7.
6. Double click on the program icon on desktop.
Or
click on Start | All Programs | Carl Zeiss
Microscopy | ZEN | ZEN (blue edition) entry
(blue icon).
7. Click on “ZEN system” button.
8. Check that you have:
*took out sample frame*
**adjusted objective lens to 10X or lower**
before you proceed with “Calibrate Now”
button.
9. (Optional)
Live cell incubator procedure as follows:
i. Turn ON Tokai HIT controller module ○ 8.
ii. Turn gas tank valve counter-clockwise ○ 9.
Read second regulator gauge to confirm gas
supply pressure is at ~17 psi (blue line). Do not
adjust the knob if it is at desired pressure.
8
9

Imaging and Flow Cytometry Core CPOS LSM 800 SOP C-5
ver. 2.1 (Updated: 11/13/2020)
iii. Select desired objective. Put on lens heater by
first unravelling Velcro tape and then re-attach
around the objective lens.See image for
reference.
iv. Mock-up on-stage incubator and adjust
motorized stage position to confirm objective
lens is centred with sample frame.
v. Position the incubator to the right of wire next
to red dot indicator on microscope stage. Wires
should be on the right hand side and gas tubing
should be on your left hand side.
vi. Slide incubator to the left while gently pushing
downwards.
vii. Insert live cell sample.
viii. Add appropriate amount of MQ water via Leuer-
lock capped tubing.
ix. Replace incubator cover.
x. Arrange the gas tubing so that they will not be
trapped under condenser.
C9iv…Before
C9iv…After
xi. Return condenser back to above microscope
stage. ****Observe very carefully that the
condenser does not press onto cover of the
incubator. Push back condenser and find
technical assistance. ***
xii. Close doors on the acrylic box around the
microscope.
C9xi

Imaging and Flow Cytometry Core CPOS LSM 800 SOP D-6
ver. 2.1 (Updated: 11/13/2020)
D. Graphical User Interface Description
The terms will be used throughout this document to locate different buttons. Reference to this part from time to
time when you read the rest of this SOP.
i. Menu bar
ii. Tool bar
iii. Left tool area
(Locate mode= 1 panel; Acquisition mode = 2 panels)
iv. Centre screen area
v. Dimension tab
vi. Display tab
vii. Status bar
viii. Image library

Imaging and Flow Cytometry Core CPOS LSM 800 SOP E-7
ver. 2.1 (Updated: 11/13/2020)
E. Locate specimen, focus, find cells / region of interest
1. You should be on "Locate" tab if the software
has just finished calibration. If not navigate to
"Locate" tab.
2. Select objective lens with software / on
microscope control panel.
Consult Chapter B to see which objective lens
needs immersion oil.
3. Push back condenser column.
4. Mount your sample on sample carrier. Ensure
your specimen holder is level.
5. [Optional] Click "BF" on software for easier
sample finding and bright-field observation.
6. Move the area of the slide you wish to observe
above objective lens with X-Y manipulator on air
table.
7. Focus onto your specimen. Notice "Z-position"
value shown on the microscope control panel
and software, larger number means higher focus
height.
Typical focus height for glass slides ~ 1200 –
1500 µm. Confirm sample placement if out of
range.
8. Click "B" = Blue; "G" = Green; "R" = Red; "Far-
red" on software for fluorescent observation.
**Note: fluorescent dye will eventually be
bleached under intense excitation light. Find
your region of interest quickly. When you turn
to other tasks turn of ALL illumination by clicking
on "All off" button. **
9. Click "Acquisition" tab when you arrive at region
of interest.
X
Y

