The frontier of live tissue imaging across space and time

Abstract

What you see is what you get-imaging techniques have long been essential for visualization and understanding of tissue development, homeostasis, and regeneration, which are driven by stem cell self-renewal and differentiation. Advances in molecular and tissue modeling techniques in the last decade are providing new imaging modalities to explore tissue heterogeneity and plasticity. Here we describe current state-of-the-art imaging modalities for tissue research at multiple scales, with a focus on explaining key tradeoffs such as spatial resolution, penetration depth, capture time/frequency, and moieties. We explore emerging tissue modeling and molecular tools that improve resolution, specificity, and throughput.

Figure 1:. Imaging methods and adjunct technologies depending on the scale.

Abbreviations used: MRI (magnetic resonance imaging), CT (computed tomography), NIRS (near infrared spectroscopy), uULM (ultrafast ultrasound localization microscopy), OCT (optical coherence tomography), MIMS (multi isotope mass spectroscopy), PAI/PAT (photoacoustic imaging/photoacoustic tomography), TAI (thermoacoustic imaging), LSCM/MPSC (laser scanning confocal microscopy/multipoint scanning confocal microscopy), AFM/ARFI (atomic force microscopy/acoustic radiation force impulse imaging), FISH (fluorescence in situ hybridization), FRET (Forester resonance energy transfer), DREADDs (Designer Receptor Exclusively Activated by Designer Drugs).

Figure 2:. Resolution and Penetration Depth.

Imaging modalities vary in imaging depth, spatial resolution, temporal resolution, and availability of molecular probes.

Figure 3:. Imaging technologies for organ visualization.

(A) Fluorescence and organ architecture can be imaged using different technical modalities in various tissues. Representative images shown here include multiphoton microscopy of the developing optic cup (Nakano et al., 2012), lightsheet microscopy of an optically cleared embryo (unpublished image courtesy of Dr. Qiang Huang and Dr. Xiling Shen at Duke University), ultrasound imaging of the kidney, and photoacoustic imaging of vasculature (unpublished images courtesy of Dr. Junjie Yao at Duke University). (B) Imaging windows placed in the cranial, dorsal, abdominal, and thoracic regions enable intravital microscopy. (Clockwise from the top) Representative imaging windows (IWs) and intravital microscopy (IM) images are from the following sources: brain IW and IM (Luckner et al., 2018); bone marrow IW (unpublished image courtesy of Dr. Gabi Bixel from Max-Planck-Institute for Molecular Biomedicine); bone marrow IM (Bixel et al., 2017); skin IW and IM (unpublished images courtesy of Haohua Tu and Stephen Boppart at the University of Illinois Biophotonics Imaging Laboratory); embryonic IW and IM, colonic IW and IM, and intestinal IW (unpublished images courtesy of Dr. Qiang Huang and Dr. Xiling Shen at Duke University); intestinal IM (Nobis et al., 2017); ovary IW and IM (Bochner et al., 2015); liver IW (unpublished image courtesy of Dr. Max Nobis and Dr. Paul Timpson at the Garvan Institute of Medical Research); liver IM (Nobis et al., 2017); kidney IW and IM (van den Berg et al., 2018b); pancreas IW (unpublished image courtesy of Dr. Qiang Huang and Dr. Xiling Shen at Duke University); pancreas IM (Nobis et al., 2017); breast IW (unpublished image courtesy of Dr. Max Nobis and Dr. Paul Timpson at the Garvan Institute of Medical Research); breast IM (Nobis et al., 2017); lung IW and IM (Lelkes et al., 2014).

Figure 4:. Imaging of in vitro tissue models.

(A) Tissue explant, organoid cultures, and organs-on-a-chip provide in vitro tissue models. (B) Organoids recapitulate the morphology of the tissue from which cells are derived. Representative brightfield (BF), H&E, and/or fluorescent microscopy (FM) images from the following sources: brain BF and FM (Xiang et al., 2017); lung H&E and FM (McCauley et al., 2017); breast BF and H&E (Sachs et al., 2018); pancreatic TEM and FM (Seino et al., 2018); kidney BF and FM (van den Berg et al., 2018a); liver BF and FM (Hu et al., 2018); ovary BF and H&E (Maenhoudt et al., 2020); gastric, intestinal, and colonic illustration (Bruens and Snippert, 2017); gastric H&E (Yan et al., 2018); intestinal and colonic FM (Múnera et al., 2017).

Figure 5:. Imaging techniques used to interrogate tissues.

Tissue imaging methods focus on architecture, fluorescence, subcellular dynamics, and isotope incorporation. (Clockwise from the left): Optical coherence tomography (OCT) is particularly well suited for imaging superficial tissues including the retina (unpublished image courtesy of Dr. Junjie Yao at Duke University). Confocal microscopy can allow specific imaging of fluorescent markers (unpublished image courtesy of Aliesha O’Raw and Dr. Xiling Shen at Duke University). Multi-isotope mass spectrometry imaging (MIMS) of the murine intestinal crypt reveal sulfur-rich granules (arrows) surrounded by proliferating stem cells (Zhang et al., 2020). At the cellular level, spatial visualization of RNA transcripts is accomplished by MERFISH (Barbash et al., 2019) and cellular status indicators like Sytox can be imaged via high-throughput imaging methods like IncuCyte (Gong et al., 2017).

Figure 6:. Combinatorial approaches used to answer biological questions.

Through using different technologies in consort, it is possible to interrogate different biological functions at an unprecedented resolution.