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. 2022 Jun;286(3):201-219.
doi: 10.1111/jmi.13109. Epub 2022 May 5.

Challenges and advances in optical 3D mesoscale imaging

Sebastian Munck  1   2 , Christopher Cawthorne  3 , Abril Escamilla-Ayala  1   2 , Axelle Kerstens  1   2 , Sergio Gabarre  1   2 , Katrina Wesencraft  4 , Eliana Battistella  4 , Rebecca Craig  4 , Emmanuel G Reynaud  5 , Jim Swoger  6 , Gail McConnell  4
Affiliations

Challenges and advances in optical 3D mesoscale imaging

Sebastian Munck et al. J Microsc. 2022 Jun.

Abstract
in English, Spanish

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence.

This review discusses the state of the art of an emerging field called mesoscale imaging. Mesoscale imaging refers to the trend towards imaging ever-larger samples that exceed the classic microscopy domain and is also referred to as ‘mesoscopic imaging’. In optical imaging, this refers to objects between the microscopic and macroscopic scale that are imaged with subcellular resolution; in practice, this implies the imaging of objects from millimetre up to cm size with μm or nm resolution. As such, the mesoscopy field spans the boundary between classic ‘biological’ imaging and preclinical ‘biomedical’ imaging, typically utilising lower magnification objective lenses with a bigger field of view. We discuss the types of samples currently imaged with examples, and highlight how this type of imaging fills the gap between microscopic and macroscopic imaging, allowing further insight into the organisation of tissues in an organism. We also discuss the challenges of imaging such large samples, from sample handling to labelling and optical phenomena that stand in the way of quantitative imaging. Finally, we put the current state of the art into context within the neighbouring fields and outline future developments, such as the use of ‘phantom’ test samples and artificial intelligence for image analysis that will underpin the quality of mesoscale imaging.

Keywords: 3D imaging; Mesolens; absorption; clearing; light sheet microscopy; mesoscale; optical projection tomography; scattering.

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Figures

FIGURE 1
FIGURE 1
Examples of optical mesoscale imaging using the Mesolens, Light‐Sheet Microscopy and OPT. (A) Brightfield Mesolens image of a section of mouse embryo at full term, stained with haematoxylin and eosin. Scale bar = 0.5 mm. (B) Digital zoom into the snout of the embryo (shown with a black box in A). The individual cell nuclei are revealed at this level of digital zoom, and the individual chromatin granules can be seen in the nuclei. Scale bar = 150 μm. (C) A confocal fluorescence Mesolens image of a whole mount of fixed mouse ileum that has been prepared with the nuclear marker Propidium Iodide and cleared using Murray's Clear. The image shows the mesoscale architecture of the ileum at a depth of 350 μm into the specimen, and a region of interest is shown with a box. Scale bar = 1 mm. (D) Region of interest boxed in C after a software zoom, with the cell nuclei in the crypts now clearly visible. Scale bar = 50 μm. (E) Tribolium castaneum, treated with RNAse and stained with Propidium Iodide and cleared with benzyl alcohol/benzyl benzoate (BABB), imaged with the Mesolens in confocal mode. This image is composed by maximum intensity projection colour‐coded by depth of 160 optical sections taken with an axial separation of 4 μm, forming a z‐stack 640 μm deep. Scale bar = 500 μm. (F) An antibody labelled E10.5 mouse embryo, cleared with BABB, and imaged with Light‐Sheet Microscopy. Cyan: neurofilament. Red: E‐cadherin. The image is a maximum‐value projection through the 3D data set. Scale bar = 500 μm. (G) Autofluorescence of the skin and transmitted light shape of a Xenopus tropicalis frogling imaged by OPT displayed in false colours. Scale bar = 2 mm
FIGURE 2
FIGURE 2
Schematic of optical configurations. (A) Mesolens, a confocal approach using a lens with a unique combination of low magnification and high numerical aperture. (B) Light‐Sheet Microscopy using two lenses with perpendicular orientation, one for illumination and the other for readout. (C, D) Optical Projection Tomography (OPT) approaches. (C) Transmission OPT. (D) Fluorescence OPT. Schematics not to scale, for example, the Mesolens setup is much larger than the other depicted devices
FIGURE 3
FIGURE 3
Schematic for absorption and scattering. (A) Absorption. The intensity of incident light is reduced by absorption. (B) Shadowing. For a number of incident photons shadowing is induced by complete absorption by the particle as indicated by the arrows. (C) Scattering. The direction of incident light is changed by a scattering angle by interaction with a particle indicated by the arrows. (D) Depending on the size, shape and density of the particles the light will be scattered differently. Here a Mie type of scattering is depicted, where more forward scattering is happening, indicated by the thickness of the arrows. (E) Sample complexity can lead to unrecoverable regions marked here by the white space in the middle, where absorption and/or scattering block the region from all possible imaging directions
FIGURE 4
FIGURE 4
Reconstructed images from a cylinder containing a uniform activity, with corresponding line profiles. (A) Reconstructed PET image without attenuation or correction for scattering. (B) Attenuation map derived from CT image of the same cylinder. (C) Reconstructed PET image corrected for attenuation but uncorrected for scattering effects. (D) Final corrected PET image. Adapted from Ref. (109)
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