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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 # 34992) in 2015

 

Applications

  • Inverted microscope
  • Airyscan super-resolution with joint deconvolution
  • Point scanning confocal
    • Brightfield
    • Phase contrast
    • Interference Contrast (DIC)
    • Fluorescence
  • Targeted photo-modulation FRAP
  • Incubation
  • Timelapse imaging
  • Deconvolution
  • Extended Depth of Focus

Description

Light sources

  • LED lamp for transmitted light

  • Laser 405nm, 488nm, 561nm, 640nm

Emission (nm)

Nominal Power (mW)

Measured Power (mW)

405

5


488

10

561

10

640

5
  • X-Cite 120LED mini for visible fluorescence

Filter (nm)

Emission Peak (nm)

Nominal Power (mW)

Measured Power (mW)

DAPI

365



GFP

470

mPlum

587

XCite_120LEDmini_Datasheet.pdf

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
Air

Zeiss

5x/0.15 Ph1
N-AchroPlan

420931-9911-000

5x

0.15

Air

N-AchroPlan

12.0

Not Available

BF, PhC, Fluo

0.17

2

10x/0.25
Air

Zeiss

10x/0.25 Ph1
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
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
Plan-Apochromat

420660-9970-000 

40x

0.95

Air

Plan Apochromat

 0.25

>80%
[410-820]

 BF, DIC, Fluo

0.13-0.21

5

 40x/1.4
Oil

Zeiss

40x/1.4
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
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

 Filters

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

Position

Name

Brand

ID

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

-------

6

-

-----

-

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

User Guide

  1. If not already done, turn on the computer (#1) and log in to Windows using your UdeM credentials
  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 the instrument for the first time, it is necessary to import the microscope configuration into the software. See the First Use section below.
  6. Start Zen

When using the instrument for the first time, it is necessary to import the microscope configuration into the software. This procedure is usually carried out during the training session. However, it can also be used to reset the software if it does not display correctly, for example. Running this procedure will erase all your experiment protocols and reset the software to its original settings (ask for support if you are not sure).

  1. If Zen is open, close it and wait until it has completely shut down (this may take up to 30 seconds)

  2. On the Desktop, open the Softwares folder

  3. Double-click Zen Settings for LSM 900

  4. A script will run and a black window will appear briefly

  5. When the message Settings for Zen have been imported successfully appears, click OK to close it

  6. You can now open Zen

During this procedure, you will:

  • Set the microscope to a safe configuration
  • Perform a calibration
  • Load your sample
  • Find and adjust the focus

Once completed, your sample will be ready for acquisition.

This step is required to calibrate the microscope in XY and Z. Performing this calibration will significantly reduce the time needed to locate and focus on your sample.

  • If not already done, select the lowest-magnification objective. On the microscope touch screen Home > Microscope > Control > Objectives > 5x
  • In Zen, once it has started a calibration dialog should appear. Simply click Calibrate Now.
    The microscope will lower the objectives, perform an XY calibration first, followed by a Z calibration, and then return to its original position.

Once calibrated, the focus is typically found at Z = 1.7 mm for a microscope slide.
The Z position can be viewed on the microscope touch screen under Home > Z-Position, as well as in Zen within the Focus tab, located on the right side of the screen.

The system may already be calibrated. This can occur if a previous user calibrated the system and left it on.

In Zen, within the Focus tab, located on the right side of the screen:

  1. Click Load to lower the objective

  2. The Z position is indicated below Current
  3. If Z position value is less than 100 um the system is calibrated

On the microscope touch screen:

  1. Lower the objective by pressing Home > Load Position

  2. Press Set Work Position to store this position

  3. If necessary, slightly adjust the focus upward to clear the Lower Z Limit Reached message displayed on the touch screen

  4. The Z position value is indicated
  5. If the value is less than 0.1 mm then the system is calibrated

In Zen, within the Stage tab, located on the right side of the screen:

  1. If not already done, check the Show All option

  2. At the bottom of the tab click Calibrate

  3. A warning message will show up click Continue

  4. The microscope will lower the objectives and perform a XY stage calibration and then return to its original position.

In Zen, within the Focus tab, located on the right side of the screen:

  1. If not already done, check the Show All option

  2. At the bottom of the tab click Calibrate

  3. A warning message will show up click Continue

  4. The microscope will then perform a Z calibration and then return to its original position

XY and Z calibrations are now complete

On the microscope touch screen:

  1. Navigate to Home > Microscope > XYZ > Position > Z-Position > Set Zero > Auto to perform focus calibration

  2. Press OK to start the calibration procedure

  3. Wait a few seconds for the calibration to complete

  4. Lower the objective by pressing Home > Load Position

  5. Press Set Work Position to store this position

  6. If necessary, slightly adjust the focus upward to clear the Lower Z Limit Reached message displayed on the touch screen

  7. Navigate to Home > Microscope > XYZ > Position > XY-Position > Set Zero > Auto to perform a stage calibration

  8. Press OK to start the calibration procedure

  9. Wait a few seconds for the calibration to complete

XY and Z calibrations are now complete

In Zen:

  1. In the menu bar, navigate to Tools > Options

  2. Select Startup/Shutdown

  3. Under Stage/Focus Calibration, ensure Request Stage/Focus Calibration on Startup is checked

  4. Click OK to close the Options dialog

Ensure that the calibration has been completed beforehand. Calibration will significantly reduce the time required to locate and focus on your sample.

On the microscope touch screen:

  1. If not already done, select the lowest-magnification objective Home > Microscope > Control > Objectives > 5x

The 5× objective is the safest to use due to its  long working distance (12 mm). The sample will appear in sharp focus well before the objective approaches it. It is recommended to always focus using the safest objectives first. Since the objectives are parafocal, focusing with the safest objectives will facilitate locating the sample when switching to higher-magnification objectives.