Imaging and Flow Cytometry Core CPOS LSM 800 SOP F-8
ver. 2.1 (Updated: 11/13/2020)
F. Confocal imaging set up
Prerequisites: Region of interest is centred in field of view and have clicked 'Acquisition' tab.
1. Select one of the pre-set made by Imaging and
Flow Cytometry Core.
2. Go to dropdown menu in "experiment settings"
area. Select settings with prefix "0IFCore_"
3. Please note "Blue" "Green" "Red" are general
broadband wavelength setting you will need to
fine tune the detection range in Imaging set up
window. (Step F8)
4. Go to "Imaging setup" window
Decide on switch track every:
“Line” = change color every line of pixel
“Frame” = Each color per frame sequential.
“Frame Fast” = lock up pinhole and wavelength
splitter but still change color per frame.
“Full Z-stack” = (only available when ‘z-stack’ is
activated. Will change track after finishing whole
z-stack.
5. Select any one track for set up.
6. Table's rows indicate detector choice.
Row 1 = PMT1
Row 2 = Airyscan detector
(when idle will work as confocal)
Row 3 = PMT2
7. 1st column indicates activation.
8. 2nd column indicates dye reference and name
(click on for dye selection window).
9. After setting up dye selection, spectra window
will be updated. Use slider to have detector
detect most of the emission wavelength range.
10. 3rd column indicates pseudo-colour choice.
Click to change.
11. Last column indicates detection range.
12. If a bright-field image is required, check the
'ESID' option in ‘Imaging Setup’ window.
13. Go to 'Channels' window. While you make
changes please observe histogram in 'Display'
tab under preview area.
Good
14. For detailed description of 'Display' tab please
read ZEN Blue 2.3 SOP on Imaging and Flow
Cytometry Core website-> Protocols -> Imaging
F7
F8
F10
F11
F12
F6
F5
F9
F4

Imaging and Flow Cytometry Core CPOS LSM 800 SOP F-9
ver. 2.1 (Updated: 11/13/2020)
..fix this later
15. Activate all channels you need by ticking
checkboxes.
16. Highlight a track you wish to adjust.
17. You may use the '1 AU' hotkey to adjust
pinhole size. Alternatively balance pinhole size
so that every channel's optical thickness are the
same (applicable when multi-channel imaging is
required).
18. When adjusting laser intensity and master gain
values pay attention to the 'Display' histogram.
Lower both settings if you see a peak at 'White'
indicating PMT over-saturation. Turn on 'Auto'
checkbox in 'Display' tab and select 'Min/Max'
option for real time display. 8-bit image do not
exceed 250 units. 16-bit image do not exceed
65500 units.
**Important information. Preventing damage to
PMT units. Avoid over-saturating the detector.
**
19. Turn on 'Live'
20. Adjust laser power. You can use value between
0.01% and 5% in normal mode.
21. Adjust 'Master Gain' to tune PMT sensitivity
(recommended range between 500V to 800V).
… F19
22. **For extremely weak fluorescent signal even at
5% laser power and 800V ‘Master Gain’ please
workout labelling parameter (preferred) or turn
on 'high intensity laser range' checkbox option.
This will open up the laser from 3.5% to 100%
range. **High chance of sample bleaching use
with caution. **
…F22
23. Other options in 'Channels' window:
24. 'Digital Gain': for mathematically enhance
apparent intensity of image. Typical setting = 1.0.
All pixel values will appear as:
(original value) * (Digital Gain factor)
Saturation
F15
F16
F20
F21
F24
F25

Imaging and Flow Cytometry Core CPOS LSM 800 SOP F-10
ver. 2.1 (Updated: 11/13/2020)
25. 'Digital Offset' for mathematically subtract
apparent intensity of image typical setting = 0.
All pixel values will appear as: (original value) -
(Digital Offset value) = image pixel intensity.
26. Go to 'Acquisition Mode' window:
27. Adjust 'Frame Size' if you are not sure and do not
mind performing 2X Nyquist sampling, click
'optimal' option.
28. Adjust 'Scan Speed' recommended value from
(slowest) ~4 µsec to (fastest) ~1 µsec 'Pixel
Dwell'
29. Switch between scan directions unidirectional
[insert image] for quality; bi-directional [insert
img] for speed.
30. Switch between bits per pixel [N.B.: different
degrees of intensities a pixel can represent] 8-
bits for presentation, representative images,
reports for faster work-flow; 16-bits for intensity
quantification / segmentation if required.
31. Adjust 'Scanner rotation' angle if applicable
32. Adjust 'Zoom' if applicable. [N.B.] This function
scans a smaller area through objective lens,
consult 'optimal' frame size after adjusting this
parameter.
33. After all parameter has been set up do the
following to acquire an image.
34. Ensure again all channels you need to acquire is
checked in 'Channels' window. (Refer to step
F15)
35. Click 'New'
36. Click 'Snap'
F27
F28
F29
F31
F32
F30
F35
F36

