Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.

High-resolution in vivo imaging is of great importance for the fields of biology and medicine. The introduction of hardware-based adaptive optics (HAO) has pushed the limits of optical imaging, enabling high-resolution near diffraction-limited imaging of previously unresolvable structures1,2. In ophthalmology, when combined with optical coherence tomography, HAO has enabled a detailed three-dimensional visualization of photoreceptor distributions3,4 and individual nerve fibre bundles5 in the living human retina. However, the introduction of HAO hardware and supporting software adds considerable complexity and cost to an imaging system, limiting the number of researchers and medical professionals who could benefit from the technology. Here we demonstrate a fully automated computational approach that enables high-resolution in vivo ophthalmic imaging without the need for HAO. The results demonstrate that computational methods in coherent microscopy are applicable in highly dynamic living systems.

Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression.

Abstract:As imaging systems become more advanced and acquire data at faster rates, increasingly dynamic samples can be imaged without concern of motion artifacts. For optical interferometric techniques such as optical coherence tomography, it often follows that initially, only amplitude-based data are utilized due to unstable or unreliable phase measurements. As systems progress, stable phase maps can also be acquired, enabling more advanced, phase-dependent post-processing techniques. Here we report an investigation of the stability requirements for a class of phase-dependent post-processing techniques -numerical defocus and aberration correction with further extensions to techniques such as Doppler, phase-variance, and optical coherence elastography. Mathematical analyses and numerical simulations over a variety of instabilities are supported by experimental investigations.

Optical coherence tomography (OCT) has become an important imaging modality with numerous biomedical applications. Challenges in high-speed, high-resolution, volumetric OCT imaging include managing dispersion, the trade-off between transverse resolution and depth-of-field, and correcting optical aberrations that are present in both the system and sample. Physics-based computational imaging techniques have proven to provide solutions to these limitations. This review aims to outline these computational imaging techniques within a general mathematical framework, summarize the historical progress, highlight the state-of-the-art achievements, and discuss the present challenges. Swanson, "Optical biopsy and imaging using optical coherence tomography," Nat. Med. 1(9), 970-972 (1995). 3. G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Boppart, C. Pitris, J. F. Southern, and J. G. Fujimoto, "In vivo endoscopic optical biopsy with optical coherence tomography," Science 276(5321), 2037-2039 (1997). 4. S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, "In vivo cellular optical coherence tomography imaging," Nat. Med. 4(7), 861-865 (1998). Sundaram, P. S. Ray, and S. A. Boppart, "Real-time imaging of the resection bed using a handheld probe to reduce incidence of microscopic positive margins in cancer surgery," Cancer Res. 75(18), 3706-3712 (2015). 9. J. G. Fujimoto and E. A. Swanson, "The development, commercialization, and impact of optical coherence tomography," Invest. Ophthalmol. Vis. Sci. 57(9), OCT1-OCT13 (2016). 10. A. M. Cormack, "Representation of a function by its line integrals, with some radiological applications. II," J.Appl. Phys. 35(10), 2722-2727 (1964). 11. G. N. Hounsfield, "Computerized transverse axial scanning (tomography). 1. Description of system," Br. J.Radiol. 46(552), 1016-1022 (1973). 12. P. C. Lauterbur, "Image formation by induced local interactions: examples employing nuclear magnetic resonance," Nature 242(5394), 190-191 (1973). 13. P. T. Gough and D. W. Hawkins, "Unified framework for modern synthetic aperture imaging algorithms," Int. J.Imaging Syst. Technol. 8(4), 343-358 (1997). shaping for optimal depth-selective focusing in optical coherence tomography," Opt. Express 21(3), 2890-2902 (2013 111-115 (1962). 77. L. Cutrona, E. Leith, C. Palermo, and L. Porcello, "Optical data processing and filtering systems," IRE Trans.Inf. Theory 6(3), 386-400 (1960). 78. L. J. Cutrona, E. N. Leith, L. J. Porcello, and E. W. Vivian, "On the application of coherent optical processing techniques to synthetic-aperture radar," Proc. IEEE 54(8), 1026-1032 (1966). 79. W. Brown and L. Porcello, "An introduction to synthetic-aperture radar," IEEE Spectr. 6(9), 52-62 (1969). 80. M. P. Hayes and P. T. Gough, "Broad-band synthetic aperture sonar," IEEE J. Oceanic Eng. 17(1), 80-94 (1992)