  1. Lower the objective by pressing Home > Load Position

  2. Press Set Work Position to store this position

  3. If necessary, slightly adjust the focus upward to clear the Lower Z Limit Reached message displayed on the touch screen

  4. Place the test slide on the microscope stage with the coverslip facing the objective

Using a test slide will significantly reduce the time required to set up the instrument.

  1. If necessary, adjust the stage to ensure that the sample is centred under the objective

In Zen:

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

Once calibrated, the focus is typically found at Z = 1.7 mm for a microscope slide.
The Z position can be viewed on the microscope touch screen under Home > Z-Position, as well as in Zen within the Focus tab, located on the right side of the screen.

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

Perform the initial focus using the safest objective before switching to higher-magnification objectives.

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

  1. Press Home>Microscope>Control>Objectives
  2. Press 10x, 20x, 40x (0.95) to select the desired objective

The 40× (0.95) Air objective is the best air objective because it has the highest number of optical corrections (Plan Apochromat) and the largest numerical aperture (0.95), but it has a smaller field of view.
The 20×(0.8) objective offers the best compromise between resolution and field of view.

There are two (2) 40x objectives, make sure you select the 40x Air (0.95)

In Zen:

  1. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP or 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 off the illumination

Your sample is ready for acquisition!

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

  1. Press Home>Microscope>Turret>Objectives
  2. Press 40x Oil (1.4) or 63x Oil (1.4) to select the desired objective. The microscope will automatically lower the stage so that the sample becomes accessible.

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

There are two (2) 40x objectives, make sure you select the 40x Oil (1.40)

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

In Zen :

  1. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP or 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 off the illumination

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 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 objectives 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 instrument with the protective dust cover
  • Take back your samples including ones in the microscope
  • Leave the microscope and the working area clean

Log

  • Quality control for Illumination, Liquid light guide and filters quality.
  • Revise Technical Datasheet
  • 20x/0.8 and 40x/0.95 with immersion oil on it. Cleaning objectives.
  • Added Troubleshooting
  • 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

Technical datasheet

Stand

  • Zeiss AxioObserver LSM800 System ID: 2633000272
    Serial 3851002662  431007-9902-000

  • Manual Field diaphragm for transmitted light

  • Manual polarizer
  • Left imaging port 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 with ESID 423053-9071
  • X-Cite 120LED mini Serial XT120LM 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 # 424242

  • 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
    • Multi-well plate

Filters

6-Position motorized Filter Turret ##424947

  1. GFP Zeiss Filter Set 38 cube 424931 000000-1031-346
  2. DAPI Zeiss Filter Set 49 cube 424931 489064-0000-000 (in MTB 488049-0000-000)
  3. mPlum Zeiss Filter Set 64 HE cube 424931  489064-0000-000
  4. DIC Analyzer Zeiss 424937-9901
  5. Empty
  6. Empty

Scanner

LSM 800 GaAsP-PMT 400102-9000-00000 confocal 2633000272 (in MTB LSM900 Scanhead)

  • 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 2263-682
  • 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 03.04.08 Rev.A 2025-07-02 
  • 1 x Intel Core i7-12700K 3.6 GHz
  • RAM 128GB DDR5 4 x 32GB no ECC
  • OS 500 GB NVMe 6 GB/s
  • Data Storage 14 TB (2 x 7 TB) HD 14 TB (2 x 7 TB) HD 213 MB/s
  • Nvidia RTX A4000 16 GB GDDR6
  • Monitor HP Z32x CNC61804QR 3840 x 2160 60 Hz CNC61804QR 3840 x 2160 60 Hz
  • Software Zen 3.10 SN=1122911743-424077 HASP=110522193
  • Zen Module Arivis Cloud Basic, Arivis Cloud Advanced, Colocalization, Data Storage, Direct Processing, Manual extended Focus, Measurements Multi-Channel. Panorama, Time Series, Advance Processing, Airyscan, Airyscan 2 Basic, Ariscan Hoiunt Deconvolution Exteneded Focus, HDR Confocal Basic, Image Analysis, Sotfware Autofocus, Tiles and Positions, Z Stack

Incubation

  • Pecon stage top incubation

Consumables

  • CO2 Tank
  • N2 Tank
  • Oil
  • Lens Cleaner

Manuals

Troubleshooting

Technical support is free and unlimited. Contact us !

This can happen when objectives are dirty. this is especially true for the 20x/0.8 and 40x/0.95 which have a short working distance. If not careful immersion oil can make them dirty. If your image seems blurry with one of these objective please contact us and we will clean it for you !

FAQ

Yes, but... This microscope has an incubation module to maintain temperature, humidity and atmosphere. Yet, it does NOT have a module which can maintain focus throughout time. Therefore, it is possible to loose the focus over long period. You can always do a software auto-focus at different time but be aware of photo-toxicity if you are using fluorescence.

Extended Focus is a method to flatten a Z-stack by keeping only the relevant information.

In Zen:

  • Select the Processing Tab
  • Select the Extended Depth of Focus Method
  • Click Apply
  • Wait until the processing is finished
  • Save the processed image

Z Stack animationMaximum Intensity projection of a Z-StackExtended Depth of Focus projection of a Z-Stack

Yes. This is a inverted microscope designed to look at sample in a dish or a multi-well plate. For long timelapse, be aware of photo-toxicity.

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Content published under CC BY-SA 4.0. Sharing allowed under the same license with attribution: "Original content by l'Institut Courtois d'innovation biomédicale, used under CC BY-SA 4.0"