Leica SP8 Confocal live imaging (2016)

The Leica SP8 confocal microscope is a spectral imaging, simultaneous multicolor, confocal system.

HyVolution is a software-based improved-resolution technique that uses integrated Huygens deconvolution software.

How does it work? First, the wizard optimizes the acquisition settings (because deconvolution uses oversampled data, it determines the best settings for the set parameters), then it performs the deconvolution automatically in the background using CUDA based processing, and then imports the resulting improved-resolution image it back into the LASX file system where one can then treat the image like any other iamge acquired with the system.

The machine also has two super-sensitive HyD detectors.

This combination achieves a lateral resolution down to 140 nm and delivers crisp multicolor images, at high fidelity.

Objectives:

HC PL FLUOTAR 10x/0.30
Plan Fluotar objective, for use with and without coverglass, no immersion.
Free working distance: 11.0 mm.

HC PL APO 20x/0.75 (multi immersion)
Plan apochromatic multi-immersion objective.
Free working distance: 0.67 mm (water immersion and 0.17 mm coverglass).
Correction collar for use with and without coverglass and use with water, glycerine and oil immersion.

HC PL APO 63x/1.40 OIL CS2 

Plan apochromat oil immersion objective.
Free working distance: 0.14 mm.

Example for methods: Microscope used was an inverted Leica DMi8 scanning confocal microscope, driven by the LASX software (Leica Microsystems, Mannheim, Germany).  Objective used was a CS2 20x/0.75 with XY pixel size of 284nm squared. Excitation for GFP was with a 488 laser and emission was detected between 495nm - 534nm, for red with a 552 laser and emission between 569nm - 640nm, with 4 times line average.  For large areas, multiple images were acquired and tiles were automatically merged into a single image with the Leica software. High resolution images were acquired with a CS2 63x/1.40 oil objective at an oversampling of XY pixel of 44nm squared, Z step of 223nm, and pinhole at 0.9 AU, and submitted to automated internal Leica Lighting deconvolution software (or Hyvolution). For display, images were single plane projected