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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).

Figure 1. A common green bottle fly (Lucilia sericata) has been imaged using a Canon-MPE 65mm macro photo lens. 85 images with sequential focusing each 23um to cover circa 2mm focus depth were acquired in RAW format with an EOS Canon 60D camera mounted on a motorised focus rail: the maximal focusing projection.

This promising approach is not yet popular in histology.         

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