Leica Sp8 Lightning Single Photon

The Biology Department Leica SP8 (Spectral Detector) Lightning LSCM was installed in December of 2018.  It is built upon a Leica DMi8 fully motorized inverted microscope and offers a resonant scanner in addition to the conventional galvanometer scanner (x2y Tandem Scanner); a piezo driven stage z drive;  two ultra-sensitive HyD detectors plus two conventional PMT detectors; and a transmitted light detector.  Channel separation for dyes is achieved via a fully-tunable prism-based spectral detector to provide unparalleled control of wavelength transmission.  The piezo drive provides ultrafast Z scan acquisition, and the resonant scanner can scan at 8000 Hz for even the fastest of live cell imaging requirements.  The instrument has 5 lasers running at 405 to 638 nm, along with 4 objectives ranging from 10X to 63X.  Three modular stage inserts accommodate slides, petri dishes, and multiwell plates.   Image acquisition and processing is run by Leica LAS X software and includes modules for Tilescanning on the fly (Navigator), FRET, FRAP, 3D viewing and analysis, and Leica’s proprietary Adaptive Deconvolution package (Lightning).  The software is extremely user-friendly, walking users through complicated experimental designs quickly and easily.

Microscope and Illumination Sources

Leica DMi8 inverted microscope with Broadband EL6000 mercury halide lightsource and 3 filter sets for DAPI, FITC and Rhodamine.  White transmitted light source with Brightfield, Polarized and DIC optics.  The stage is fully motorized with standard and Super Z galvo Z drives.  There are three piezo-driven adaptor plates for microscope slides, petri dishes, and multiwell plates, as well as a standard Z-drive adaptor plate for slides and multiwell plates. 

Objectives
Objective Magnification Numerical Aperture

HC PL APO CS2

10X dry

0.40

HC PL APO CS2

20X multi immersion

0.75

HC PL APO CS2

40X oil immersion

1.30

HC PL APO CS2

63X oil immersion

1.40

Lasers
LasersPowerPower at Objective
405 nm DMOD compact< 60 mW< 5 mW
488 nm DPSS< 12 mW< 3 mW
514 nm DPSS< 12 mW< 3 mW
552 nm DPSS< 12 mW < 3 mW
638 nm DPSS< 12 mW< 3 mW
Scanhead

SP8 LIAchroics Compact RGB x2y Tandem scanner: 8000 Hz resonant scanner + 10-1800 Hz galvanometric scanner. 

Detectors

Two PMT confocal detectors + Two HyD GaAsP-Detectors for gated imaging.  One brightfield transmitted light detector. 

Software

LAS X software with modules:

Module Uses

Navigator

GUI for point scanning and Tiled images

Dye Finder

Dye setup wizard for multicolor imaging

3D visualization

3D viewer, processor and analysis wizard for Z stacks

MicroLab

Wizards for FRAP, FLIP and FRET

FRAP Zoomer

FRAP beam control for enhanced photobleaching

Lightning

Adaptive Deconvolution software

Applications and Output

Applications:

FRAP, FRET, photoactivation, 3D rendering and analysis, Timelapse, Deconvolution, colocalization, point scanning, Tiling, Z plane acquisition.

Output:

.lei, .tiff, .jpg, .avi, .mov, .mpeg4