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Zeiss LSM-900 confocal Airyscan microscope

Desmarais Building, Room 2234
Advanced Microscope Tier 2 usage price

Instrument awarded to Dr. Jean-Philippe Gratton by the Canadian Foundation for Innovation (CFI)

  • Transmitted light
  • Phase Contrast
  • Interference Contrast (DIC)
  • Fluorescence
  • Laser scanning confocal
  • Airyscan super-resolution

Zeiss LSM900

Light sources

  • LED lamp for transmitted light

  • Laser 405nm, 488nm, 561nm, 640nm

Emission (nm)

Nominal power (mW)

405

30

488

25

561

25

640

45

  • X-Cite 120LED mini for visible fluorescence

    Filter (nm)

    Emission Peak (nm)

    Nominal power (mW)

    Power at the sample (mW)

    DAPI

    365



    GFP

    		470

    mPlum

    		587

Objectives

  1. 5x/0.15 Ph1 Air
  2. 10x/0.25 Ph1 Air
  3. 20x/0.80 Air
  4. 40x/0.95 Air
  5. 40x/1.4 Oil
  6. 63x/1.4 Oil

Position

Name

BrandFull name

identifier

Magnification

Numerical aperture

Immersion

Type

Working distance (mm)

Transmittance

(% [nm])

Applications

Coverglass thickness (mm)

1

5x/0.15 Ph1 Air

Zeiss

5x/0.15 Ph1 Air
N-AchroPlan

420931-9911-000

5x

0.15

Air

N-AchroPlan

12.0

Not Available

BF, PhC, Fluo

0.17

2

10x/0.25 Ph1 Air

Zeiss

10x/0.25 Ph1 Air
N-AchroPlan

420941-9911-000

10x

0.25

Air


N-AchroPlan

6.5

Not Available

BF, PhC, Fluo

0.17

3

20x/0.8 Air 

Zeiss

20x/0.8 Air
Plan-Apochromat

 420650-9901-000

20x

0.8

Air

Plan-Apochromat

0.55 

>90% [400-800]

BF, DIC, Fluo

0.17

4

 40x/0.95 Air

Zeiss

40x/0.95 Air
Plan-Apochromat

420660-9970-000 

40x

0.95

Air

Plan-Apochromat

 0.25

>80% [410-820]

 BF, DIC, Fluo

Variable
0.13-0.21

5

 40x/1.4 Oil

Zeiss

40x/1.4 Huile
Plan-Apochromat

420762-9900-000

40x

1.4

Oil

Plan-Apochromat

 0.13

>80% [500-850] 

 BF, DIC, Fluo

0.17

6

63x/1.4 Oil

Zeiss

63x/1.4 Huile
Plan-Apochromat

 420782-9900-799

63x

1.4

Oil

Plan-Apochromat 

 0.19

 >80% [440-710]

 BF, DIC, Fluo, Super-Resolution
Strehl ratio >90%.

0.17

BF: Bright-field
PhC: Contraste de phase
DIC: Interference contrast

 Filter cubes

  1. GFP
  2. DAPI
  3. mPlum
  4. DIC Analyzer
  5. Empty
  6. Empty

Position

Name

Brand

Identifier

Excitation

Dichroic

Emission

Graph

1

GFP
Filter Set 38		Zeiss000000-1031-346BP 470/40FT 495BP 525/50

Zeiss Filter Set 38 GFP

2

DAPI
Filter Set 49		Zeiss

488049-9901-000

G 365FT 395BP 445/50

Zeiss Filter Set 49 DAPI

3

mPlum
Filter Set 64 HE		Zeiss489064-0000-000BP 587/25 (HE)FT 605 (HE)BP 647/70 (HE)

Zeiss Filter Set 64 HE mPlum

4

DIC AnalyzerZeiss424937-9901-000----

5

Vide------

6

Vide

-----

-

Detectors

  • 2x Gallium Arsenide Phosphid (GaAsP) Photomultiplier Tube (PMT)
  • 1x transmitted light Electronically Switchable Illumination and Detection module (ESID)
  • 1x Airyscan detector for 63x objective
  1. If not already done, turn on the computer (#1) and use your UdM credentials to log in to Windows
  2. Remove the dust cover from the microscope

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

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

  5. 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 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.

