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Review
. 2020 Aug 21;23(8):101432.
doi: 10.1016/j.isci.2020.101432. Epub 2020 Aug 2.

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Maria Victoria Gómez-Gaviro  1 , Daniel Sanderson  2 , Jorge Ripoll  2 , Manuel Desco  3
Affiliations
Review

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Maria Victoria Gómez-Gaviro et al. iScience. .

Abstract

Three-dimensional (3D) optical imaging techniques can expand our knowledge about physiological and pathological processes that cannot be fully understood with 2D approaches. Standard diagnostic tests frequently are not sufficient to unequivocally determine the presence of a pathological condition. Whole-organ optical imaging requires tissue transparency, which can be achieved by using tissue clearing procedures enabling deeper image acquisition and therefore making possible the analysis of large-scale biological tissue samples. Here, we review currently available clearing agents, methods, and their application in imaging of physiological or pathological conditions in different animal and human organs. We also compare different optical tissue clearing methods discussing their advantages and disadvantages and review the use of different 3D imaging techniques for the visualization and image acquisition of cleared tissues. The use of optical tissue clearing resources for large-scale biological tissues 3D imaging paves the way for future applications in translational and clinical research.

Keywords: Biomedical Discipline; Imaging Methods in Chemistry; Medical Imaging; Optical Imaging.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic Showing the Fundamental Physics and Functioning of the Imaging and Spectroscopic Techniques Considered in This Review The biological sample is represented by a central brown rectangle, the objective by a blue ellipsoid, the photodetectors by purple boxes, and the laser emitters and detector by a black box. Blue: LSFM. Red: confocal microscopy. Green: Brillouin spectroscopy. Yellow: Multiphoton microscopy. Brown: SRS microscopy. Brillouin and Raman graphs represent laser intensity versus frequency. Confocal and Raman microscopes may irradiate the sample from the opposite direction too. (A) Input from the different 3D imaging systems. Each color represents the laser beam produced by the source of each imaging system. Discontinuous lines in the color spectrum represent commonly used excitation wavelengths (nm) in LSFM, and continuous lines represent in the same color the peak wavelength of the corresponding emission filters used. Raman microscope image was adapted from Stimulated Raman Scattering Microscopy (https://www.castl.uci.edu/sites/default/files/Min%20SS%20presentation.pdf), Confocal microscope image was adapted from Zeiss microscopy (https://www.zeiss.com/microscopy/int/products/confocal-microscopes/lsm-900-for-materials-non-contact-surface-topography-in-3d.html). (B) Output of the 3D imaging systems. Each color represents the laser beam obtained from the sample. Below each imaging system, the physical mechanism on which each one is based is graphically shown. Confocal, multiphoton, and LSFM microscopies are based on fluorescence, whereas Brillouin is based on phonon detection and Raman microscopy on stimulated Raman scattering. Magnification objective range from MP was retrieved from Singh et al. (2015).
Figure 2
Figure 2
Workflow Summarizing the Different Possible Steps that an Optical Clearing Method Can Follow to Render a Whole Organ Transparent The chosen pipeline will depend on the properties of the tissue that is going to be treated and on the specific goals desired. Each box represents a specific physical or chemical procedure and the most common substances used to achieve it.
Figure 3
Figure 3
Mouse Tissues Cleared with the CUBIC Protocol Mice were perfused with 4% PFA and organs were extracted. The CUBIC clearing protocol was adapted to each tissue by changing the clearing time. Cleared and uncleared kidney, spleen, lung, brain, liver, heart, and intestine are shown.
Figure 4
Figure 4
Mouse Hearts Cleared with CUBIC and Vasculature 3D Image (A and B) Mice were perfused with PFA 4%, and the hearts were removed. The CUBIC clearing protocol was applied in (B); (A) heart without clearing. Length of background squares: 3.85 mm. (C) CUBIC cleared heart inside the cuvette. (D and E) SPIM images of mouse heart vasculature. Mice were perfused with PFA 4% and Lectin-649Alexa, and the hearts were removed and cleared with CUBIC protocol. 3D imaging of the heart was acquired with SPIM microscopy using 635 nm excitation laser, and 670 nm emission filter. Stripes artifact was removed with VSNR Fiji plugin, and 3D image rendering was performed with ImageJ. Both images represent different perspectives of the same rendering. Scale bar: 100 microns.
Figure 5
Figure 5
Graphical Summary of Optical Clearing Methods that Have Been Applied So Far to Murine Organs The mouse image was adapted from Science Photo Library (https://www.sciencephoto.com/media/385863/view/mouse-anatomy#:∼:text=This%20image%20is%20not%20available%20for%20purchase%20in%20your%20country).
Figure 6
Figure 6
Graphical Summary of All the Optical Clearing Methods that Have Been Applied So Far to Each of the Human Organs or Tissues Mentioned in the Text The human image was adapted from Shutterstock (https://www.shutterstock.com/es/video/clip-2318627-human-body-internal-organs-loop-rotation).
See this image and copyright information in PMC

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