Quality control in molecular optical sectioning microscopy is indispensable for transforming acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, we develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle. Demonstrated in a prototypical multiphoton microscope, our tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal-to-noise frames with either gentle illumination or low-concentration fluorescence labeling.
Label-free multiphoton imaging has been a powerful tool to study the microstructure and specific chemical distributions in biological tissue, especially in tumors and their microenvironments. Thus, this technique has great potential to assist in cancer-related clinical studies. A portable label-free multiphoton imaging system was constructed with four imaging modalities: two- and three- photon fluorescence, and second and third harmonic generation. Mosaicked multimodal images can be acquired with dual-channel detection and galvo-mirror scanning. This system was demonstrated during animal surgeries for real-time, label-free assessment of tumor tissue samples acquired via core-needle biopsy and fine-needle aspiration.
Tumor-associated extracellular vesicles (TEVs), which represent a unique kind of inter-cellular communication carrier, have been found to play vital roles in directing the invasion and metastasis of tumor cells. However, because the human tumor microenvironment and TEVs significantly degrade or lose vitality over relatively brief periods of time after breast cancer surgical excision, lab-based studies with fresh human tissue specimens cannot provide accurate TEV information. By designing and building a portable label-free nonlinear imaging system, we have been able to conduct intraoperative imaging of fresh, unstained breast tissue specimens immediately after excision. Various features of the breast tumor microenvironment from multimodal nonlinear images were characterized to indicate tumor progression, invasiveness, and tumor grade, such as tumor-accommodating collagen structure visualized using second harmonic generation imaging, fibroblasts shown by two photon auto-fluorescence, and TEVs highlighted using third harmonic generation imaging. In particular, we found TEV count as a promising biomarker of tumor aggressiveness and margin distance. A decreasing trend of TEV counts with larger margin distance and lower cancer aggressiveness grades was revealed among 18 breast cancer cases. In addition, clear differences in TEV counts between images collected from breast cancer cases and healthy breast reduction cases, in another aspect, validate the potential of identifying TEVs using our imaging method. Acquisition and interpretation of these intraoperative image data not only provided assessment of the human tumor microenvironment, but also offered the potential to intraoperatively assess tumor margin distance and determine tumor aggressiveness.
In contrast to a broadband Ti:sapphire laser that mode locks a continuum of emission and enables broadband biophotonic applications, supercontinuum generation moves the spectral broadening outside the laser cavity into a nonlinear medium, and may thus improve environmental stability and more readily enable clinical translation. Using a photonic crystal fiber for passive spectral broadening, this technique becomes widely accessible from a narrowband fixed-wavelength mode-locked laser. Currently, fiber supercontinuum sources have benefited single-photon biological imaging modalities, including light-sheet or confocal microscopy, diffuse optical tomography, and retinal optical coherence tomography. However, they have not fully benefited multiphoton biological imaging modalities with proven capability for high-resolution label-free molecular imaging. The reason can be attributed to the amplitude/phase noise of fiber supercontinuum, which is amplified from the intrinsic noise of the input laser and responsible for spectral decoherence. This instability deteriorates the performance of multiphoton imaging modalities more than that of single-photon imaging modalities. Building upon a framework of coherent fiber supercontinuum generation, we have avoided this instability or decoherence, and balanced the often conflicting needs to generate strong signal, prevent sample photodamage, minimize background noise, accelerate imaging speed, improve imaging depth, accommodate different modalities, and provide user-friendly operation. Our prototypical platforms have enabled fast stain-free histopathology of fresh tissue in both laboratory and intraoperative settings to discover a wide variety of imaging-based cancer biomarkers, which may reduce the cost and waiting stress associated with disease/cancer diagnosis. A clear path toward intraoperative multiphoton imaging can be envisioned to help pathologists and surgeons improve cancer surgery.
Label-free multi-photon imaging has been a powerful tool for studying tissue microstructures and biochemical distributions, particularly for investigating tumors and their microenvironments. However, it remains challenging for traditional bench-top multi-photon microscope systems to conduct ex vivo tumor tissue imaging in the operating room due to their bulky setups and laser sources. In this study, we designed, built, and clinically demonstrated a portable multi-modal nonlinear label-free microscope system that combined four modalities, including two- and three- photon fluorescence for studying the distributions of FAD and NADH, and second and third harmonic generation, respectively, for collagen fiber structures and the distribution of micro-vesicles found in tumors and the microenvironment. Optical realignments and switching between modalities were motorized for more rapid and efficient imaging and for a light-tight enclosure, reducing ambient light noise to only 5% within the brightly lit operating room. Using up to 20 mW of laser power after a 20x objective, this system can acquire multi-modal sets of images over 600 μm × 600 μm at an acquisition rate of 60 seconds using galvo-mirror scanning. This portable microscope system was demonstrated in the operating room for imaging fresh, resected, unstained breast tissue specimens, and for assessing tumor margins and the tumor microenvironment. This real-time label-free nonlinear imaging system has the potential to uniquely characterize breast cancer margins and the microenvironment of tumors to intraoperatively identify structural, functional, and molecular changes that could indicate the aggressiveness of the tumor.
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