AdvancedImagingUIC

• 

  Location : UIC Room 5 (0B05), Bartolomeu Dias Wing

  Manufacturer : Zeiss

  Model : LSM 980

  Year Installed: 2019

  Optics : Confocal fluorescence and DIC

  Detectors : 2 PMT + 1 GaAsP + 1 TLD (Transmitted light - brightfield) + Airyscan2

  Keywords:

• Confocal microscopy allows one to optically section a fluorescently labelled sample with superior contrast and even higher resolution by using a pinhole to reject light that originates above and below the focal plane. By collecting a series of image planes (“optical slices”) through the full thickness of a sample,it is possible to assemble a three-dimensional reconstruction of the entire sample.

  The Zeiss LSM 980 is an inverted microscope equipped with a laser scanning head with a total of 7 detectors:

  • 1x blue-green multialkali PMT

  • 4x GaAsP PMT spectral detectors (tunable between 450-750 nm)

  • 1x green-far-red multialkali PMT

  • 1x Airyscan2 detector, that consists of an array of 32 GaAsP detectors instead of a pinhole, each with the size of approximately 0.2 Airy units. Using Zeiss patented technology, the LSM980 can achieve non-diffraction limited optical sectioning with resolutions down to 140nm. The AiryScan2 also allows acquisition in “Multiplexing” mode(s), which accelerates the acquisition speed (up to 8x faster than normal confocal) and a sensitivity mode that enhances the signal-to-noise acquisition. Different combinations of these 3 modes of acquisition with the AiryScan2 module can be selected by the user.

  • The microscope is also equipped with a dark heating and athmospheric control chamber to allow live imaging of mammalian cells.

Magnification		NA1	Immersion	Working distance (mm)	FOV (mm)	Theoretical resolution / @Nyquist	Link
2.5x		0.085	-	8.8	10	3.95 / 1.58	ref
10x		0.30	-	5.5	2.5	1.12 / 0.45	ref
20x		0.8	-	0.55	1.25	0.42 / 0.17	ref
25x		0.8	water, silicone oil, glycerine or oil	0.57	0.72	0.42 / 0.17	ref
40x		1.1	water	0.62	0.625	0.31 / 0.12	ref
63x		1.4	oil	0.14	0.397	0.24 / 0.10	ref

(1) NA - Numerical Aperture. For more information, follow this link.

Theoretical resolution for each objective, which might differ from the actual pixel size of acquired image, calculated as 0.61*550nm/NA.

Nyquist = max resolution with sampling at 2.5x the theoretical resolution; must be used for images to be deconvolved.

The pixel size in microns of your picture is saved automatically. Use Bio-formats to recognize it with ImageJ/Fiji (already installed in the Fiji package).

Filters for sample observation.

Note: *contains reference number for the corresponding filters. 1 - Mirror used for Confocal imaging.

First Filter Wheel

Second Filter Wheel

Turn on procedures

1. Verify if the fluorescent lamp is not hot (touch it), otherwise wait until it’s cold (10-15min). Afterwards, turn on the switch (it should be already turned on, because it’s controlled by the master control switch).

   

2. Turn on the componentes switch next to the computer keyboard.

   

3. Turn on laser key (downward position) in the unit below the table.

   

4. Login into your pGina account on the computer.
5. Start ZEN Blue 3.0.

Turn off procedures

1. Remove the sample and immediately pull apart the sample holders in the stage insert.
2. Gently wipe with optic cleaning paper and EtOH paper the immersion objective(s) used. If you suspect the objective needs deep cleaning please inform the staff.
3. Close ZEN and logout from windows (if there’s an user after you, you can now leave the microscope).
4. If there is no one in the next 2h, turn off the fluorescence lamp (behind the computer screen).
5. If you are the last user of the day (check booking in Agendo): Turn off Laser key (to horizontal position) in the large unit under the table and turn off the “components switch” in the table near the keyboard.

Acquisition

Acquiring images in a confocal is a complex procedure, first time users are required to contact the UIC staff to discuss the use of confocal microscopes and fluorescent imaging in general. If you need assistance don’t forget to press “with assistance” at reservation time.

Zeiss LSM 980 manual is in construction.