GIMM Bioimaging Wiki

Microscope overview

Spinning disk confocal microscopy (SDC) is a technique well suited for observation of cells and organoids. Compared to conventional widefield microscopy, SDC provides instant optical sectioning, allowing the imaging of cells and shallow tissues (typically a maximum of a few tens of micrometres deep) in 3D. Compared to laser scanning confocal microscopes (such as Leica Stellaris, ZEISS Airyscan), the Dragonfly SDC provides much faster acquisition (up to 400 fps, with limited ROI (2048×128), 40 fps full frame), with lower phototoxicity and photobleaching. A spinning disk with multiple small pinholes is installed between the light source and specimen to generate point-like illumination covering the whole area “simultaneously”. Each small aperture serves as a detecting pinhole to remove out-of-focus fluorescence. This system was built to be versatile, and allow imaging of different sample sizes. It includes both high and low magnification objectives (which require different pinhole sizes, 25 or 40um). The scanhead allows 1x or 2x magnification (for extra magnification and to allow Nyquist sampling and deconvolution - see table below).

Microscope: Nikon Ti2 Eclipse
Confocal scanner: Andor Dragonfly 200
Camera: Andor Sona-4BV11

Data files older than 1 month will be automatically deleted on this system, please copy your data to the GIMM server using the desktop link.

System components

LASERs

LASER	Wavelength	Maximum Power	Measured after objective @ 100%
Violet	405 nm	100 mW	7 mW
Blue	488 nm	100 mW	17 mW
Yellow-Green	561 nm	100 mW	20 mW
Red	640 nm	100 mW	17 mW

Objectives

Magnification	Model	Immersion	NA	WD (mm)	Max FoV (1600x1350px)	ideal CSU pinhole	Pixel Size (zoom 1x)	PFS Compatible	Reference
10x	CFI Plan Fluor	Air	0.30	16	1.3 mm	25 μm	1.0561 μm	Yes	MRH00105
20x	CFI Plan Apochromat	Air	0.8	0.8	0.68 mm	40 μm	0.5097 μm	Yes	MRD70270
40x	CFI Plan Apochromat λD	Air	0.95	0.21	0.34 mm	40 μm	0.2580 μm	Yes	MRD70470
60x	CFI Plan Apochromat VC	Water	1.2	0.28-0.31	0.23 mm	40 μm	0.1719 μm	Yes	MRD07602

Upon request:

Magnification	Model	Immersion	NA	WD (mm)	Max FoV (1600x1350px)	ideal CSU pinhole	Pixel Size (zoom 1x)	PFS Compatible	Reference
20x	CFI Plan Fluor	0.75	multi	0.33-0.51	0.68 mm	25 μm	0.5097 μm	No	MRH07241
40x	CFI Apochromat LWD λS	1.15	Water	0.59-0.61	0.34 mm	40 μm	0.2580 μm	Yes	MRD77410
100x	CFI Plan Apochromat λD	1.45	Oil	0.13	0.13 mm	40 μm	0.1038 μm	Yes	MRD71970

Emission Filters Spinning Disk

Position	Filter name	Dichroic	Transmission
0	QUAD	405/488/561/640	445 nm
			521 nm
			594 nm
			698 nm
1	405	405/488/561/640	422-468 nm
2	488	405/488/561/640	502-540 nm
3	561	405/488/561/640	573-616 nm
4	637	405/488/561/640	660-724 nm
5	Pol	–	–
8	561 extra	405/488/561/640	574-638 nm

Turn On Procedure

Turn on power bar #1.
Turn on power bar #2.
Turn on the LASER key
Turn on the incubation power bar (if you are planning on using the incubator and/or CO2).
Turn on the computer.
Log in Windows with your Agendo credentials.
Start the Fusion software

If the system is already on:

Log in Windows with your Agendo credentials.
Start the Fusion software

Turn Off Procedure

If there is another user in the next hour:

Close Fusion
Log off the computer
Clean up immersion objectives

Else:

Close Fusion
Turn off the computer
Turn off the incubation controller (if used)
Switch the LASER key off
Turn off power bars #1 and #2
Clean up immersion objectives

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