  1. If open, close the Zen software and wait for it to close completely (up to 30 seconds)
  2. On the Desktop open the Documentation folder
  3. Double-click Zen Settings for LSM900
  4. A script will run and a black window will appear briefly
  5. Click OK to close the message Settings for Zen have been imported successfully.
  6. You can then open 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:

  1. Press Home>Load Position to lower the objectives to the 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. If not already done, 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

Once calibrated, the focus can be found at Z = 1.7 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:

  1. If not already done, press Home>Microscope>Turret>Objectives>5x to select the 5x objective

    The 5x objective is the safest because it has the longest working distance (12mm). 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 parafocal, focusing with the safest objective will then allow you to easily find your sample with another objective. The 10x objective is also safe because its working distance is 6.5 mm.

  2. If not done already, press Home>Load Position to lower the objective to the lowest position and 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. 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.

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

On the computer:

  1. Open Zen
  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

    Once calibrated, the focus can be found at Z = 1.7 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

Important

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

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

  1. Press Home>Microscope>Control>Objectives, press 10x, 20x or 40x to select the desired objective
    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) but has a smaller field of view.
    The 20x/0.8 objective offers the best compromise between Resolution and Field of View

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


In Zen software :

  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!

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 objective. The microscope will automatically lower the stage so that the sample is accessible.

    The 40x and 63x oil objectives provide the same spatial resolution because they have the same numerical aperture (1.4).
    The 40x oil objective offers a larger field of view and transmits light slightly better beyond 700nm.
    The 63x oil objective transmits light slightly better in the visible spectrum (440-710 nm) and has a better Strehl ratio (90%). It is particularly suited for super-resolution imaging, but its field of view is smaller.

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


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

In Zen software:

  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!
  • 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.

  1. Save your data
  2. Close the software Zen
  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. Select the 5x objective and press load position to place the objectives in a safe position
  6. Turn off the laser key (#4) in the rack below the microscope
  7. Turn off the Components (#3) and System (#2) switches in the rack below the microscope

  8. Turn off the computer

  9. Cover the microscope

Important Reminders

  • Take back your samples including ones in the microscope
  • Leave the microscope and the working area clean
  • Quality control for Illumination, Liquid light guide and filters quality.
  • Joystick replaced
  • Installation Airyscan upgrade. New workstation. New detector. New casing.
  • Issue when the joystick is at the rest position. If it is slightly to the left, the stage will move while the image is acquired. Temporary fix: Make sure the joystick is slightly to the right when it is resting.
  • User guide added to Wiki French and english version

  • Zen 2.6 updated hotfix 12
  • Microsoft Windows updated

  • Added to wiki

Stand

  • Zeiss AxioObserver LSM900 Serial: 2633000272

  • Manual Field diaphragm for transmitted light

  • Manual polarizer
  • Left imaging port with LSM900 confocal 
  • Manual Aperture diaphragm
  • Manual Fluorescence field diaphragm
  • 6x motorized nosepiece
  • 6x Motorized reflector changer
  • 3x Motorized sideport turret (100% Visible, 100% Left (confocal), 100% right (empty)
  • TL Motorized shutter
  • RL Motorized shutter

Light sources

  • Transmitted LED light
  • X-Cite 120LED mini Serial XT120LM-0279 RS232 COM Port 6
  • Laser URGB 400102-9300-000 Serial 2631000466  Toptica iChrome-ZLE-4_40407 v3.1 Aug 2016

