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.

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.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

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.

Fluorescent nanodiamonds for characterization of nonlinear microscopy systems.

Characterizing the performance of fluorescence microscopy and nonlinear imaging systems is an essential step required for imaging system optimization and quality control during longitudinal experiments. Emerging multimodal nonlinear imaging techniques require a new generation of microscopy calibration targets that are not susceptible to bleaching and can provide a contrast across the multiple modalities. Here, we present a nanodiamond-based calibration target for microscopy, designed for facilitating reproducible measurements at the object plane. The target is designed to support day-to-day instrumentation development efforts in microscopy laboratories. The images of a phantom contain information about the imaging performance of a microscopy system across multiple spectral windows and modalities. Since fluorescent nanodiamonds are not prone to bleaching, the proposed imaging target can serve as a standard, shelf-stable sample to provide rapid reference measurements for ensuring consistent performance of microscopy systems in microscopy laboratories and imaging facilities.

In vivo characterization of minipig skin as a model for dermatological research using multiphoton microscopy.

Minipig skin is one of the most widely used non-rodent animal skin models for dermatological research. A thorough characterization of minipig skin is essential for gaining deeper understanding of its structural and functional similarities with human skin. In this study, three-dimensional (3-D) in vivo images of minipig skin was obtained non-invasively using a multimodal optical imaging system capable of acquiring two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy (FLIM) images simultaneously. The images of the structural features of different layers of the minipig skin were qualitatively and quantitatively compared with those of human skin. Label-free imaging of skin was possible due to the endogenous fluorescence and optical properties of various components in the skin such as keratin, nicotinamide adenine dinucleotide phosphate (NAD(P)H), melanin, elastin, and collagen. This study demonstrates the capability of optical biopsy techniques, such as TPEF and FLIM, for in vivo non-invasive characterization of cellular and functional features of minipig skin, and the optical image-based similarities of this commonly utilized model of human skin. These optical imaging techniques have the potential to become promising tools in dermatological research for developing a better understanding of animal skin models, and for aiding in translational pre-clinical to clinical studies.

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.

Automated fast computational adaptive optics for optical coherence tomography based on a stochastic parallel gradient descent algorithm.

The transverse resolution of optical coherence tomography is decreased by aberrations introduced from optical components and the tested samples. In this paper, an automated fast computational aberration correction method based on a stochastic parallel gradient descent (SPGD) algorithm is proposed for aberration-corrected imaging without adopting extra adaptive optics hardware components. A virtual phase filter constructed through combination of Zernike polynomials is adopted to eliminate the wavefront aberration, and their coefficients are stochastically estimated in parallel through the optimization of the image metrics. The feasibility of the proposed method is validated by a simulated resolution target image, in which the introduced aberration wavefront is estimated accurately and with fast convergence. The computation time for the aberration correction of a 512 × 512 pixel image from 7 terms to 12 terms requires little change, from 2.13 s to 2.35 s. The proposed method is then applied for samples with different scattering properties including a particle-based phantom, ex-vivo rabbit adipose tissue, and in-vivo human retina photoreceptors, respectively. Results indicate that diffraction-limited optical performance is recovered, and the maximum intensity increased nearly 3-fold for out-of-focus plane in particle-based tissue phantom. The SPGD algorithm shows great potential for aberration correction and improved run-time performance compared to our previous Resilient backpropagation (Rprop) algorithm when correcting for complex wavefront distortions. The fast computational aberration correction suggests that after further optimization our method can be integrated for future applications in real-time clinical imaging.

Development of a fast calibration method for image mapping spectrometry.

An image mapping spectrometer (IMS) is a snapshot hyperspectral imager that simultaneously captures both the spatial (x, y) and spectral (λ) information of incoming light. The IMS maps a three-dimensional (3D) datacube (x, y, λ) to a two-dimensional (2D) detector array (x, y) for parallel measurement. To reconstruct the original 3D datacube, one must construct a lookup table that connects voxels in the datacube and pixels in the raw image. Previous calibration methods suffer from either low speed or poor image quality. We herein present a slit-scan calibration method that can significantly reduce the calibration time while maintaining high accuracy. Moreover, we quantitatively analyzed the major artifact in the IMS, the striped image, and developed three numerical methods to correct for it.

Assessing the severity of psoriasis through multivariate analysis of optical images from non-lesional skin.