Imaging and Flow Cytometry Core CPOS LSM 800 SOP G-11
ver. 2.1 (Updated: 11/13/2020)
G. Saving an Image file
1. Go to right hand side Image Library column to
find your latest acquired image. Double click on
the image you wish the save. Click on 'Save'
button.
2. Alternatively,
Right click on the image you wish to save and
then click 'save selected' from the pull down
menu appeared.
3. Navigate to D:\\ (Hard drive 'D') User\
4. Create a folder with your PI's name and + 'Lab'
wording suffix. Applicable if no such folder exists
and you are the first one in your lab using LSM
800.
5. Navigate into the folder you have just created.
6. Choose file format as '.czi' acronym for 'Carl Zeiss
Image'
7. Type in name (preferably no spacing).
8. Click 'Save'
9. Warning Icon or indicates unsaved
or changes has been made to your .czi file.
10. Image upload.
Please see guide on Desktop 'Upload guide'.
11. Image Export.
Please see ZEN Blue 2.3 SOP.
12. The following content describes additional
function on the LSM 800 setup.
G1
G2

Imaging and Flow Cytometry Core CPOS LSM 800 SOP H-12
ver. 2.1 (Updated: 11/13/2020)
H. Removing sample from microscope stage.
1. On control panel press 'load position' button.
Objective lens should be moved to lowest
position allowing clearance for you to operate
with the stage area.
2. Move slider on sample carrier apart to retrieve
your specimen, then install a new specimen.
3. Press 'return to work position' on control panel.
H1

Imaging and Flow Cytometry Core CPOS LSM 800 SOP I-13
ver. 2.1 (Updated: 11/13/2020)
I. Z-stack set up
Pre-requisite: you have setup all necessary channels according to previous chapters including laser power, master
gain, pin hole size.
1. **Note: image intensity might increase beyond
saturation if the z-position you chose to setup
laser power / master gain is not the brightest
structure in the z-volume. Decrease laser power
/ gain accordingly. **
2. click checkbox to activate 'Z-stack' module.
3. select one channel with the most representative
structures / most robust in the group.
4. turn ON 'Live'.
…i4
5. hands on **Fine Focus** knob. Please note
current z-position value (greater numeric value =
higher focus position)
or
6. cursor over preview window. Press 'Control' key
on keyboard. Scroll with mouse wheel while
'Control' key is pressed.
7. decrease focus height until you cannot see your
cell / structure. stop and click 'Set First'.
8. Increase focus height until you cannot see your
cell / structure. stop and click 'Set Last'.
9. Click 'Stop'. to pause laser scanning.
10. note on 'Range' value to confirm if your desired
structure is roughly the same size as stated.
Readjust 'First' and 'Last' section positions
accordingly.
11. note optical thickness under pinhole slider in
'Channels' window. This is your maximum
interval if you wish to capture all structure in a
certain z-volume.
…i10
i2
i7
i8
i11

Imaging and Flow Cytometry Core CPOS LSM 800 SOP I-14
ver. 2.1 (Updated: 11/13/2020)
12. Most application “keep interval” is useful.
13. If you select 'optimal' in 'Z-stack' window. LSM
will perform optical sections where each section
will have 50% overlap with the previous section
(2x Nyquist sampling). Choose this option if 3D
rendering / volumetric calculation is needed.
14. For a more graphical representation of the z-
sections, you may view them in 'optimize
Sectioning and Step'.
15. Z-stack setup finished.
16. Select all channels you wish to capture.
17. Click 'Start Experiment'.
18. If you wish to view your Z-stack 3D rendered.
Click on '3D' tab on the side of preview window.
19. Please refer to step F4. Decide if you wish to
accelerate scan by “switch track every z-stack”.
Slight compromise on height accuracy.
i12
i14
i13

