Multiphoton microscopy (MPM) provides high-resolution imaging of deep tissue structures while allowing for the visualization of non-labeled biological samples. However, photon generation efficiency of intrinsic biomarkers is low and this, coupled with inherent detection inaccuracies in the photoelectric sensors, leads to an introduction of noise in acquired images. Higher dwelling times can reduce noise but increase the likelihood of photobleaching. To combat this, deep learning methods are being increasingly employed to denoise MPM images, allowing for a more efficient and less invasive process. However, machine learning models can hallucinate information, which is unacceptable for critical scientific microscopy applications. Uncertainty quantification, which has been demonstrated for image-to-image regression tasks, can provide confidence bounds for machine learning-based image reconstruction tasks, adding confidence to predictions. In this work, we discuss incorporating uncertainty quantification into an optimized denoising model to guide adaptive multiphoton microscopy image acquisition. We demonstrate that our method is capable of maintaining fine features in the denoised image, while outperforming other denoising methods by adaptively selecting to reimage the most uncertain pixels in a human endometrium tissue sample.
SWIR-based microscopy has opened up windows to cellular and extracellular dynamics in deep tissues and living biological systems. New generation of laser sources, with high pulse energy, wide continuous tunable range, and a compact form, are in high demand to advance nonlinear microscopy and SWIR-based imaging to its full potential for deep-tissue imaging.
This talk will discuss our recently developed approach that exploits the spatial and temporal degrees of control of nonlinear effects in step-index MMFs using a 3D-printed programmable fiber piano. By leveraging the rich spatiotemporal degrees of freedom and the high spectral brilliance in SI MMF, We have achieved broadband high-peak-power spanning 560–2200 nm, resulting from combined spectral energy reallocation (up to 166-fold) and temporal shortening (up to 4-fold) uniquely enabled by the fiber shaper. Its potential as a nonlinear imaging source is further demonstrated by applying the MMF source to multiphoton microscopy, where multi-fold signal enhancement is achieved for label-free tissue imaging with adaptive optimization.
Multiplex imaging facilities biological studies in multicellular dynamics in living organisms due to its molecular specificity, 3D subcellular resolution, and deep tissue penetration. However, one major bottleneck is detecting and resolving multiplexed signals of weak fluorescence due to a tradeoff between signal throughput and spectral resolution. Here, we demonstrate high-speed, programmable, and broadband excitation encoding to enhance sensitivity without sacrificing signal throughput in multiplex multiphoton imaging. We utilize a 22-kHz programmable digital micromirror device to modulate the spectrum of a high-power broadband laser, achieving versatile excitation encoding schemes with a 750-nm bandwidth in the NIR regime. The proposed method will benefit applications that demand high-speed and high-content performance, including hyperspectral multiphoton microscopy and computational spectroscopy.
Achieving high-precision light manipulation is crucial for delivering information through complex media with high fidelity. Digital micromirror devices (DMDs) have emerged as a promising candidate as high-speed wavefront shaping devices but at the cost of compromised fidelity, largely due to the limited degrees of freedom and the challenge of optimizing a binary amplitude mask. Here we leverage the properties of sparse-to-dense transformation in complex media and introduce a sparsity-constrained optimization framework. The proposed optimization framework could enhance existing holographic setups without changes to the hardware, and enable high-fidelity and high-speed wavefront shaping through different scattering media and platforms.
Label-free nonlinear microscopy allows for high-resolution and three-dimensional imaging of live biological specimens without the need for exogenous labels. The integration of multiple modalities further enhances molecular specificity and visualization diversity for metabolic and structural mapping of heterogeneous tissue architectures. In this work, we introduce high-speed simultaneous label-free autofluorescence-multiharmonic (hSLAM) microscopy, where a high-peak-power adaptive fiber source based on multimode fiber (MMF) is employed with a nonlinear fiber piano. We will also talk about how the higher speed SLAM enables multicellular dynamics in living tissues with higher spectral flexibility and peak power, providing new possibilities for bioimaging.
In this talk, I will present optical imaging platforms and methodologies that aim to empower label-free in vivo microscopy. Label-free in vivo microscopy promises to be a versatile tool for studying and diagnosing diseases in living animals and humans. Part of the challenge of label-free in vivo microscopy lies in the lack of simultaneous contrast, limited signal generation efficiency, and nonintuitive interpretation. This talk will cover how we attempt to address these challenges by resorting to light engineering and algorithms.
For investigating the potential for personalized treatment of pancreatic cancer, two- and three-photon autofluorescence and second and third harmonic generation images were simultaneously generated from in vivo pancreatic tumor patient-derived xenograft (PDX) mice. Two different treatment regimens were administered to PDX mice for 5 weeks and compared. Control mice were treated only with saline. Mice were classified into three groups according to their response to the treatment: responsive, resistant, and control. Optical redox ratios were calculated from tumor regions which showed differences between the responsive group compared to the resistant and control groups.
Significance: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation.
Aim: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively.
Approach: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity.
Results: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging.
Conclusions: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.
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.
Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.
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.
We investigate and demonstrate the feasibility of using a combined Raman scattering (RS) spectroscopy and low-coherence interferometry (LCI) probe to differentiate microbial pathogens and improve our diagnostic ability of ear infections [otitis media (OM)]. While the RS probe provides noninvasive molecular information to identify and differentiate infectious microorganisms, the LCI probe helps to identify depth-resolved structural information as well as to guide and monitor positioning of the Raman spectroscopy beam for relatively longer signal acquisition times. A series of phantom studies, including the use of human middle ear effusion samples, were performed to mimic the conditions of in vivo investigations. These were also conducted to validate the feasibility of using this combined RS/LCI probe for point-of-care diagnosis of the infectious pathogen(s) in OM patients. This work establishes important parameters for future in vivo investigations of fast and accurate determination and diagnosis of infectious microorganisms in OM patients, potentially improving the efficacy and outcome of OM treatments, and importantly reducing the misuse of antibiotics in the presence of viral infections.
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