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Advances in Cell Microscopy

Rectangle Image

Advances in Cell Microscopy

Credit: iStock

We all know that seeing is believing. This has led scientists to image cells with microscopes as early as 1658.1 Since then, microscopy has significantly modernized, and has become a ubiquitous tool in cell biology labs with the use of fluorescent and 3D microscopy.

Does seeing more mean believing more? It certainly looks like there is a push towards being able to see ever more with the latest advances in cell imaging. It also looks like quantity can be combined with quality: microscope tools can see at higher resolutions to obtain more information with staggering precision, and more importantly, in live cells under minimal perturbations. Scientists can now routinely visualize individual organelles, map the movement of chromosomal loci, sense mechanical forces, and image cells continuously for days in a high-throughput manner. Read on to learn about five key advances in cell microscopy to understand where the field is moving to!

1. Brillouin microscopy

Brillouin microscopy is a non-invasive, label-free method which can probe the viscoelastic properties of biological samples with diffraction-limited resolution in 3D.2 It has the advantage of being contact free, unlike atomic force microscopy. Mechanical properties of cells and tissues are often altered in diseased tissues and are therefore of acute interest towards the understanding of mechanisms underlying pathologies. Brillouin light scattering revolves around the interaction of light with spontaneous, thermally induced density fluctuations. Mechanical properties, such as stiffness, can be inferred from the frequency shifts of the scattered light spectrum.2  This has enabled numerous types of biological measurements, such as the 3D mapping of the intracellular biomechanical properties in whole cells,3 or the imaging of intestinal organoid in 3D.2  There are, however, still challenges to be solved with this microscopy technique, and data requires particularly careful interpretation, as some have suggested that Brillouin measurements might be dominated by hydration and not stiffness effects.4

2. CRISPR-labeled fluorescence imaging

CRISPR has undoubtedly revolutionized gene editing and regulation, and in doing so, it also has facilitated cell microscopy. Groups have used it to label defined chromosomal loci to image the 3D structure of the genome in live cells,5 as opposed to conventional in situ hybridization studies that images only fixed cells. Using Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins, Ma et al. recently achieved simultaneous imaging of up to six chromosomal loci in individual live cells, a technique they call CRISPRainbow. In principle, they explain that adding just one more color to CRISPRainbow would increase the number of simultaneous live-cell detection of genomic loci to 15.6

3. Light sheet microscopy

Light sheet fluorescence microscopy (LSFM) illuminates only the thin imaging focal plane of a sample and detects the fluorescence from this specific plane, thus minimizing out-of-focus fluorescence and photobleaching.7 This means the dynamics of organisms and cells can be observed, without the need to section a sample. Until recently, the resolution did not permit subcellular imaging in fields of view that were large enough to contain several cells. Indeed, the optical heterogeneity of tissues can cause aberrations that quickly compromise resolution, signal, and contrast with increasing imaging depth. Progress had been made with Bessel light beams, and lattice-light sheet but this technique remained complex and expensive to set up. In 2019, Chang et al. described the new method of field synthesis which facilitates the use of light sheet with much simpler optics.8 It combines LSFM with adaptive optics, which compensates for optical distortions by changing the shape of a mirror to create an equal but opposite distortion. It requires less power, minimizing photobleaching and allows for the imaging of multiple colors at the same time at high resolution. This enabled Liu et al. to image endocytosis over time at the nanoscale by detecting the diffusion of clathrin-coated pits in a human stem cell-derived organoid or in the dorsal tail region of a zebrafish.9 It also helped them visualize with exquisite details organelle dynamics during zebrafish embryogenesis, and 3D cell migration of neuron, cancer or immune cells in vivo. This breakthrough promises to revolutionize quantitative subcellular 4D cell biology.

4. Holo-tomographic microscopy

Holo-tomographic microscopy (HTM) is a quantitative phase microscopy method where the object’s complex wave field is encoded into a hologram and is combined with rotational scanning of the specimen.9 This results in fast 3D reconstruction of live sample’s refractive index distribution at a resolution below the diffractive limit of light defined by the Rayleigh criterion. A key asset of the technique is the low energy it transfers to the sample, ensuring low phototoxicity, allowing for subcellular dynamics to be studied while going unperturbed. Scattering-free systems have now been developed allowing for subcellular high-resolution imaging such as that of individual mitochondria going through fusion and fission cycles. Using this technique, Sandoz et al. report for the first time organellar spinning implicated in cellular reorganization before mitosis in mouse embryonic stem cells (mESCs). 80 minutes before mitosis, they observe the nucleus, nucleoli, the nuclear membrane, lipid droplets and mitochondria rotating, which suggests a potential mechanism of redistribution of the cellular material before division to be investigated in additional studies.10

5. High content-analysis microscopy

Seeing more is not only about reaching high resolution: it can also mean seeing for longer periods of time. Scientists are realizing that imaging cells for short periods of time, or at discrete time points can mean they miss out on crucial cellular dynamics. This is why systems are now being developed to allow continuous imaging of samples. On the one hand, some microscopes are being developed such that they can be integrated inside incubators, to track cells in 2D over time as they grow continuously. Similarly, some incubators have been designed so that they contain cameras that can image any assay sequentially, to be able to image more than one sample in the same incubator. On the other hand, high throughput systems are being developed so that many cellular assays in well-plates can be studied over long times, in an effort to design data-rich experiments. For example, Anastasov et al. grew numerous tumor spheroids made of cancer and stromal cells that were imaged for 14 days to quantify their growth under different combinations of radiation and chemotherapy.11 This high content set-up allowed them to screen a large panel of chemotherapeutic agents, and their combination with radiation, identifying vinblastine as a radio sensitizing agent which is more efficient at decreasing the size of spheroids compared to assays where vinblastine was used alone.


1. Hajdu SI. A note from history: The discovery of blood cells. Ann Clin Lab Sci. 2003;33(2):237-238.

2. Prevedel R, Diz-Muñoz A, Ruocco G, Antonacci G. Brillouin microscopy - a revolutionary tool for mechanobiology? arXiv:190102006 [physics]. Published online January 7, 2019. Accessed June 18, 2021. http://arxiv.org/abs/1901.02006

3. Antonacci G, de Turris V, Rosa A, Ruocco G. Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS. Commun Biol. 2018;1(1):1-8. doi:10.1038/s42003-018-0148-x

4. Wu P-J, Kabakova IV, Ruberti JW, et al. Water content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materials. Nat Methods. 2018;15(8):561-562. doi:10.1038/s41592-018-0076-1

5. Chen B, Gilbert LA, Cimini BA, et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell. 2013;155(7):1479-1491. doi:10.1016/j.cell.2013.12.001

6. Ma H, Tu L-C, Naseri A, et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016;34(5):528-530. doi:10.1038/nbt.3526

7. Forero-Shelton M. Peering into cells at high resolution just got easier. Nat Methods. 2019;16(4):293-294. doi:10.1038/s41592-019-0373-3

8. Chang B-J, Kittisopikul M, Dean KM, Roudot P, Welf ES, Fiolka R. Universal light-sheet generation with field synthesis. Nat Methods. 2019;16(3):235-238. doi:10.1038/s41592-019-0327-9

 9. Liu T-L, Upadhyayula S, Milkie DE, et al. Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science. 2018;360(6386). doi:10.1126/science.aaq1392

10. Sandoz PA, Tremblay C, Equis S, et al. Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells. bioRxiv. Published online September 4, 2018:407239. doi:10.1101/407239

Anastasov N, Höfig I, Radulović V, et al. A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment. BMC Cancer. 2015;15(1):466. doi:10.1186/s12885-015-1481-9