SP8X (Leica-microsystems, Mannheim, Germany) Short check ...

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General Instrumentation. Contact: [email protected] tel 0243652199 Version August 15, 2017

SP8X (Leica-microsystems, Mannheim, Germany)

Short check list switch on

ALWAYS ACTIVATE EXTERNAL COOLING/VENTILATION box on the corridor when working outside

office hours (weekend, evenings, Christmas break)

Knob PC-Microscope (panel 15)

Knob Scanner Power (panel 15)

Knob Laser Power (panel 15)

Key laser emission (panel 15)

Fluorescence lamp on (panel 11)

Samples on microscope stage

Choose objective ( objective icon, panel 2)

XYZ adjustment (xyz icon, panel 2)

TCS-User login om pc

Optional: CO2 and air flow on (conducts 19), temperature (NEEDS IDEALLY 12 HRS TO STABILIZE!)

(box 7), CO2 (The Brick; unit 16), water pump for 40x(unit 12), humidifier

Start LAS-X (with/without environmental control)

Configuration> 405/WLL Lasers ON

Optional 90 degree rotation

Define laser lines + intensity (START WITH LOW INTENSITY)

Choose range HyDs (Very sensitive;) and/or PMTs (NEVER PUT UNDER A LASER LINE TO NOT

OVER-IRRADIATE THE DETECTORS) and PMT trans

Adjust offset and gain PMTs and HyD standard/Bright/Photon counting

Adjust frame size (pixels), pinhole, (optional) zoom, (optional) bidirectional scanning, speed

scanning

Define settings for time-lapse t, and/or z-stack and/or lambda series, and/or sequential scan,

single or multiple position

Optional Autofocus: hardware AFC and / or software best focus etc

Name of project and correct path on D> Users> …

mailto:[email protected]

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Short check list switch off

Save your projects ON D-DRIVE D > USERS> INST> … PC

UPLOAD YOUR DATA ON A SERVER, NEVER ON A USB STICK/EXTERNAL HARD DRIVE! (VIRUSES)

Close all laser lines

405 nm and WLL from ON to Off in software

All HyDs and PMTs on OFF

Clean any immersion medium from objectives

Objective 10x in upright position

Remove slide

Last users of the day:

close LAS-X

Turn off Windows

Turn WLL key to off (# 4 on panel 15)

Switch fluorescence lamp off (panel 11)

Switch off (options) Temperature inside cube (box 7), the Brick (unit 16), Bartels water pump (unit 12), close air flow (wall), close CO2 flow (wall)

WLL key 90 degree counter clockwise

Laser power off ( panel 15)

Wait 5 minutes (post-cooling)

Scanner Power off ( panel 15)

PC-Microscope off ( panel 15)

Optional: bring external cooling timer to 0


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SHORT MANUAL ON HOW TO SWITCH ON OR OFF THE SP8X CONFOCAL MICROSCOPE 1. Equipment setup

Basic components of the Leica SP8x microscope 1. White light laser (WLL)

2. DMi8 inverted microscope

3. The Cube, cell culture box

4. Humidifier

5. Monochrome camera for wide field imaging

6. Anti-vibration table

7. Temperature box (under the table)

8. Table with container for electronics and 405 nm laser

9. Xyz navigation/focus

10. main control box

11. Power unit fluorescence lamp wide field microscopy

12. Bartels water pump unit for 40x objective

13. Monitor for hardware settings, data acquisition and image processing

14. Knobs for adjustment of hardware (HyDs, PMTs, laser angle rotation, z-focus etc)

15. Main power switches

16. The Brick for CO2 control

17. Computer steering hardware and imaging

18. Air pump for anti-vibration table

19. Conducts for air flow and CO2 flow


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2. Spectral detection system (schematic)

3. System startup

- On panel 15 (see details here right) Switch on PC-Microscope (knob 1)

(Windows start up)

- Wait 1 min. Switch on Scanner Power (knob 2),

- Switch on Laser Power (knob 3)

- Turn the key (4) 90 degree clockwise for the WLL

- When Windows has finished to load, choose entry TCS-User. No password required

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- Switch on fluorescence lamp (unit 11). Note that the intensity can be varied. (Do not press shutter).

Start with a low intensity.

