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Button Hyperlink
iconbuild
titleFrançais
typeprimary
urlhttps://wiki.umontreal.ca/display/Microscopie/Zeiss+LSM-800


Tabs Container
idInstrument Tabs
titleZeiss Axio-Imager Z2
directionhorizontal
Tabs Page
idDescription
titleDescription

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

      Développer
      titleX-Cite 120LED mini complete specifications

      Emission peak (nm)

      Power (mW)

      334

      5



  • Objectives

  1. 5x/0.15 Ph1 Air WD 5.30 N-AchroPlan 420931-9911

  2. 10x/0.25 Ph1 WD TBD N-AchroPlan 420941-9911

  3. 20x/0.80 Air WD 0.61 Plan ApoChromat 420650-9901

  4. 40x/1.4 Oil WD TBD Plan ApoChromat 420762-9900

  5. 63x/1.40 Oil WD 0.19 Plan ApoChromat 420782-9900

  6. 40x/0.95 Air WD TBD Plan ApoChromat 420660-9970 Variable coverslip 0.13-0.21
  7. Développer
    titleComplete lens specifications

    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
    Air

    Zeiss

    20x/0.8 DIC II

    Plan-Apochromat
    W0.8x1/36"

    440640-9903-000

    20x

    0.8

    Air

    Plan Apochromat

    0.55

    >90% [410-800]

    BF, DIC, Fluo

    0.17

    4

    63x/1.40

    Huile

    Zeiss

    63x/1.4 DIC III

    Plan-Apochromat

    M27

    420782-9900-000

    63x

    1.4



    Remarque
    Huile



    Plan Apochromat

    0.19

    >80% [440-710]

    BF, DIC, Fluo

    0.17

    BF: Bright-field
    DIC: Interference contrast

  • Filter cubes
  1. 38 GFP
  2. 49 DAPI
  3. 64 mPlum
  4. DIC Analyzer
  5. Empty
  6. Empty
    Développer
    titleComplete filter specifications


    Position

    Nom

    Marque

    Identifiant

    Filtre d'excitation 

    Miroir dichroïque

    Filtre d'émission

    Commentaire



    1

    DAPI

    Filter Set 49

    Zeiss

    488049-9901-000

    365/50

    [340-390]

    395LP

    445/50

    [420-470]




    2

    GFP

    Filter Set 38

    Zeiss

    000000-1031-346

    470/40
    [450-490]

    495LP

    525/50

    [500-550]


    FT 495

    BP 525/50



  • Detector
    • 2 GaASP PMT


Tabs Page
idUser Guide
titleUser Guide


UI Expand
expandedtrue
titleStartup
  1. Remove the dust cover from the microscope
  2. Turn on the computer (#1)

  3. Turn on the System (#2) and Components (#3) switches in the rack on the left of the microscope

  4. Turn on the laser key (#4) in the rack on the left of the microscope

  5. Use your UdM credentials to log in to Windows

    Remarque
    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.

  6. Start the Zen Blue software


UI Expand
titleFirst Use

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.

Remarque

Please note, this procedure will delete all your experiment protocols and restore the software to its original settings.

  1. If open, close the Zen Blue software and wait for it to close completely (up to 30 seconds)
  2. On the Desktop open the Documentation folder
  3. Double-click Settings for LSM800
  4. A script will run and a black window will appear briefly
  5. You can then reopen the Zen Bluesoftware


UI Expand
titleLoading samples

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.

UI Expand
titleFocus Calibration Z

On the microscope touch screen:

  1. Press Home>Load Position to lower the stage to its lowest position
  2. Press Set Work Position to store this position
  3. If necessary, move the focus slightly up to remove the “Lower Z limit reached” message displayed on the touchscreen
  4. Press Home>Microscope>Control>Objectives>5x to select the 5x objective
  5. If asked, tap Done to remove the oil lens cleaning warning
  6. Press Home>Microscope>XYZ>Position>Z-Position>Set zero>Auto to perform focus calibration
  7. Press OK to start the focus calibration procedure
  8. Wait a few seconds for the calibration to be completed
Remarque

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


UI Expand
titleFirst focus
Avertissement
titleImportant

Make sure to calibrate the focus before performing the first focus.

On the microscope touch screen:

  1. Press Home>Microscope>Turret>Objectives
  2. Press 5x to select the 5x lens
    Info

    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.

  3. Press Home>Load Position to lower the stage to its lowest position
  4. Press Set Work Position to store this position
  5. If necessary, move the focus slightly up to remove the “Lower Z limit reached” message displayed on the touchscreen
  6. Place the test slide on the microscope stage with the coverslip toward the objective
    Remarque
    titleImportant

    Always use the test slide to perform the first focus.

