EV imaging

EVs are nano-sized protein, nucleic acid, lipid and metabolites containing membranous lipid bilayer vesicles that are secreted by multiple cell types. They are generally divided into two categories: ectosomes and exosomes. Ectosomes are vesicles formed from the cell plasma membrane sprouting outwards. These include microvesicles, microparticles and large vesicles with a size ranging from 50 to 1000 nm in diameter. Exosomes are small EVs in a size range of 40-160 nm in diameter with an endosomal origin.

EVs have biological activities, mediate intercellular communication and moderate various signaling pathways in the recipient cells that internalize EVs under physiological or pathological conditions using different mechanisms of particle uptake. A wide range of stimulatory or inhibitory functional outcomes are shown to be induced following cellular interactions with EVs, including proliferation, angiogenesis, apoptosis, cytokine production, immune system modulation, cell motility, invasion or metastasis.

There is an increasing amount of scientific evidence that the cargo present in EVs reflects the pathophysiological state of the cell. Therefore, EVs may serve as diagnostic or prognostic biomarkers of many diseases. Additionally, EVs has gained attention for their potential use as therapeutic agents. EVs may be exploited in clinical settings for targeted drug delivery and to block disease progression.

Our laboratory is currently interested in evaluating the biological activity of EVs originating from different biological sources using different imaging modalities as well as biochemical methods. We have invested significant time and efforts in optimizing the isolation, quantification, and visualization protocols of serum, urine, cell culture and tissue-derived EVs.


  • Sorrells JE, Martin EM, Aksamitiene E, Mukherjee P, Alex A, Chaney EJ, Marjanovic M, Boppart SA. Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy. Scientific Reports, 11:3308. 2021. https://doi.org/10.1038/s41598-020-80813-0 
  • Sun Y, Chen E, Thomas J, Liu Y, Tu H, Boppart SA. K-means clustering of coherent Raman spectra from extracellular vesicles visualized by label-free multiphoton imaging. Optics Letters, 45:3613-3616, 2020. https://doi.org/10.1364/OL.395838
  • You S, Barkalifa R, Chaney EJ, Tu H, Park J, Sorrells JE, Sun Y, Liu YZ, Yang L, Chen DZ, Marjanovic M, Sinha S, Boppart SA. Label-free visualization and characterization of extracellular vesicles in breast cancer. PNAS, 116(48):24012-24018. 2019. https://doi.org/10.1073/pnas.1909243116 
  • Sun Y, You S, Tu H, Spillman Jr. DR, CHaney EJ, Marjanovic M, Li J, Barkalifa R, Wang J, Higham AM, Luckey NN, Cradock KA, Liu G, Boppart SA. Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging. Science Advances, 4(12): eaau5603. (2018). https://doi.org/10.1126/sciadv.aau5603

SLAM and EV imaging

EVs were imaged with SLAM with the need of capturing EVs label-free and revealing various metabolic information at the same time. SLAM is capable of imaging isolated EVs derived from cell culture media, urine, serum, and other biofluids. Moreover, SLAM can image EVs inside biological tissues in vivo and ex vivo. SLAM uses 3 optical channels for the EV analysis: THG, 2PF and 3PF. Optical redox ratio FAD/(FAD+NADH) is used for differentiating subpopulations of EVs through metabolic and structural differences. Subpopulation of EVs enable the diagnosis and progression of cancer. NAD(P)H-rich EV ratio is one of the markers for diagnosing and staging cancer of parent cells. Vesicle dynamics in cells are known to be cooperating in carcinogenesis and SLAM can visualize this step. Dynamics of EVs (release, uptake, and migration of EVs) were found in cell and tissue level imaging with SLAM.  

EV imaging with SLAM (a) SLAM images of EVs isolated from cell cultured media. (b) Zoomed-in image of 1 representative EV with 2D (upper) and 3D (lower) visualization maps. Each pixel size is 500 nm. (c) TEM image of EVs. (d) Release of EV from a cell. (e) In vivo visualization of EVs in tumor-bearing rat.

  • You, S., Barkalifa, R., Chaney, E.J., Tu, H., Park, J., Sorrells, J.E., Sun, Y., Liu, Y.Z., Yang, L., Chen, D.Z. and Marjanovic, M., 2019. Label-free visualization and characterization of extracellular vesicles in breast cancer. Proceedings of the National Academy of Sciences, 116(48), pp.24012-24018.
  • Sun, Y., You, S., Tu, H., Spillman, D.R., Chaney, E.J., Marjanovic, M., Li, J., Barkalifa, R., Wang, J., Higham, A.M. and Luckey, N.N., 2018. Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging. Science advances, 4(12), p.eaau5603.
  • You, S., Tu, H., Chaney, E.J., Sun, Y., Zhao, Y., Bower, A.J., Liu, Y.Z., Marjanovic, M., Sinha, S., Pu, Y. and Boppart, S.A., 2018. Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy. Nature communications, 9(1), pp.1-9.
  • Tu, H., Liu, Y., Marjanovic, M., Chaney, E.J., You, S., Zhao, Y. and Boppart, S.A., 2017. Concurrence of extracellular vesicle enrichment and metabolic switch visualized label-free in the tumor microenvironment. Science advances, 3(1), p.e1600675.