Zeiss LSM 800 Manual de usuario

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

Magnification

10x

20x

EMPTY

Numerical Aperture

0.3

0.8

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

Immersion

Air

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. Turn on PSU # ○

3and ○

3. Turn on Laser module “LM” turn-key ○

4. Turn on power for Metal halide lamp for finding

your specimen under fluorescent light ○

5. Turn ON computer ○

6. Double click on the program icon on desktop.

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 ○

ii. Turn gas tank valve counter-clockwise ○

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.

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.

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

F10

F11

F12

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

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