Exploring horizons in 3D microscopy: what the future holds
The microscope slide is flat (2D), but the world around us is not – despite the flat-earth theories. We need volume information about our samples, ideally with high resolution in all three dimensions as well as over time – the fourth dimension…
3D-microscopy is booming; profiting from an increase in computation power. However, improvements in the resolution power of the non-optical in vivo 3D imaging – such as ultrasound tomography, micro-computed tomography (CT), micro-positron emission tomography (PET), photoacoustic imaging and others – will certainly progress,1,2,3 thereby narrowing the gap between these originally 3D, large volume, low-resolution technologies, and the originally high resolution but only 2D microscopy. Classically, samples for microscopy were prepared as thinly as possible to prevent any blurriness from an out-of-focus signal. The standard histological section is 5mm thick,4 which is too thick to be considered flat. Students analysing histological sections realise one could learn more about a sample by simply scrolling though the focusing. Unfortunately, the integrated 3D picture occurs only in the investigator’s brain. So, how best to capture only the in-focus information from each focusing plane and disregard the out-of-focus information? Thankfully, this became possible with motorised focusing enabling precisely stepped acquisition, followed by an appropriate image analysis. For the transmitted and reflected light macro- and microscopy, the Zerene stacker5 is a useful analysis tool for reconstruction of complex surfaces, such as an insect eye or a flower6 (Figure 1).
This promising approach is not yet popular in histology.
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