Optional side equipment

Temperature control (the blue air heater called the CUBE and the transparent polycarbonate incubator

around the microscope, called The Box, both from Life Imaging Services, Basel, Switzerland; control 7):

by default left on at 37°C, to prevent expansion and shrinking (drift) of the parts. But when switching

between room temp and 37°C needs 12 hours to stabilize!

- Bartels water pump (12); fill the water container with CLEAN MILLI_Q. Software can be operated from

AOIContro icon on desktop.

- If CO2 and air bubbling are needed, switch on the orange gas mixer appliance, called The Brick (16), gas,

pressure and open the valves for CO2 and pressurized air (Se values in a spate menu)

- Humidifier


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4. Sample insertion, the DMi8 microscope and objectives

Sample

Insert the sample on the stage with the coverslip directed towards the objective. Take care that nothing

can leak out of the sample and that the coverslip is clean and free of buffer crystals

The microscope overview (touchscreen)

Provides a summary of the status and allows one to adjust the fluorescence intensity (cumulative with

the knob on the power box of the fluorescence lamp)

Wide-field microscopy: bright-field and fluorescence.

Possibility to switch between Bright field (BF), Fluorescence (FLUO) and Confocal Scan (CS). Operate

fluorescence shutter (IL-shutter) or transmission light shutter (TL-shutter). Choice filter cubes DAPI, FITC,

RHOD(amine), (C )Y5.

Name Excitation [nm] DM [nm] Emission [nm]

DAPI 340-380 400 425LP

FITC 450-490 510 515LP

RHOD 517-563 580 590

Y5 590-650 660 662-738

The Objectives

Name Magnification Num. aperture Dry- Immersion

15506285 HCX PL APO CS 10x 0.40 Dry

15506242 HCX PL Fluotar L(ong distance) C, 6.9

20x coverslip correction 0-2

0.40 Dry

15506201 HCX PL Fluotar L(ong), C, obj.

40x corr 0-2 0.60 Dry

15506357 HC PL APO CS2 40x 0-2 mm coverslip correction

1.10 Water (with pump)

15506194 HCX PL APO CS 63x coverslip correction

1.30 at 21°C Glycerol

15506350 HC PL APO CS2 63x 1.40 Oil


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To switch objective, either press 2x on the desired magnification

on the touchscreen, or click the objective from the list in the LASX

software (see explanations on starting LAS-X further up).

xyz navigation of the stage

The xy position and the z-level can be adapted/monitored through several ways: with the joystick wheel,

on the touchscreen, focus knobs on the microscope (z focus), knobs on the USB panel (steers galvo z-


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motor) and through the LAS-X software.

Koehler illumination

- Focus on sample

- Close field diaphragm

- Adjust height of (open) condenser until the edge

of the field diaphragm polygon is sharp

- Center the view of the field diaphragm

- Open the field diaphragm up to the edge of the

field of view

- Give just a little bit contrast

5. LAS-X software start up

Launch the LAS-X software from the icon on the desktop. Wait until all hardware and licences

are initialized

Options to initialize (see also screenshot on next page):

-When no temperature control is required : “machine without Environmental control” with Microscope

“DMi8”

- When temperature control, and/or CO2 control, and/or water pump is needed, switch ALL THREE

DEVICES ON, WAIT UNTIL THE BRICK START UP IS FINISHED and then choose: - “machine with

Environmental control” with Microscope “DMi8”


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-The third option is a simulator (does not need lasers, but the scan head power should be on):

“SimulatorSP8” DM-Manual7

6. Lasers startup and configuration

Under Configuration, go to Laser

Config. and set WLL and/or Diode

405 nm laser on. Standard power of

WLL when on now is 50%, when

stand-by 8%.

Under Acquire, go to the rainbow

WLL panel, add a laser line (from 1 up

to 8 lines possible in the range from

470 to 670 nm) and drag the slider of

the wavelength to the desired value

in nm or type the wavelength in nm

after a double-click (here value 487

nm). Adjust the intensity of single

laser lines (405nm or WLL laser)

dragging the round spot (see below)

to the desired percentage (here 7.15%

for the 405nm). Start with a low value

(1%) to prevent over-irradiation, in

particular of the HyD.


