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titleFrançais
typeprimary
urlhttps://wiki.umontreal.ca/display/Microscopie/Zeiss+Elyra

Tabs Container
idInstrument Tabs
titleZeiss Elyra PS.1
directionhorizontal
This illumination mode only works to localize samples - NOT for acquisition

(only for sample localisation)

  •  4 lasers 405, 488 561 638


    • Tabs Page
    idDescription
    titleDescription

    Zeiss Elyra PS.1 structured illumination microscope

    J-A Bombardier Building, Room 3223-03
    Advanced Microscope Tier 2 usage price

    Instrument awarded to Dr. Daniel Zenklusen and Pascal Chartrand by the Canadian Foundation for Innovation (CFI)

    • Applications
      • Fluorescence
      • Super-resolution

    • Light sources

      • halogen lamp for transmitted light

      • X-Cite Xylis for visible fluorescence

    Note
    titleX-Cite Xylis
    Note
    titleLaser stability

    For quantitative imaging, turn lasers ON or STANDBY at least 60-90 before acquisition

    Expand
    titleLaser complete specifications

    Emission peak (nm)

    Nominal Output Power (mW)

    Power at sample plane in mW (2024/07/15)

    SIM mode

    Power at sample plane in mW (2024/07/15)

    TIRF mode (WF-EPI)

    405

    50

    2.15

    3.6

    488

    100

    5.52

    7.7

    561

    100

    7.9

    10.3

    639

    100

    2.64

    3.2

    • Objectives

    1. 10x/0.30 Air WD 5.30

    2. 63x/1.40 Oil WD 0.19

    3. Empty


    Expand
    titleFull 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)

    1

    10x/0.30
    Air

    Zeiss

    10x/0.3 DIC I

    EC Plan-Neo Fluar

    M27

    420340-9901-000

    10x

    0.3

    Air

    Plan Neofluar

    5.2

    >90% [480-780]

    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



    Note
    Huile



    Plan Apochromat

    0.19

    >80% [440-710]

    BF, DIC, Fluo

    0.17

    BF: Bright-field
    DIC: Interference contrast

    • Filter cubes
    1. Transmitted light
    2. Filter set 77 HE (GFP/Cy3.5/Cy5)
    3. BP420-490 + LP750
    4. LP 570
    5. BP490-520 + LP750
    6. LP640
    Expand
    titleComplete filter specifications


    Position

    Name

    Brand

    ID

    Excitation filter

    Dichroic mirror

    Emission filter

    Filter spectra

    Fluorophore examples

    1

    Transmitted light

    Zeiss







    2

    Filter Set 77 HE

    Zeiss

    		489077-0000-000

    TBP 483 + 564 + 642 (HE)

    TFT 506 + 582 + 659 (HE)

    TBP 526 + 601 + 688 (HE)

    GFP, FITC, Alexa488, Cy3.5, DsRed, Calcium Orange, mCherry, Cy5, Alexa647

    3

    BP420-490 + LP750

    Zeiss







    4

    LP 570

    Zeiss







    5

    BP490-520 + LP750

    Zeiss







    6

    LP640

    Chroma

    SP102v1

    546/11
    [541-551]

    557LP




    • Detector
      • TV1 for PALM/STORM (side port): EMCCD iXon EM+ DU-897D-CS0-#BV-462 (Andor)
        • Back illuminated, >90% QE; single photon detection
        • 512x512, 16 µm pixels (i.e. 1 pixel = 266 nm2of the sample when using the 63x Objective and 1x adapter), 35 fps (full chip) to hundreds fps (cropped chip)
        • 14 bits; 10, 5, 3 et 1 MHz
        • Cooled to -80oC, Readout noise 49 e- @ 10 MHz or 32 e- @ 3 MHz

      • TV2 for SR-SIM (bottom port) : EMCCD iXon3 DU-885K CSO VP461 (Andor)

        • Quantum Efficiency:  50-65% between 400nm and 750nm
        • Full chip acquisition: 1004x1002, 8 µm pixels (i.e. 1 pixel = 133 nm2of the sample when using the 63x Objective and 1x adapter), 31 fps (full chip) à 13 812 fps
        • Picture after SIM reconstruction: 1904x1900 px
        • 14 bits; 35, 27 et 13 MHz
        • Cooled to -80oC, Readout noise 25 e- @ 35 MHz or 12 e- @ 13 MHz


    Tabs Page
    idUser Guide
    titleUser Guide
    UI Expand
    expandedtrue
    titleStartup
    1. Turn on the computer (#1)

    2. Turn on the microscope power bar on the left of the microscope (#2)

    3. Turn on the microscope power bar on the right of the microscope (#3)

    4. Use your UdeM credentials to log in to Windows

      Note

      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.