Patients with psoriasis represent a heterogeneous population with individualized disease expression. Psoriasis can be monitored through gold standard histopathology of biopsy specimens that are painful and permanently scar. A common associated measure is the use of non-invasive assessment of the Psoriasis Area and Severity Index (PASI) or similarly derived clinical assessment based scores. However, heterogeneous manifestations of the disease lead to specific PASI scores being poorly reproducible and not easily associated with clinical severity, complicating the efforts to monitor the disease. To address this issue, we developed a methodology for non-invasive automated assessment of the severity of psoriasis using optical imaging. Our analysis shows that two-photon fluorescence lifetime imaging permits the identification of biomarkers present in both lesional and non-lesional skin that correlate with psoriasis severity. This ability to measure changes in lesional and healthy-appearing skin provides a new pathway for independent monitoring of both the localized and systemic effects of the disease. Non-invasive optical imaging was conducted on lesions and non-lesional (pseudo-control) skin of 33 subjects diagnosed with psoriasis, lesional skin of 7 subjects diagnosed with eczema, and healthy skin of 18 control subjects. Statistical feature extraction was combined with principal component analysis to analyze pairs of two-photon fluorescence lifetime images of stratum basale and stratum granulosum layers of skin. We found that psoriasis is associated with biochemical and structural changes in non-lesional skin that can be assessed using clinically available two-photon fluorescence lifetime microscopy systems.

IsoSense: frequency enhanced sensorless adaptive optics through structured illumination.

We present IsoSense, a wavefront sensing method that mitigates sample dependency in image-based sensorless adaptive optics applications in microscopy. Our method employs structured illumination to create additional high spatial frequencies in the image through custom illumination patterns. This improves the reliability of image quality metric calculations and enables sensorless wavefront measurement even in samples with sparse spatial frequency content. We demonstrate the feasibility of IsoSense for aberration correction in a deformable-mirror-based structured illumination super-resolution fluorescence microscope.

Rapid adaptive remote focusing microscope for sensing of volumetric neural activity.

The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

Closed loop tracked Doppler optical coherence tomography based heart monitor for the Drosophila melanogaster larvae.

This paper presents a novel instrument for biosciences, useful for studies of moving embryos. A dual sequential imaging/measurement channel is assembled via a closed-loop tracking architecture. The dual channel system can operate in two regimes: (i) single-point Doppler signal monitoring or (ii) fast 3-D swept source OCT imaging. The system is demonstrated for characterizing cardiac dynamics in Drosophila melanogaster larva. Closed loop tracking enables long term in vivo monitoring of the larvae heart without anesthetic or physical restraint. Such an instrument can be used to measure subtle variations in the cardiac behavior otherwise obscured by the larvae movements. A fruit fly larva (top) was continuously tracked for continuous remote monitoring. A heartbeat trace of freely moving larva (bottom) was obtained by a low coherence interferometry based doppler sensing technique.

Aberrations and adaptive optics in super-resolution microscopy.

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy - or rather nanoscopy - to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem.

Multiplexing-based polarization sensitive en-face optical coherence tomography.

We present a time-domain polarization-sensitive (PS) optical coherence tomography configuration operating at 830 nm, equipped with multichannel acousto-optic deflectors and single photodetectors. The system is used to simultaneously acquire interference information from multiple PS channels and to enable measurement and imaging of backscattered intensity to create both PS and polarization insensitive images. Our approach enables multiple channel imaging without need to divide the object signal. Here, we employ our system in order to demonstrate PS imaging of a thermally damaged muscle tissue.

Frequency multiplexed long range swept source optical coherence tomography.

We present a novel swept source optical coherence tomography configuration, equipped with acousto-optic deflectors that can be used to simultaneously acquire multiple B-scans originating from different depths. The sensitivity range of the configuration is evaluated while acquiring five simultaneous B-scans. Then the configuration is employed to demonstrate long range B-scan imaging by combining two simultaneous B-scans from a mouse head sample.

Simultaneous multiple-depths en-face optical coherence tomography using multiple signal excitation of acousto-optic deflectors.

We present a novel low-coherence interferometer configuration, equipped with acousto-optic deflectors that can be used to simultaneously acquire up to eight time domain optical coherence tomography en-face images. The capabilities of the configuration are evaluated in terms of depth resolution, signal to noise ratio and crosstalk. Then the configuration is employed to demonstrate simultaneous en-face optical coherence tomography imaging at five different depths in a specimen of armadillidium vulgare.