Imaging and Flow Cytometry Core CPOS LSM 800 SOP J-15
ver. 2.1 (Updated: 11/13/2020)
J. Airyscan imaging set up
1. Follow air to oil immersion lens change
procedure in training. Switch to 63x objective.
2. Select pre-set with prefix '0IFCore_Airyscan...'
or
3. Click 'Smart setup'
4. select fluorescent dyes and then click '+' button.
5. Select 'Airyscan' mode.
6. click 'OK'
7. Confirm the following settings in 'Acquisition
Mode' window:
8. 'Frame Size' = 256 x 256 px. (This is to facilitate
intensity adjustment preventing pre-mature
sample bleaching)
9. 'bit' = Either 8 or 16 but follow carefully in step
[J19] when adjusting intensity parameter.
10. 'Scan Speed' = maximum
11. 'Zoom' > 1.3x
(Please refer to steps F26 to F32 for graphical
guidance)
12. Start by only checking √ONE channel in the
'channels' window.
13. Adjust detection wavelength range to suit your
particular fluorescent dye.
[Refer to Step F9 for graphical guide.]
14. Turn on 'Continuous' scan mode
15. ! Important! Go to 'Airyscan' tab in centre
preview area.
16. Click on 'Display' tab under centre preview area.
17. Turn on 'Auto' checkbox in 'Display' tab and
select 'Min/Max' option for real time intensity
display.
18. Adjust 'Master Gain' between 500V to 850V.
J2
J3
J17
J15

Imaging and Flow Cytometry Core CPOS LSM 800 SOP J-16
ver. 2.1 (Updated: 11/13/2020)
19. Adjust laser power so that max image intensity
reach ~ 5000 - 8000 intensity units (around 50%
of detector dynamic range) under 16-bit mode or
~30 intensity units under 8-bit mode.
20. **Important information. For proper
downstream Airyscan processing and preventing
damage to detector. Avoid over-saturating the
Airyscan detector. (intensity must be <8000
units). **
21. ‘Stop’Continuous Scan.
22. Select a new channel unselect previous channel.
Repeat step [J12] to step [J19] until all channel
has been optimized.
23. Check ALL channels.
24. Turn on 'Continuous' scan mode.
…J24
25. Go to 'Airyscan' tab in centre preview area.
26. Check 'Detector' under Airyscan tab. Wait for
the detector to be aligned.
*Advanced: you may do this step during intensity
setup.*
If cannot perform this step seek help from
technical staff.
[Refer to step J15]
example for aligned detector
27. Please check if software shows any warning
about Airyscan acquisition.
28. Slower scan speed if required
29. Change back to unidirectional scanning if
required.
30. Click 'New' and 'Snap' for a single optical section.
Or 'Start Experiment if you have any other
modules activated.
31. Refer to 'Saving an Image File' (Insert Chapter
number).
J26
J30

Imaging and Flow Cytometry Core CPOS LSM 800 SOP K-17
ver. 2.1 (Updated: 11/13/2020)
K. Tile / Position set up (Simple)
(With multi-position function included in this chapter)
1. click checkbox to activate 'Tile' module.
Tile Simple set up
2. Scroll to “tile Set up” window.
3. Select scan mode: 'Tiles' / 'Size' / 'Stake'.
4. Tiles: Enter the number of X and Y to define the
tile region
5. Example: X=3 and Y=5 equals to tile region
containing 15 tiles
6. Size: Define the size of tile region
7. Stake: User define two opposite corner markers
with x-y motorized control and '+' button. The
software will define the total number of tiles
based on your input region. Keep on pressing
“+” if you are not happy with the 2nd stake.
Please see illustration.
Click “Done” to confirm tile area.
8. Check all tiles you wish to capture in one
experiment (applies when multiple tiles are
defined. Ignore this step otherwise).
9. Goto Options tab to adjust % overlap.
10. Press ‘Start Experiment’.
Position Simple set up
11. switch between 'Locate' and acquisition to setup
positions with eyepiece.
K4
K6
K7
K5
1st stake
2nd stake
Additional
stake
K8
K9

