Inspiring a convergent engineering approach to measure and model the tissue microenvironment.

Understanding the molecular and physical complexity of the tissue microenvironment (TiME) in the context of its spatiotemporal organization has remained an enduring challenge. Recent advances in engineering and data science are now promising the ability to study the structure, functions, and dynamics of the TiME in unprecedented detail; however, many advances still occur in silos that rarely integrate information to study the TiME in its full detail. This review provides an integrative overview of the engineering principles underlying chemical, optical, electrical, mechanical, and computational science to probe, sense, model, and fabricate the TiME. In individual sections, we first summarize the underlying principles, capabilities, and scope of emerging technologies, the breakthrough discoveries enabled by each technology and recent, promising innovations. We provide perspectives on the potential of these advances in answering critical questions about the TiME and its role in various disease and developmental processes. Finally, we present an integrative view that appreciates the major scientific and educational aspects in the study of the TiME.

Dispersion mismatch correction for evident chromatic anomaly in low coherence interferometry.

The applications of ultrafast optics to biomedical microscopy have expanded rapidly in recent years, including interferometric techniques like optical coherence tomography and microscopy (OCT/OCM). The advances of ultra-high resolution OCT and the inclusion of OCT/OCM in multimodal systems combined with multiphoton microscopy have marked a transition from using pseudo-continuous broadband sources, such as superluminescent diodes, to ultrafast supercontinuum optical sources. We report anomalies in the dispersion profiles of low-coherence ultrafast pulses through long and non-identical arms of a Michelson interferometer that are well beyond group delay or third-order dispersions. This chromatic anomaly worsens the observed axial resolution and causes fringe artifacts in the reconstructed tomograms in OCT/OCM using traditional algorithms. We present DISpersion COmpensation Techniques for Evident Chromatic Anomalies (DISCOTECA) as a universal solution to address the problem of chromatic dispersion mismatch in interferometry, especially with ultrafast sources. First, we demonstrate the origin of these artifacts through the self-phase modulation of ultrafast pulses due to focusing elements in the beam path. Next, we present three solution paradigms for DISCOTECA: optical, optoelectronic, and computational, along with quantitative comparisons to traditional methods to highlight the improvements to the dynamic range and axial profile. We explain the piecewise reconstruction of the phase mismatch between the arms of the spectral-domain interferometer using a modified short-term Fourier transform algorithm inspired by spectroscopic OCT. Finally, we present a decision-making guide for evaluating the utility of DISCOTECA in interferometry and for the artifact-free reconstruction of OCT images using an ultrafast supercontinuum source for biomedical applications.

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy.

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

Programmable hyperspectral coherent anti-Stokes Raman scattering microscopy.

Hyperspectral coherent Raman scattering microscopy provides a significant improvement in acquisition time compared to spontaneous Raman scattering yet still suffers from the time required to sweep through individual wavenumbers. To address this, we present the use of a pulse shaper with a 2D spatial light modulator for phase- and amplitude-based shaping of the Stokes beam to create programmable spectrally tailored excitation envelopes. This enables collection of useful spectral information in a more rapid and efficient manner.

Large field-of-view metabolic profiling of murine brain tissue following morphine incubation using label-free multiphoton microscopy.

BACKGROUND: Although the effects on neural activation and glucose consumption caused by opiates such as morphine are known, the metabolic machinery underlying opioid use and misuse is not fully explored. Multiphoton microscopy (MPM) techniques have been developed for optical imaging at high spatial resolution. Despite the increased use of MPM for neural imaging, the use of intrinsic optical contrast has seen minimal use in neuroscience. NEW METHOD: We present a label-free, multimodal microscopy technique for metabolic profiling of murine brain tissue following incubation with morphine sulfate (MSO4). We evaluate two- and three-photon excited autofluorescence, and second and third harmonic generation to determine meaningful intrinsic contrast mechanisms in brain tissue using simultaneous label-free, autofluorescence multi-harmonic (SLAM) microscopy. RESULTS: Regional differences quantified in the cortex, caudate, and thalamus of the brain demonstrate region-specific changes to metabolic profiles measured from FAD intensity, along with brain-wide quantification. While the overall intensity of FAD signal significantly decreased after morphine incubation, this metabolic molecule accumulated near the nucleus accumbens. COMPARISON WITH EXISTING METHODS: Histopathology requires tissue fixation and staining to determine cell type and morphology, lacking information about cellular metabolism. Tools such as fMRI or PET imaging have been widely used, but lack cellular resolution. SLAM microscopy obviates the need for tissue preparation, permitting immediate use and imaging of tissue with subcellular resolution in its native environment. CONCLUSIONS: This study demonstrates the utility of SLAM microscopy for label-free investigations of neural metabolism, especially the intensity changes in FAD autofluorescence and structural morphology from third-harmonic generation.