    5. Start the Zen Black 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.

    Note

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

    1. If open, close the Zen Black 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 Zeiss Elyra
    4. Click Yes
    5. Click OK
    6. A script will run and a black window will appear briefly
    7. You can then reopen the Zen Black software


    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>Turret>Objectives>10x to select the 10x 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
    Note

    Once calibrated, the focus can be found Z=1.7 mm for the adjustable insert and Z=3.1 mm for simple inserts. The Z value can be found on the microscope touch screen Home>Z-Position


    UI Expand
    titleFirst focus
    Warning

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

    On the microscope touch screen:

    1. Press Home>Microscope>Turret>Objectives
    2. Press 10x to select the 10x lens
      Info

      The 10x objective is the safest because it has the longest working distance (5.3 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. Select one of the 3 available insert: i) Petri, ii) Slide ou iii) Combo with tilt adjustment

    7. Place the test slide in the insert with the coverslip toward the objective
      Note

      Always use the test slide to perform the first focus.

    8. Place the insert on the microscope stage
    9. If necessary, move the stage so that the sample is centered on the objective

    On the computer:

    1. Open the Zen Black software
    2. In the Locate tab, select BF visible or Fluo visible to activate the configuration
    3. Adjust the focus with the main dial while looking through the eyepieces until the image is perfectly sharp

      Note

      Once calibrated, the focus can be found Z=1.7 mm for the adjustable insert and Z=3.1 mm for simple inserts. 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
    Warning
    titleImportant

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


    UI Expand
    titleFocusing with air objectives

    This microscope does not have additional air objectives. However, if there were, the procedure would be as follows:

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

    1. Press Home>Microscope>Turret>Objectives
    2. Press on the desired lens

    In Zen Black software:

    1. In the Locate tab, select BF visible or Fluo visible 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
    titleFocusing with oil lenses

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

    1. Press Home>Microscope>Turret>Objectives

    2. Press 63x Oil (1.4) to select the 63x lens. The microscope will automatically lower the stage so that the sample is accessible.

    3. Remove the insert from the microscope stage
    4. Place a drop of oil on the objective
    5. Replace the insert on the microscope stage
    6. Press Done. The microscope will automatically return the sample to its original position

    In Zen Black software:

    1. In the Locate tab, select BF visible or Fluo visible 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).
    Note

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

    UI Expand
    titleShutdown
    1. Save your data
    2. Close the software Zen Black
    3. Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
    4. Clean oil lenses with lens cleaner and paper

    5. Turn off the microscope power bar on the right of the microscope (#3)

    6. Turn off the camera and laser power bar on the left of the microscope (#2)
    7. Turn off the computer
    Note
    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
    Check Loss of 488nm laser power
    Tabs Page
    idTechnical Datasheet
    titleTechnical Datasheet

    Stand

    • Zeiss AxioObserver Z1 upright Serial:
      Part Number: 
      System ID

    • Camera adapter Model 60N-C, 1", 1x, Model: 426114

    • Motorized Neutral density filters for transmitted light

    • Manual Field diaphragm for transmitted light

    • Manual polarizer
    • Left imaging port with manual splitter camera adapter Model 60N-C, 1", 1x, Model: 426114
    • Trinocular with 100% ocular 40% occular/70% camera and 100% manual splitter
    • 3mm liquid light guide #805-0038
    • Zeiss 423302-0000 Collimator
    • Motorized Aperture diaphragm
    • Motorized Fluorescence field diaphragm

    Light sources

    • Transmitted Halogen light 12V 100W HAL 100 #423000

    Condenser

    • Motorized condenser #424201-9902

    • Lens NA 0.9 WD TBD Part Number: TBD

    • Manual polarizer

    • Filter turret 6 positions manual

    Objectives

    1. 10x/0.30 Air WD 5.30 DIC I  Plan-NeoFluar M27 420340-9901-000

    2. 63x/1.40 Oil WD 0.19 DIC III Plan-Apochromat M27 420782-9900-000

    3. 100x/1.40 Oil WD 0.17 DIC III Plan-Apochromat M27 420792-9900-000 

    Stage

    • Motorized stage Zeiss AIM System #2502000124
    • Remote control joystick
    • Inserts
      • Slide only
      • Plate

    Filters

    10-positions motorized filter wheel #

    1. DAPI Zeiss Filter Set 49 cube 424933

    Detector

    • 2 camera Evolve 512 Serial

    Workstation

    • HP Z800 Workstation Serial: CZC1473Y0Q Part number: WJ112ECJ#AK6
    • 2 x Intel Xeon X5650 2.66 GHz
    • RAM 24 GB DDR3 1333 MHz ECC (12 x 2 GB)
    • OS 500 GB SSD 410 MB/s
    • 2 TB HD Data Storage (2 x 1 TB spanned volume) 110 MB/s
    • Video Card ATI FirePro V5800 1 GB DDR5
    • Monitor Dell ST2410  24' 1920 x 1080
    • Software Zen Blue 2.6 Hotfix 12

    Incubation

    • Zeiss 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 look at cell in a dish?

    Yes. It is an inverted microscope designed for the observation of living specimens. The spinning disk is particularly appreciated for its limited phototoxicity. This is an inverted microscope designed to look at specimen in a dish or a multi-well plate. The objectives are optimized to image through thin glass bottom multi-well plates. You may also image specimen mounted between a slide and a 0.17mm thick coverslip.



    Tabs Container
    idDemo Image
    titleZeiss Elyra
    directionhorizontal


    Tabs Page
    idDemo Image
    titleDemo Image




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