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7. Overview menu

Option: click on the + buttons to get UV laser and White laser start menus. Then click ON

Note: this view is after setup of laser lines and detectors

ad A Acquisition window

Acquisition mode

- xyz, xzy, xyt, xylambda and derived,

- Mosaic scan or multi-positions,

- Autofocus

- Seq for sequential scan

Imaging properties

- Format: image size in pixels (optimize option)

- Scan speed

- Single direction or bidirectional

- Zoom factor: centric zoom in or out (from 0.75x on)

- Zoom In : excentric zoom ON

- Averaging (Line, frame)

- Accu: Accumulation (line, frame)

- Paning zoomed images

- Rotation of scan axis (90 degree: same orientation eye vision, DFXC365 FX camera and confocal

scan)


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Pinhole settings

- Airy or other

Z-stacks

- Set focal plane

- Begin - End: define start - final level

- Option: go to center

- Nr Steps -/ Step size

- Series from low to high or high to low

- Galvo flow For rapid z-stacks (<(1500)500 µm total distance)

- Advanced: z-compensation

ad B Excitation lines

- Configure, Acquire, Process, Quantify, Analyze

ad C Excitation lines – dye finder

- 405nm and up to 8 lines freely selectable for the WLL

- Define single laser line intensity

Also: change objective

Also: dye finder, dye assistant, own dye settings

ad D Detectors

- 2 (time-gating compatible) HyDs and 2 GaAsP-PMTs

- free selection of HyDs and PMTs windows through AOBS. However, NEVER PUT A DETECTION

WINDOW BELOW A LASER LINE, BECAUSE OF OVER-IRRADIATION RISKS

- HyDs in Photon counting (linear), Standard (linearized) or bright (non-linear) mode with or without

time gating

ad E Channels

-Visualization of single channels + overlay

ad F Post processing

- rendering and projections (3D reconstruction, stereo, surface, orthogonal)


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8. USB-Control panel and AOBS configuration, e.g. for reflection imaging

In confocal mode most of the time the SP8x is utilized to make fluorescence images, but it is also

possible to make reflection images using the AOBS at half way strength. Ask assistance to do so.

The gain and offset of the detectors, as well as the z (galvo or wide) position of the stage, and other

freely selectable functions can be attributed to the USB control panel. The sensitivity of the knobs can

be adapted.


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9. Dye finder and dye database

Dye finder

Dye finder gives an indication on overlapping among multiple dyes, and how to handle the settings (e.g.

sequential scans) for most selective, best performance. However,

the program is not fully adapted to the advanced WLL

possibilities. Therefore, check best settings manually through

own reasoning.

Dye database

This database showing excitation and emission spectra can be reached through Configuration > icon

here below

10. Wide-field and camera imaging

Switch to monochrome wide field-camera (DFC365FX) view by clicking the camera icon:

Note that the intensity of the metal halide lamp can be regulated with a knob on the power box itself

and –cumulative- inside the software as shown here below under FIM (in this example 30%)


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The camera can be utilized

both for fluorescence imaging and for transmission microscopy in monochrome views. One can switch

among filter cubes for DAPI, FITC, Rhodamine, and Cy5.

11. Shut down

Check first in bookings whether another user comes just after you.

Save your projects ON D-DRIVE D > USERS> INST> … PC

UPLOAD YOUR DATA ON A SERVER, BUT NEVER ON A USB STICK/EXTERNAL HARD DRIVE!!

Close all laser lines

405 nm and WLL from ON to Off in software

All HyDs and PMTs on OFF

Clean any immersion medium from objectives

Objective 10x in upright position

Remove slide Last user of the day (if for any reason you cannot come to the facility make sure that the microscope can be switched off by an experienced user) :

close LAS-X

Turn off Windows

Turn WLL key to off (# 4 on panel 15)

Switch fluorescence lamp off (panel 11)

Switch off (options) Temperature inside cube (box 7), the Brick (unit 16), Bartels water pump (unit 12), close air flow (wall), close CO2 flow (wall; unit 19)

WLL key 90 degree counter clockwise

Laser power off ( panel 15)

Wait 5 minutes (post-cooling)

Scanner Power off ( panel 15)

PC-Microscope off ( panel 15)

Optional: bring external cooling timer to 0


SP8X (Leica-microsystems, Mannheim, Germany) Short check ...

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