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Zeiss LSM800 confocal microscope
Roger Gaudry Building, Room R-421
Advanced Microscope Tier 2 usage price
Instrument awarded to Dr. Jean-Philippe Gratton by the Canadian Foundation for Innovation (CFI)
- Applications
- Transmitted light
- Interference Contrast (DIC)
- Fluorescence
- Laser scanning confocal
Light sources
12V 100W halogen lamp for transmitted light
X-Cite 120LED mini for visible fluorescence
Emission peak (nm)
Power (mW)
334
5
Objectives
5x/0.15 Ph1 Air WD 5.30 N-AchroPlan 420931-9911
- 10x/0.25 Ph1 WD TBD N-AchroPlan 420941-9911
20x/0.80 Air WD 0.61 Plan ApoChromat 420650-9901
- 40x/1.4 Oil WD TBD Plan ApoChromat 420762-9900
63x/1.40 Oil WD 0.19 Plan ApoChromat 420782-9900
- 40x/0.95 Air WD TBD Plan ApoChromat 420660-9970 Variable coverslip 0.13-0.21
Position
Nom
Marque
Nom complet
Identifiant
Grossissement
Ouverture numérique
Immersion
Type
Distance de travail (mm)
Transmittance
(% [nm])
Technique
Épaisseur du couvre-objet (mm)
2
20x/0.80
AirZeiss
20x/0.8 DIC II
Plan-Apochromat
W0.8x1/36"20x
0.8
Air
Plan Apochromat
0.55
BF, DIC, Fluo
0.17
4
63x/1.40
Huile
Zeiss
63x/1.4 DIC III
Plan-Apochromat
M27
63x
1.4
HuilePlan Apochromat
0.19
BF, DIC, Fluo
0.17
BF: Bright-field
DIC: Interference contrast
- Filter cubes
- 38 GFP
- 49 DAPI
- 64 mPlum
- DIC Analyzer
- Empty
- Empty
Position
Nom
Marque
Identifiant
Filtre d'excitation
Miroir dichroïque
Filtre d'émission
Commentaire
1
DAPI
Zeiss
365/50
[340-390]
395LP
445/50
[420-470]
2
GFP
Zeiss
470/40
[450-490]495LP
525/50
[500-550]
FT 495
BP 525/50
- Detector
- 2 GaASP PMT
- Remove the dust cover from the microscope
- Turn on the computer (#1)
Turn on the System (#2) and Components (#3) switches in the rack on the left of the microscope
Turn on the laser key (#4) in the rack on the left of the microscope
Use your UdM credentials to log in to Windows
When using for the first time, it is necessary to import the microscope-specific parameters BEFORE starting the software. See the First Use section below.Start the Zen software
When using for the first time, it is necessary to import the microscope-specific parameters into the software. This procedure is usually carried out during the training session. However, it is also possible to use it to reset the software if it is not displayed correctly, for example.
Please note, this procedure will delete all your experiment protocols and restore the software to its original settings.
- If open, close the Zen software and wait for it to close completely (up to 30 seconds)
- On the Desktop open the Documentation folder
- Double-click Settings for LSM800
- A script will run and a black window will appear briefly
- You can then reopen the Zen software
This procedure puts the microscope in a safe configuration and performs a focus calibration. At the end of this procedure the microscope will be ready for acquisition.
On the microscope touch screen:
- Press Home>Load Position to lower the stage to its lowest position
- Press Set Work Position to store this position
- If necessary, move the focus slightly up to remove the “Lower Z limit reached” message displayed on the touchscreen
- Press Home>Microscope>Control>Objectives>5x to select the 5x objective
- If asked, tap Done to remove the oil lens cleaning warning
- Press Home>Microscope>XYZ>Position>Z-Position>Set zero>Auto to perform focus calibration
- Press OK to start the focus calibration procedure
- Wait a few seconds for the calibration to be completed
Once calibrated, the focus can be found at Z = 1.6 mm). The Z value can be found on the microscope touch screen Home>Z-Position
Important
Make sure to calibrate the focus before performing the first focus.
On the microscope touch screen:
- Press Home>Microscope>Turret>Objectives
- Press 5x to select the 5x lens
The 5x objective is the safest because it has the longest working distance (TBD mm). The sample will appear perfectly sharp long before the lens approaches it. It is recommended to always first focus with the safest lens. The objectives are para-focal, focusing with the safest objective will then allow you to easily find your sample with another objective.
- Press Home>Load Position to lower the stage to its lowest position
- Press Set Work Position to store this position
- If necessary, move the focus slightly up to remove the “Lower Z limit reached” message displayed on the touchscreen
- Place the test slide on the microscope stage with the coverslip toward the objective
Important
Always use the test slide to perform the first focus.
- If necessary, move the stage so that the sample is centered on the objective
On the computer:
- Open the Zen
- In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, mPlum) to activate the configuration
- Adjust the focus with the main dial while looking through the eyepieces until the image is perfectly sharp
Once calibrated, the focus can be found at Z = 1.6 mm). The Z value can be found on the microscope touch screen Home>Z-Position
- In the Locate tab, select Off to turn off the illumination
Important
First focus with the safest lens before selecting another lens and continuing with secondary focus.
