Cancer metabolism

Cancer is characterized by many different physical and biochemical properties, many of which can be examined and studied with optical imaging. It’s well documented that cancer cells undergo a metabolic reprogramming, called the Warburg Effect, which can be observed through label free multiphoton imaging of autofluorescence metabolic cofactors NADH and FAD. Additionally, structural changes in the collagen of tumors can be observed with second harmonic generation (SHG). Simultaneous label-free autofluorescence multiharmonic (SLAM) imaging can be used to image NADH, FAD, SHG, and third harmonic generation of tumors in vivo. NADH can also be examined using fluorescence lifetime imaging microscopy (FLIM). Additionally, cancer associated extracellular vesicles can be characterized with our nonlinear optical microscopy techniques. 

  • 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.
  • You S, Tu H, Chaney EJ, Sun Y, Zhao Y, Bower AJ, Liu Y-Z, Marjanovic M, Sinha S, Pu Y, Boppart SA. Intravital imaging by simultaneous label-free autofluorescence multiharmonic microscopy. Nature Communications, 9:2125. 2018.
  • Bower AJ, Sorrells JE, Li J, Marjanovic M, Barkalifa R, Boppart SA. Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy. Biomedical Optics Express, 10(12):6408-6421. 2019.
  • Sun Y, You S, Tu H, Spillman DR Jr, 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. 2018.

Intravital SLAM imaging of the tumor microenvironment 

Simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy is a powerful tool that elucidates pathways in biological processes, which gives major advantages to intravital imaging as no extrinsic agents are involved.  SLAM is a single-excitation source nonlinear imaging platform using a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast, simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third-harmonic generation from a wide range of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissues. In addition, in vivo tracking of cellular events can also be allowed.  

One of the fascinating applications of SLAM is that it can be used for cancer prognosis. SLAM can see the morphological and metastatic alternations in tumor microenvironment during the chemotherapy. In vivo study with PDX mice showed that SLAM can find the effective chemotherapy regimen leading to personalized therapy. 

  • You S, Tu H, Chaney EJ, Sun Y, Zhao Y, Bower AJ, Liu Y-Z, Marjanovic M, Sinha S, Pu Y, Boppart SA. Intravital imaging by simultaneous label-free autofluorescence multiharmonic microscopy. Nature Communications, 9:2125. 2018.
  • Park, J., Chaney, E., You, S., Abdelrahman, A.M., Leiting, J.L., Yonkus, J.A., Groves, P.D., Harrington, J.J., Spillman, D.R., Lynch, I.T. and Marjanovic, M., 2020, April. Characterizing Treatment Response of Pancreatic Tumor Patient-Derived Xenografts in Mice by Simultaneous Label-Free Autofluorescence Multi-Harmonic (SLAM) Microscopy. In Clinical and Translational Biophotonics (pp. TM2B-4). Optical Society of America. 

Leukocyte-swarming visualized and characterized by SLAM microscopy. Images acquired at (a) the beginning and (b) the end of the swarming. Collagen rearrangement was marked by the white boundary (Cluster 1, C1) while lipid interaction was marked by cyan boundary (Cluster 2, C2). (c)(e) Zoomed-in images of multi-nucleated neutrophils. (f) Traces of Cells 1–14, which were tracked to travel via similar routes to the same cluster at different time points, as shown in the velocity map (j)(g) Traces of Cells 15–16, which were both tracked for > 30 min and exhibited different behavior, with Cell 15 migrating towards the cluster with high speed and high directionality throughout the entire time course and Cell 11 mostly making random walk, as shown in the corresponding velocity map (k)(h) Leukocytes leaving the site (Cells 17–18 in Video). The series of snapshots were taken every 2 s and shown for every 20 s. The first three snapshots showed the deformation of the leukocyte, changing from a round shape to a stretched cell elongated along the direction of travel, which typically precedes the acceleration process shown in the last three snapshots. (i) Quantification of collagen clearance, cell accumulation, and lipid deformation within the marked clusters in a and b (C1 and C2). (j)(k) Velocity and directionality map of Cells 1–16. The color of the curves matches the color of the traces in (f) and (g) and the Video. D directionality. Scale bar: 50 µm 

SLAM image of an in vivo tissue after drug treatment. (a) Representative image of tumor tissue in channel of (a) 2PF, (b) 3PF, (c) SHG, (d) THG, (e) merged. Different aspects of THG/3PAF compared between (f) chemotherapy treated and (g) control mice. 

