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. 

PUBLICATIONS:

• 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. 

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.  

Histopathology has been widely adopted in tissue assessment in clinical decision making and in research. However, its complex procedure of tissue preparation and analysis prevent histopathology from intraoperative assessment and cause delays in treatment planning. Our lab developed an Artificial Intelligence (AI)-driven real-time intraoperative diagnosis system based on stain-free slide-free multimodal multiphoton microscopy. This system captures rich molecular and structural information from living or freshly excised tissue and use Deep Neural Networks (DNN) to extract meaningful features and generate diagnoses. Compared to traditional histopathology, which may take several days to process the tissue and generate analysis reports, our highly automated system only requires minimal tissue processing and generates reliable diagnosis within minutes, which makes this technique an attractive alternative or adjunct to histochemistry in clinical settings, especially in time-sensitive scenarios such as the intraoperative assessment of breast tissue during surgical oncology procedures.

Histology imaging, invented centuries ago, serves an essential clinical imaging method to provide evidence for cancer-related diagnosis. However, the time- and labor- intense tissue preparation processes of histology causes delay in cancer diagnosis and increases the cost of cancer treatment. Our lab uses a novel imaging method, the simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy, to generate histology-like images from label-free tissues, saving the time and labor of tissue processing. By imaging fresh human and rat tissues without any tissue processing or staining, various biological tissue features are effectively visualized by one or multiple imaging modalities of the SLAM microscope and converted to histology-like contrasts. In particular, we realized for the first time the real-time 3D histology-like imaging. 

PUBLICATIONS:

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.