After performing the first focus, on the microscope touch screen:
- Press Home>Microscope>Turret>Objectives
- Press 10x, 20x or 40x to select the desired lensThe 40x objective is the best Air objective because it has the greatest number of optical corrections (Plan Apochromat) and the largest numerical aperture (0.95). It offers a lateral resolution of 420nm at a wavelength of 550nm.
There are two (2) 40x objectives, make sure you select the Air one
- Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
- In the Locate tab, select Off to turn the illumination off
- Your sample is ready for acquisition!
After performing the first focus, on the microscope touch screen:
- Press Home>Microscope>Turret>Objectives
Press 63x Oil, 40x Oil to select the desired lens. The microscope will automatically lower the stage so that the sample is accessible.
The 40x objective is the best oil objective because it has the most optical corrections (Plan Apochromat) and the largest numerical aperture (1.4). It offers a lateral resolution of 240nm at a wavelength of 550nm.
There are two (2) 40x objectives, make sure you select the OIL one
- Place a drop of oil on the objective
- Press Done. The microscope will automatically return the sample to its original position
In Zen software:
- In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, mPlum) to activate the configuration
- Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
- In the Locate tab, select Off to turn the illumination off
- Your sample is ready for acquisition!
- Files can be saved temporarily (during acquisition) on the local C: drive (desktop)
- At the end of each session, copy your data to your external drive and delete it from the local C: drive
- You can store your files on the D: drive (Data Storage). If you do, please create a folder per laboratory using the principal investigator last name. Within, create one folder per user (Firstname_Lastname).
In any case, your files should be removed from the C: drive.
- Save your data
- Close the software Zen
- Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
- If used, clean oil lenses with lens cleaner and paper
- Select the 5x objective and press load position to place the objectives in a safe position
- Turn off the laser key (#4) in the rack on the left of the microscope
- Turn off the System (#2) and Components (#3) switches in the rack on the left of the microscope
Turn off the computer
- Cover the microscope
Important Reminders
- Take back your samples including ones in the microscope
- Leave the microscope and the working area clean
The following diagrams allow you to follow the light path in transmitted light (bright field, DIC) and in reflected light (fluorescence).
- Quality control for Illumination, Liquid light guide and filters quality.
- Camera sensor clean up
- User guide added to Wiki French and english version
- Objective 10-0.3 Plan NeoFluor #420340-9901-000 added
- Parafocality adjustment using 100x-1.4 as reference
- Condenser removed and cleaned (broken glass)
- Zen 2.6 updated hotfix 12
- Microsoft Windows updated
- X-Cite Liquid light guide replaced (Transmittance was 75% remaining)
- Fluorescence Light bulb replaced (was 2593 hours)
- Power output at the sample using 20x objective GFP cube 488nm is 116mW
- Objective parafocality adjusted
- Objective Focus speed was changed 10x: 7; 20x: 6; 4-x: 5; 63x: 3; 100x-1.3: 3; 100x-1.4: 3.
- Condenser lens cleaned (oil)
- Data storage OK
- Added to wiki
Stand
- Zeiss AxioObserver LSM800 Serial:
System ID 1022265893 Manual Field diaphragm for transmitted light
- Manual polarizer
- Left imaging port with LSM800 confocal
- Motorized Aperture diaphragm
- Motorized Fluorescence field diaphragm
Light sources
- Transmitted Halogen light 12V 100W HAL 100 #423000
- TBD Filters
- X-Cite 120LED mini
Condenser
- Motorized condenser #424201-9902
Lens NA 0.9 WD TBD Part Number: TBD
- Manual polarizer
Filter turret 6 positions manual
H Empty
- Ph1
- Ph2
- Ph3
- DICII #426702
- DIC III #426706
Objectives
20x/0.80 Air WD 0.61 DIC II Plan-Apochromat W0.8x1/36" 440640-9903-000
- 63x/1.40 Oil WD 0.19 DIC III Plan-Apochromat M27 420782-9900-000
Stage
- Motorized stage Marhauser
- Remote control joystick
- Inserts
- Combo slide and 30mm dish
Filters
6-positions motorized filter wheel #
- GFP Zeiss Filter Set 38 cube 424933
- DAPI Zeiss Filter Set 49 cube 424933
- mPlum Zeiss Filter Set 64
- DIC Analyzer Zeiss 424932-01
- Empty
- Empty
Detector
- 2 GaASp PMT
Workstation
- Fujitsu Espimo P920 90+
- 2 x Intel Core i5
- RAM 32GB D
- OS 1 TB SSD 410 MB/s
- 2 TB HD Data Storage
- Video Card
- Monitor HP
- Software Zen Blue 2.6 Hotfix 12
Incubation
Consumables
- 12V 100W halogen lamp OSRAM XenoPhot #64623 HLX
Troubleshooting
The best way to solve a problem in Microscopy is to follow the light path. You will find in the Light path tab of this page, the diagrams which will allow you to follow the light all along its path through the microscope.
- Open the light path file
- Starting from the light source and moving towards the detector, verify that there is indeed light after each component of the microscope
FAQ
- Aucune étiquette