Temporally optimized and spectrally shaped hyperspectral coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy offers label-free chemical contrasts based on molecular vibrations. Hyperspectral CARS (HS-CARS) microscopy enables comprehensive microscale chemical characterization of biological samples. Various HS-CARS methods have been developed with individual advantages and disadvantages. We present what we believe to be a new temporally optimized and spectrally shaped (TOSS) HS-CARS method to overcome the limitations of existing techniques by providing precise control of the spatial and temporal profiles of the excitation beams for efficient and accurate measurements. This method uniquely uses Fourier transform pulse shaping based on a two-dimensional spatial light modulator to control the phase and amplitude of the excitation beams. TOSS-HS-CARS achieves fast, stable, and flexible acquisition, minimizes photodamage, and is highly adaptable to a multimodal multiphoton imaging system.

Analog multiplexing of a laser clock and computational photon counting for fast fluorescence lifetime imaging microscopy.

The dynamic range and fluctuations of fluorescence intensities and lifetimes in biological samples are large, demanding fast, precise, and versatile techniques. Among the high-speed fluorescence lifetime imaging microscopy (FLIM) techniques, directly sampling the output of analog single-photon detectors at GHz rates combined with computational photon counting can handle a larger range of photon rates. Traditionally, the laser clock is not sampled explicitly in fast FLIM; rather the detection is synchronized to the laser clock so that the excitation pulse train can be inferred from the cumulative photon statistics of several pixels. This has two disadvantages for sparse or weakly fluorescent samples: inconsistencies in inferring the laser clock within a frame and inaccuracies in aligning the decay curves from different frames for averaging. The data throughput is also very inefficient in systems with repetition rates much larger than the fluorescence lifetime due to significant silent regions where no photons are expected. We present a method for registering the photon arrival times to the excitation using time-domain multiplexing for fast FLIM. The laser clock is multiplexed with photocurrents into the silent region. Our technique does not add to the existing data bottleneck, has the sub-nanosecond dead time of computational photon counting based fast FLIM, works with various detectors, lasers, and electronics, and eliminates the errors in lifetime estimation in photon-starved conditions. We demonstrate this concept on two multiphoton setups of different laser repetition rates for single and multichannel FLIM multiplexed into a single digitizer channel for real-time imaging of biological samples.

Bichromatic tetraphasic full-field optical coherence microscopy.

Significance: Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast. Aim: We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts. Approach: BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast. Results: BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors. Conclusions: BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.

Supercontinuum intrinsic fluorescence imaging heralds free view of living systems.

Optimal imaging strategies remain underdeveloped to maximize information for fluorescence microscopy while minimizing the harm to fragile living systems. Taking hint from the supercontinuum generation in ultrafast laser physics, we generated supercontinuum fluorescence from untreated unlabeled live samples before nonlinear photodamage onset. Our imaging achieved high-content cell phenotyping and tissue histology, identified bovine embryo polarization, quantified aging-related stress across cell types and species, demystified embryogenesis before and after implantation, sensed drug cytotoxicity in real-time, scanned brain area for targeted patching, optimized machine learning to track small moving organisms, induced two-photon phototropism of leaf chloroplasts under two-photon photosynthesis, unraveled microscopic origin of autumn colors, and interrogated intestinal microbiome. The results enable a facility-type microscope to freely explore vital molecular biology across life sciences.

Tunable image-mapping optical coherence tomography.

We present tunable image-mapping optical coherence tomography (TIM-OCT), which can provide optimized imaging performance for a given application by using a programmable phase-only spatial light modulator in a low-coherence full-field spectral-domain interferometer. The resultant system can provide either a high lateral resolution or a high axial resolution in a snapshot without moving parts. Alternatively, the system can achieve a high resolution along all dimensions through a multiple-shot acquisition. We evaluated TIM-OCT in imaging both standard targets and biological samples. Additionally, we demonstrated the integration of TIM-OCT with computational adaptive optics in correcting sample-induced optical aberrations.