Condenser

  • Manual condenser

  • Condenser Lens NA 0.55 WD 26mm

  • Manual polarizer

  • Filter turret 6 positions manual

    1. H Empty

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

Objectives

  1. 5x/0.15 Ph1 Air WD 5.6 N-AchroPlan 420931-9911-000

  2. 10x/0.25 Ph1 WD 6.0 N-AchroPlan 420941-9911-000

  3. 20x/0.80 Air WD 0.61 Plan-Apochromat 420650-9901-000

  4. 40x/0.95 Air WD 0.25 Plan-Apochromat 420660-9970-000 Variable coverslip 0.13-0.21
  5. 40x/1.4 Oil WD 0.13 Plan-Apochromat 420762-9900-000

  6. 63x/1.40 Oil WD 0.19 Plan Apochromat 420782-9900-799

Stage

  • Motorized stage Marzhauser Scan IM 130x100 Serial 14032630
  • Remote control joystick Marzhauser  2-Schsen-Joystick CZ Serial 2420142128 Article 90-76-200-0820 Zeiss Serial 432903-9011-000
  • Inserts
    • Slide combo
    • 6-well plate
    • 35 mm dish
    • Multi-well plat

Filters

6-Position motorized Filter Turret 424947

  1. GFP Zeiss Filter Set 38 cube 424931 000000-1031-346 BP 470/40 450-490 FT 495 BP525/50 500-550
  2. DAPI Zeiss Filter Set 49 cube 424931 489064-0000-000 G365 FT 395 BP 445/50 335-383 395 420-470
  3. mPlum Zeiss Filter Set 64 HE cube 424931  489064-0000-000 BP587/25 FT605 BP647/70 574-599 605 612-682
  4. DIC Analyzer Zeiss 424937-9901
  5. Empty
  6. Empty

Scanner

LSM900 Scan head

  • 8x Motorized Filter wheel 1
  1. Empty
  2. SP 470 000000-2090-296 410-470 93% 480-750 0.001%
  3. SP 545 000000-2090-297 410-546 93% 557-750 0.001%
  4. SP 620 000000-2090-298 410-617 93% 630-750 0.001%
  5. Empty
  6. Empty
  7. Empty
  8. Empty
  • 8x Motorized Filter wheel 2
  1. Empty
  2. LBF 640 0000000-2090-299 410-617 93% 630-644 0.001%
  3. LP 575 0000000-2090-294 576-750 93% 410-565 0.001%
  4. LP 655 0000000-2090-295 656-750 93% 395-643 0.001%
  5. SP 620 000000-2090-298 410-617 93% 630-750 0.001%
  6. Empty
  7. Empty
  8. Empty

 Main Beam splitter

MBS 405+488+561+640 T10/R90

400102-9600-000

Transmission

  • 420-471 93%
  • 502-546 93%
  • 575-618 93%
  • 656-800 93%
  • 631-642 10%

Reflection

  • 399-410 99%
  • 481-492 99%
  • 558-564 99%
  • 631-642 90%

Detector

  • 2 GaASp PMT
  • 1 Airyscan 2 63x Serial 2657000116
  • 1 transmitted ESID Motorized Left TL ESID LED, Right ESID Detector

Workstation

  • HP Z2 G9 Serial CZC4027PF7 829W6EC#ABB

  • I155440-CIB

  • Motherboard HP 895C
  • BIOS version 2024-09-03
  • 1 x Intel Core i7-12700K 3.6 GHz
  • RAM 128GB DDR5 4 x 32GB no ECC
  • OS 500 GB NVMe 6 GB/s
  • 7 TB HD Data Storage 213 MB/s
  • Nvidia RTX A4000 16 GB GDDR6
  • Monitor HP Z32x
  • Software Zen 3.10

Incubation

Consumables

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.

  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

Yes. It is an inverted microscope designed for observing specimens in culture. The objectives are optimized for viewing through glass-bottom multi-well plates. It is also possible to observe samples between a slide and coverslip (with a thickness of 0.17mm).

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