  7. If necessary, move the stage so that the sample is centered on the objective

On the computer:

  1. Open the Zen Blue software
  2. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, mPlum) to activate the configuration
  3. Adjust the focus with the main dial while looking through the eyepieces until the image is perfectly sharp

    Remarque

    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


  4. In the Locate tab, select Off to turn off the illumination


UI Expand
titleSeconday focus
Avertissement
titleImportant

First focus with the safest lens before selecting another lens and continuing with secondary focus.


UI Expand
titleFocusing with air objectives

After performing the first focus, on the microscope touch screen:

  1. Press Home>Microscope>Turret>Objectives
  2. Press 10x, 20x or 40x to select the desired lens
    Info
    The 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.
    Remarque

    There are two (2) 40x objectives, make sure you select the Air one


  3. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
  4. In the Locate tab, select Off to turn the illumination off
  5. Your sample is ready for acquisition!


UI Expand
titleFocusing with oil lenses

After performing the first focus, on the microscope touch screen:

  1. Press Home>Microscope>Turret>Objectives

  2. Press 63x Oil, 40x Oil to select the desired lens. The microscope will automatically lower the stage so that the sample is accessible.

    Info

    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.

    Remarque

    There are two (2) 40x objectives, make sure you select the OIL one


  3. Place a drop of oil on the objective
  4. Press Done. The microscope will automatically return the sample to its original position

In Zen Bluesoftware:

  1. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, mPlum) to activate the configuration
  2. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
  3. In the Locate tab, select Off to turn the illumination off
  4. Your sample is ready for acquisition!
UI Expand
titleStorage management
  • 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).
Remarque

In any case, your files should be removed from the C: drive.

UI Expand
titleShutdown
  1. Save your data
  2. Close the software Zen Blue
  3. Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
  4. If used, clean oil lenses with lens cleaner and paper
  5. Turn off the laser key (#4) in the rack on the left of the microscope
  6. Turn off the System (#2) and Components (#3) switches in the rack on the left of the microscope

  7. Turn off the computer

  8. Cover the microscope
Remarque
titleImportant Reminders
  • Take back your samples including ones in the microscope
  • Leave the microscope and the working area clean
Tabs Page
idLight Path
titleLight Path

The following diagrams allow you to follow the light path in transmitted light (bright field, DIC) and in reflected light (fluorescence).



Tabs Page
idManuals
titleManuals


Tabs Page
idLog
titleLog
UI Expand
titleTo do
  • Quality control for Illumination, Liquid light guide and filters quality.
  • Camera sensor clean up
UI Expand
title2024-02-14 Added to wiki
  • User guide added to Wiki French and english version
UI Expand
title2022-03-28
  • Objective 10-0.3 Plan NeoFluor #420340-9901-000 added
  • Parafocality adjustment using 100x-1.4 as reference
  • Condenser removed and cleaned (broken glass)
UI Expand
title2021-10-20
  • Zen 2.6 updated hotfix 12
  • Microsoft Windows updated
UI Expand
title2021-10-18
  • 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
UI Expand
title2021-09-27
  • Added to wiki


Tabs Page
idTechnical Datasheet
titleTechnical Datasheet

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

    1. H Empty

    2. Ph1
    3. Ph2
    4. Ph3
    5. DICII #426702
    6. DIC III #426706

Objectives

  1. 20x/0.80 Air WD 0.61 DIC II Plan-Apochromat W0.8x1/36" 440640-9903-000

  2. 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 #

  1. GFP Zeiss  Filter Set 38 cube 424933
  2. DAPI Zeiss Filter Set 49 cube 424933
  3. mPlum Zeiss Filter Set 64
  4. DIC Analyzer Zeiss 424932-01
  5. Empty
  6. 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


Tabs Page
idFAQ
titleTroubleshooting & FAQ

Troubleshooting

UI Expand
titleI don't see any fluorescence!

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.

  1. Open the light path file
  2. Starting from the light source and moving towards the detector, verify that there is indeed light after each component of the microscope

FAQ

UI Expand
titleCan I use this microscope to observe cells in culture?
Yes it is an inverted microscope. Point scanning confocal induce damage so care should be taken while imaging live cells. a coverslip (thickness 0.17mm).




Tabs Container
idDemo Image
titleZeiss LSM-800
directionhorizontal


Tabs Page
idDemo Image
titleDemo Image




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