Phase-sensitive detection of anomalous diffusion dynamics in the neuronal membrane induced by ion channel gating.

Non-ergodicity of neuronal dynamics from rapid ion channel gating through the membrane induces membrane displacement statistics that deviate from Brownian motion. The membrane dynamics from ion channel gating were imaged by phase-sensitive optical coherence microscopy. The distribution of optical displacements of the neuronal membrane showed a Lévy-like distribution and the memory effect of the membrane dynamics by the ionic gating was estimated. The alternation of the correlation time was observed when neurons were exposed to channel-blocking molecules. Non-invasive optophysiology by detecting the anomalous diffusion characteristics of dynamic images is demonstrated.

Computational Photon Counting Using Multithreshold Peak Detection for Fast Fluorescence Lifetime Imaging Microscopy.

Time-resolved photon counting methods have a finite bandwidth that restricts the acquisition speed of techniques like fluorescence lifetime imaging microscopy (FLIM). To enable faster imaging, computational methods can be employed to count photons when the output of a detector is directly digitized at a high sampling rate. Here, we present computational photon counting using a hybrid photodetector in conjunction with multithreshold peak detection to count instances where one or more photons arrive at the detector within the detector response time. This method can be used to distinguish up to five photon counts per digitized point, whereas previous demonstrations of computational photon counting on data acquired with photomultiplier tubes have only counted one photon at a time. We demonstrate in both freely moving C. elegans and a human breast cancer cell line undergoing apoptosis that this novel multithreshold peak detection method can accurately characterize the intensity and fluorescence lifetime of samples producing photon rates up to 223%, higher than previously demonstrated photon counting FLIM systems.

Tracking the binding of multi-functional fluorescent tags for Alzheimer's disease using quantitative multiphoton microscopy.

A recent theranostic approach to address Alzheimer's disease (AD) utilizes multifunctional targets that both tag and negate the toxicity of AD biomarkers. These compounds, which emit fluorescence with both an activation and a spectral shift in the presence of Aß, were previously characterized with traditional fluorescence imaging for binary characterization. However, these multifunctional compounds have broad and dynamic emission spectra that are dependent on factors such as the local environment, presence of Aß deposits, etc. Since quantitative multiphoton microscopy is sensitive to the binding dynamics of molecules, we characterized the performance of two such compounds, LS-4 and ZY-12-OMe, using Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy and Fast Optical Coherence, Autofluorescence Lifetime imaging and Second harmonic generation (FOCALS) microscopy. This study shows that the combination of quantitative multiphoton imaging with multifunctional tags for AD offers new insights into the interaction of these tags with AD biomarkers and the theranostic mechanisms.

Ultra-parallel label-free optophysiology of neural activity.

The electrical activity of neurons has a spatiotemporal footprint that spans three orders of magnitude. Traditional electrophysiology lacks the spatial throughput to image the activity of an entire neural network; besides, labeled optical imaging using voltage-sensitive dyes and tracking Ca2+ ion dynamics lack the versatility and speed to capture fast-spiking activity, respectively. We present a label-free optical imaging technique to image the changes to the optical path length and the local birefringence caused by neural activity, at 4,000 Hz, across a 200 × 200 µm2 region, and with micron-scale spatial resolution and 300-pm displacement sensitivity using Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM). The undulations in the optical responses from mammalian neuronal activity were matched with field-potential electrophysiology measurements and validated with channel blockers. By directly tracking the widefield neural activity at millisecond timescales and micrometer resolution, SPoOF OCM provides a framework to progress from low-throughput electrophysiology to high-throughput ultra-parallel label-free optophysiology.

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics.

Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo. Fast analog detection of fluorescence lifetime and real-time processing on a graphical processing unit enables longitudinal imaging of biological dynamics. We detail the effect of optical aberrations on the accuracy of FLIM beyond the context of undistorting image features. To compensate for the sample-induced aberrations, we implemented a closed-loop single-shot sensorless adaptive optics solution, which uses computational adaptive optics of OCM for wavefront estimation within 2 s and improves the quality of quantitative fluorescence imaging in thick tissues. Multimodal imaging with complementary contrasts improves the specificity and enables multidimensional quantification of the optical signatures in vitro, ex vivo, and in vivo, fast acquisition and real-time processing improve imaging speed by 4-40 × while maintaining enough signal for quantitative nonlinear microscopy, and adaptive optics improves the overall versatility, which enable FOCALS microscopy to overcome the limits of traditional label-free imaging techniques.

