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

Tabs Container
idInstrument Tabs
titleZeiss Elyra PS.1
directionhorizontal
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



Info
titleMain applications
  • Fluorescence
  • Super-resolution:
    • SIM (Structured Illumination Microscopy)
    • PALM/STORM (Stochastic Optical Reconstruction Microscopy)
  • TIRF, HiLo


  • Light sources

    • halogen lamp for transmitted light

    • X-Cite Xylis for visible fluorescence (only for sample localisation)

    •  4 lasers 405, 488 561 638

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)

Image Modified

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




  • DetectorsDetector
    • 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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