High Resolution 3D Stereo Light Microscopy

Conventional high resolution 2D light microscopy is routinely used in clinical research,  pathomorphological diagnostics and immunochemical diagnostics. In high resolution microscopes, the depth of focus is very shallow. Recent developments allow specimens as thick as 1mm or more to be photographed  in multiple "slices" and computationally rendered into a sharply focused image, much like computed tomography x-ray (CT) or magnetic resonance images (MRI) in man-sized diagnostics. By splitting the photographed image into a left and right image, the flat 2D image can be viewed as a realistic 3D-image, which is very helpful to understand complex morphology, or to resolve co-localization problems.

The application of 3D (stereo) light microscopy has so far been reported for neuroscientific applications (large neurons), developmental biology (whole embryos) and pathology (Pap smear). In cooperation with Dr. Martina Böttner and Prof. Dr. med. Thilo Wedel in the Anatomy Department, we are currently establishing vibratome based cutting techniques and non-fluorescent as well as fluorescent staining techniques for thick specimens from many different tissues. We will also explore the 3D microscopy of haematological pathodiagnostic blood smears (in analogy to Pap smears) and of whole uncut needle biopsies (e.g. breast cancer or prostate cancer tissue). By using 3D on unsliced needle biopsies, we aim to reduce the work of cutting cancer tissue samples for subsequent next generation sequencing of RNA and DNA. The current techniques of scratching tissue samples from thin (10-20 micrometer) slices is very labor intensive.

Stereo 3D microscopy images can be very beautiful and great fun compared to flat 2D images. Publications are no problem: Stereo images can be published as left/right pairs of color images in papers and on web sites, which give the reader a realistic 3D-illusion.  Other methods to view these images in color: on a 2D display with red/cyan anaglyph glasses, on a 3D display with 3D glasses. With the mentoring of Dr. Gary Greenberg, himself mentored by neuroscience legend Prof. Geoffrey Burnstock, we are able to generate fascinating 3D stereo microscope images.

Our 3D stereo microscopy platform consists of an Edge-3D RMV3 with four Apo Plan air objectives: N.A. 0.15 / W.D. 20mm, N.A. 0.35 / W.D. 4.2mm, N.A. 0.65 / W.D. 0.7mm and N.A. 0.95 / W.D. 0.1mm. The working distance (WD) limits specimen thickness, or their penetrated thickness. We currently have white light illumination and will shortly also have 4-color fluorescent illumination. For highest resolution, we have N.A. 1.35 / W.D. 0.25mm Apo Plan oil immersion optics.