AdvancedImagingUIC

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• The Leica Stellaris 5 system is mounted on a Leica DM6 upright microscope, equipped with 405 nm, 448 nm, 488 nm, 561 nm and 638 nm laser lines, and with two of Leica’s new Power HyD S spectral detectors. The Stellaris5 is equipped also to perform FRET (CFP-YFP AB or SE), FRAP, and Lightning (Super-resolution).

  Location : Room 0B03

  Manufacturer: Leica

  Model: Stellaris 5

  Year Installed: 2021

  Optics: Stellaris5 confocal scanhead

  Detectors: 2x Power HyD S (spectral) + 1T-PMT (Transmitted light)

  Operating conditions : Room temp (22ºC)

  Keywords: Confocal, HyD, upright, Lightning

Magnification		Objective name	NA1	Immersion	Working distance (mm)	Resolution @488 nm in XY (µm)	Nyquist Sampling2 @488 nm in XY (µm)	Resolution @488 nm in Z (µm)	Nyquist Sampling2 @488 nm in Z (µm)	Link
10x dry		HC PL APO 10x/0,40 CS2	0.40	Dry	2.560	0.488	0.152	2.630	1.461	ref
10x water		HC APO L 10x/0,30 W U-V-I	0.30	Water	3.6	0.651	0.203	6.41	3.581	ref
20x dry		HC PL APO 20x/0,75 CS2	0.75	Dry	0.62	0.260	0.081	0.648	0.360	ref
40x (Optional)		HC PL APO 40x/1,30 OIL CS2	1.3	OIL	0.24	0.150	0.046	0.299	0.165	ref
40x Water (Optional)		HC APO L 40x/0,80 W U-V-I	0.8	Water	3.3	0.244	0.046	0.821	0.119	ref
63x		HC PL APO 63x/1,40 OIL CS2	1.40	OIL	0.14	0.139	0.043	0.235	0.130	ref

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

(2) To achieve optimal lateral (XY) resolution and Nyquist Rate, please speak with the UIC personnel for optimal pixel size.

These values were calculated using the Resolution formula adapted to confocals as seen in the Olympus micro website: R = 0.4*λ/NA

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).

Turn on procedures

1. Turn buttons (1) (2) and the Key switch (3) left side of the black cabinet

   

2. Turn on the Computer.
3. Log on to the computer using your Agendo credentials
4. Start the Leica Confocal Software - “LAS AF”.

5. Ask staff if it is necessary to exchange the stage insert (glass or universal)

   

6. Allow the software to initialize the stage (remove sample).
7. Turn on the lasers you will need.

Turn off procedures

If there is someone booked after you, close the software, save your files in the server and then log off.

Leave everything else ON and clean any immersion lenses you used. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipets, slides, etc...

If there is no one booked afterwards for more than 30 minutes, turn off the lasers in the software panel. If you are the last user of the day:

1. Turn off the laser key (3) , the (1) and (2) buttons of the black cabinet.

Acquisition

Users can easily damage the microscope, their samples, themselves, or others through seemingly 'proper' use of the microscope. Newcomers to the Stellaris 5 need to be personally trained for use in the microscope and the rules of the facility to keep themselves, their samples, and the equipment safe, clean and ready for further use. If you need assistance don’t forget to press “with assistance” when booking this microscope.

Tips:

To quickly setup a new experiment we recommend using the “dye assistant”, which allows the user to interactively configure the best detector settings for every combination of fluorophores.

Operation in Lightning mode (Super-resolution) requires switching to a different mode of the LAS-X software

FRET and FRAP modules are also accessible as additional modules of LAS-X.

Note: one of the major changes of this version of LAS-X is the multi-position acquisition. Consult with the staff to learn how to use this new “Mosaic” feature. This allows the setting up of automated acquisition of multiple positions in full auto mode.