Intraoperative label-free multimodal nonlinear optical imaging

The majority of the current intraoperative optical imaging techniques involve labeling which perturbs the tissue microenvironment and alters the optical signatures of various biochemical processes. In contrast, multiple nonlinear optical imaging (NLOI) modalities have been demonstrated to have the ability to visualize microstructures and provide molecular and functional information. With the development and demonstration of our lab-based simultaneous label-free autofluorescence and multi-harmonics (SLAM) microscope,  continuous efforts have been made to adapt this multimodal NLOI platform to a portable system for intraoperative cancer imaging. Previously we have demonstrated one version of our intraoperative label-free NLOI system in human breast cancer surgeries. Correlations with the histology results have shown that this intraoperative NLOI platform has the potential to provide diagnostic information at point-of-procedure without destroying the biological tissue integrity. With our recent upgrades on the imaging system, we were able to provide optical assessment of needle biopsies taken from live animal patients during veterinary surgeries. We are working towards further improvement of the intraoperative label-free NLOI system to enable real-time screening of needle biopsies and surgical specimens for cancer diagnosis at point-of-procedure. 

Label-free multimodal NLOI results (left) along with corresponding histology images (right) from human breast tissue specimens diagnosed as (A) invasive ductal carcinoma (IDC), with the red dashed arrow showing an overall orientation of collagen alignment, (B-C) ductal carcinoma in situ (DCIS), showing adipocytes (red dashed arrows), blood vessel (red solid arrow in B), and mammary duct (red solid arrow in C), and (D) healthy breast tissue from a breast reduction surgery. Scale bars represent 100 µm. Channel pseudo-colors: THG, magenta; 3PF, cyan; SHG, green; 2PF, yellow.

  • Sun, Y., You, S., Tu, H., Spillman, D. R., Chaney, E. J. Marjanovic, M. Li, J., Barkalifa, R., Wang, J., Higham, A. M., Luckey, N. N., Cradock, K. A., Liu, Z. G., & Boppart, S. A. “Intraoperative visualization of the tumor microenvironment and of extracellular vesicles by label-free nonlinear imaging,” Science Advances, vol. 4, no. 12, eaau5603, 2018.
  • Yang, L., Park, J., Marjanovic, M., Chaney, E. J., Spillman, D. R., Phillips, H. & Boppart, S. A. "Intraoperative label-free multimodal nonlinear optical imaging for point-of-procedure cancer diagnostics." IEEE JSTQE, submitted. 

Live cell imaging using SLAM microscopy

Live cell imaging using SLAM microscope has been demonstrated in two different applications: cell identification or differentiation, and metabolic profiling calibration. While  third harmonic generation (THG, magenta) highlights the cell membranes and multiphoton autofluorescence (2PAF, 3PAF) can reveal the biochemical information in the cells, the combination of the three modalities can provide an informative optical signature, which can be beneficial in tissue imaging for identifying different types of normal cells, immune cells and tumor cells. Quantitative optical redox analyses of live-cell images have been mainly used to calibrate the metabolic profiling for tissue microenvironment and the extracellular vesicles.

Live-cell imaging by SLAM microscopy. (a) Mammary epithelial cells (HMEC) cells, (b) breast cancer cells (MDA-MB-231), and (c) macrophages. Scale bar: 40 µm. 

  • Boppart, S. A, You, S., Li, L., Chen, J., and Tu, H.  "Simultaneous label-free autofluorescence-multiharmonic microscopy and beyond.” APL Photonics 4, 100901 (2019);
  • You, S., Barkalifa, R., Chaney, E. J., Tu, H., Park, J., Sorrells, J. S., Sun. Y., Liu, Y. Z., Yang, L., Chen, D. Z., Marjanovic, M., Sinha, S., and Boppart, A. B. “Label-free visualization and characterization of extracellular vesicles in breast cancer.” PNAS, 116 (48) 24012-24018 (2019).