Single-photon peak event detection (SPEED): a computational method for fast photon counting in fluorescence lifetime imaging microscopy.

Fluorescence lifetime imaging microscopy (FLIM) characterizes samples by examining the temporal properties of fluorescence emission, providing useful contrast within samples based on the local physical and biochemical environment of fluorophores. Despite this, FLIM applications have been limited in scope by either poor accuracy or long acquisition times. Here, we present a method for computational single-photon counting of directly sampled time-domain FLIM data that is capable of accurate fluorescence lifetime and intensity measurements while acquiring over 160 Mega-counts-per-second with sub-nanosecond time resolution between consecutive photon counts. We demonstrate that our novel method of Single-photon PEak Event Detection (SPEED) is more accurate than direct pulse sampling and faster than established photon counting FLIM methods. We further show that SPEED can be implemented for imaging and quantifying samples that benefit from higher -throughput and -dynamic range imaging with real-time GPU-accelerated processing and use this capability to examine the NAD(P)H-related metabolic dynamics of apoptosis in human breast cancer cells. Computational methods for photon counting such as SPEED open up more opportunities for fast and accurate FLIM imaging and additionally provide a basis for future innovation into alternative FLIM techniques.

Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is a widely used technique in biomedical optical imaging. Presently, many two-photon time-domain FLIM setups are limited by long acquisition and postprocessing times that decrease data throughput and inhibit the ability to image fast sub-second processes. Here, we present a versatile two-photon FLIM setup capable of video-rate (up to 25 fps) imaging with graphics processing unit (GPU)-accelerated pixelwise phasor analysis displayed and saved simultaneously with acquisition. The system uses an analog output photomultiplier tube in conjunction with 12-bit digitization at 3.2 GHz to overcome the limited maximum acceptable photon rate associated with the photon counting electronics in many FLIM systems. This allows for higher throughput FLIM acquisition and analysis, and additionally enables the user to assess sample fluorescence lifetime in real-time. We further explore the capabilities of the system to examine the kinetics of Rhodamine B uptake by human breast cancer cells and characterize the effect of pixel dwell time on the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) autofluorescence lifetime estimation accuracy.

Simultaneous 4-phase-shifted full-field optical coherence microscopy.

A new method is presented for full-field optical coherence tomography imaging, which permits capturing single shot phase sensitive imaging through simultaneous acquisition of four phase-shifted images with a single camera using unpolarized light for object illumination. Our method retains the full dynamic range of the camera by using different areas of a single camera sensor to capture each image. We demonstrate the performance of our method by imaging phantoms and live cultures of fibroblast, cancer, and macrophage cells to achieve 59 dB sensitivity with isotropic resolution down to 1 µm, and displacement sensitivity down to 0.1 nm. Our method can serve as a platform for developing high resolution imaging systems because when used in conjunction with broadband spatially incoherent light sources, the resolution is not affected by optical aberrations or speckle noise.

Simultaneous two-photon activation and imaging of neural activity based on spectral-temporal modulation of supercontinuum light.

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.

Full-field spectral-domain optical interferometry for snapshot three-dimensional microscopy.

Prevalent techniques in label-free linear optical microscopy are either confined to imaging in two dimensions or rely on scanning, both of which restrict their applications in imaging subtle biological dynamics. In this paper, we present the theoretical basis along with demonstrations supporting that full-field spectral-domain interferometry can be used for imaging samples in 3D with no moving parts in a single shot. Consequently, we propose a novel optical imaging modality that combines low-coherence interferometry with hyperspectral imaging using a light-emitting diode and an image mapping spectrometer, called Snapshot optical coherence microscopy (OCM). Having first proved the feasibility of Snapshot OCM through theoretical modeling and a comprehensive simulation, we demonstrate an implementation of the technique using off-the-shelf components capable of capturing an entire volume in 5 ms. The performance of Snapshot OCM, when imaging optical targets, shows its capability to axially localize and section images over an axial range of ±10 µm, while maintaining a transverse resolution of 0.8 µm, an axial resolution of 1.4 µm, and a sensitivity of up to 80 dB. Additionally, its performance in imaging weakly scattering live cells shows its capability to not only localize the cells in a densely populated culture but also to generate detailed phase profiles of the structures at each depth for long durations. Consolidating the advantages of several widespread optical microscopy modalities, Snapshot OCM has the potential to be a versatile imaging technique for a broad range of applications.