Ultramicroscopy for micrometre resolution

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This content originally appeared in the Jan 2017 issue and is a sponsored blog

Ultramicroscopy is a light-sheet illumination based system designed to image specimens of millimetre size, such as mouse brain in 3D, with micrometre resolution. All emitted fluorescence signal coming from the sample is collected, as in wide-field techniques, and bleaching is drastically reduced, since it illuminates only the section of the sample that is actually being imaged.

Formalin-induced fluorescence reliably reveals the cellular structure of tissue samples but exhibits a weak and fast fading intrinsic signal that is often missed when observed using confocal microscopy. However, even by using ultramicroscopy, the signal remains weak and it requires an ultra-sensitive camera, such as Andor iXon EMCCD cameras. The exceptional sensitivity of the EMCCD camera combined with the optimal illumination of the sample permits capture of the organization of tissues in great detail.

 

The iXion Ultra EMCCD camera, from Andor

 

3D rendering of a mouse hippocampus acquired using Andor iXon EMCCD. Courtesy of Ulrich Leischner, Max Planck Institute of Psychiatry, Munich, Germany.

The region CA1 of the mouse hippocampus was fixed and labelled using formalin, which cross-links with the proteins and lipids of the sample to forms chemical rings capable of emitting fluorescence. The sample was excited at 488 nm, imaged using a 20x objective after 600ms exposure time. X-Y field of view was 0.5 mm and Z-stack was taken by recording an image every 500 nm for a total depth of 0.3 mm.

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