Imaging and Flow Cytometry Core CPOS LSM 800 SOP K-18
ver. 2.1 (Updated: 11/13/2020)
12. Reference to Chapter [E] 'locate specimen,
focus, find cells/region of interest'
13. Go to 'Locate' tab.
14. Turn on fluorescence channel for navigation.
15. Use xy-motorized control to navigate your
specimen.
16. When you have a region of interest in view, go
to 'Acquisition' tab.
17. Turn ON 'Live' view set Focus until image is clear.
Critical
…K16
…K17
18. GoTo 'Tile' window positions section and click on
'+' under positions.
19. Repeat steps [K12] to step[K17] until you have
all of the positions required.
20. Press ‘Start Experiment’
K16

Imaging and Flow Cytometry Core CPOS LSM 800 SOP L-19
ver. 2.1 (Updated: 11/13/2020)
L. Tile / Position Advanced Setup
Advanced set up (Preview scan)
1. Common steps = Establish a preview scan ->
determine region of interest (R.O.I.) either in
area or in points -> verify positions -> Start
Experiment.
2. Select the most robust (bright, not
important enough to be bleached)
fluorescent channel in your settings.
3. Go to tile window and click on 'Advanced
setup'.
4. The software should 1) automatically turn on
'Live'; 2) replace half of the preview area
with a navigation map; and 3) added
controls for tile setups.
5. [Optional] In “Acquisitions mode” window.
Change to 5X objective lens; 0.5X zoom; bi-
directional scan; maximum scan speed.
6. You may move the specimen around with x-y motorized stage controller to figure out general are of
your specimen. Or you may double click on the navigator area to see specific area. Focus onto the
specimen.
L3
L4
Live View Area
Sample Navigation Area

Imaging and Flow Cytometry Core CPOS LSM 800 SOP L-20
ver. 2.1 (Updated: 11/13/2020)
7. Go to 'Tile Setup' and draw an R.O.I. that can
contain the whole specimen. Rectangular
/ circular / free-hand draw.
8. Preview scan tab. For maximum speed,
disable 'Use Existing Experiment Settings'.
Choose only the channels useful for
navigation / screening targets.
9. 'Start Preview Scan'
10. After preview scan is finished. An overview
images should be visible in ‘Advanced tile’
area.
11. If the preview scanned imaged is not large
enough to cover your desired area. Check
off 'Delete Existing Preview Images' option.
Draw a new R.O.I area to cover missing area
and then 'Start Preview Scan' again.
12. Switch to the objective suitable for your high
magnification scans. Red F.O.V. mark on
advanced tile area should resize to fit new
F.O.V. physical dimension.
13. (for navigating large area, an air lens is
preferred over immersion oil lenses as the
immersion medium might not be able to
follow with position of the objective.
Consult CPOS imaging team for choices of
objective lens.)
14. Go to Tiles tab or Positions Tab to setup
scan.
**Different steps between tile and positions:
Advanced Tile
15. Go to 'Tile Region Setup' tab under advanced
tile preview area.
16. Select 'Contour' (for Predefined and Carrier
is not described here, request CPOS Imaging
team for setup your plate map)
17. Use cursor to select and delete the old ROI
used to capture preview scan. Use ROI tool
to define a new area.
18. Select one of the three R.O.I. tools
(rectangular [icon] / circular [icon] /
freehand [icon])
19. Draw on map to define Tile area.
L8
L9
L11
L17
L18