This PDF file contains the front matter associated with SPIE Proceedings Volume 10499, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Three-dimensional (3D) structured illumination (SI) patterns that include lateral and axial variations have attracted more attention recently as their use in fluorescence microscope enhances the 3D resolution of the native imaging system. 3D SI patterns have already been created by interfering three mutually-coherent waves using a diffraction grating or some electro-optical devices such as spatial light modulators. Here, an interesting approach to generate a 3D SI pattern of tunable modulation frequency is shown. Our proposed illumination system is based on the incoherent illumination of a Fresnel biprism using several equidistant linear sources (i.e., slits). Previously, we investigated and compared numerically this tunable SI microscopy (SIM) system with the one achieved with three-wave interference. In this contribution, we implement our proposed incoherent 3D SIM system of tunable-frequency in an open-setup. We evaluate the axial confinement of the illumination pattern obtained with this system by recording the SI pattern using a mirror sample and different number of slits and compare these data with simulation results. Moreover, we verify that with a higher number of slits used, the axial confinement of the pattern increases, and consequently, the system’s optical sectioning capability improves.

We demonstrate a new method for two-dimensional super-resolution fluorescence imaging. Our method achieves more than 2-fold resolution enhancement over the Rayleigh resolution limit simply with a focused illumination spot and non-negative least-squares inversions, and with practical photon budgets that can be supported by common fluorophores used for biological fluorescence microscopy.

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as ~5 mm³ and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell ’leaders’, migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

Hybrid imaging modalities are becoming more popular since they utilize the benefit of both optical and ultrasound (US) imaging modalities. They use the contrast based on optical properties and negligible scattering of US waves to extend the depth of imaging. Ultrasound modulated optical tomography (UOT) and acoustic radiation force (ARF) with speckle pattern analysis, both use the idea of utilizing a focused US wave to spatially encode in information in the diffused light. We have previously shown that compared to UOT, ARF regime can result in a stronger signal and the mean irradiance change (MIC) signal can reflect the mechanical and thermal properties of the tissue non-invasively. In addition to the mechanical and thermal properties of the medium, the MIC signal is able to reveal information about the morphology of the medium. A tumor is formed by a group of cancer cells that are result of rounds of successive mutation. Cancer cell grow without control in abnormal shapes. In this study, we have modeled cells with their nuclei, assuming that the scattering events occur at the location of the nuclei of the cells. We have shown that, although the MIC signal is not sensitive to the size of the particle, it can detect the presence of the tumor base on the higher concentration of cells in a tumor.

Localization of point sources represents an integral component of microscopy data analysis in applications such as the high-accuracy tracking of single molecules and the high-resolution visualization of subcellular structures labeled with stochastically activated fluorophores. The choice of a suitable method for localization and the customization of the chosen method are both critically dependent on various factors. These factors include the characteristics of the data such as the level of the signal detected from the point source and the types and amounts of noise that are present, experimental design choices such as the optics of the microscope used and the emission wavelength of the fluorophore used, and analysis requirements such as the desired processing throughput and level of localization accuracy. Consequently, the determination and customization of a localization method necessitate experimentation with various considerations, including the underlying optimization algorithm to use, the point spread function model with which to fit a point source, and the computational and algorithmic settings that affect the performance of the localization. As there are numerous combinations to evaluate, software is needed that enables one to efficiently carry out this experimentation. We describe here a software framework and implementation that addresses this important aspect of localization analysis. As a demonstration of this software, we use it to explore ways to improve the throughput of single molecule localization without sacrificing the localization accuracy. In doing so, we also highlight tools in the software that importantly allow the examination of localization results in great detail.

Many single-pixel imaging techniques have been developed in recent years. Though the methods of image acquisition vary considerably, the methods share unifying features that make general analysis possible. Furthermore, the methods developed thus far are based on intuitive processes that enable simple and physically-motivated reconstruction algorithms, however, this approach may not leverage the full potential of single-pixel imaging. We present a general theoretical framework of single-pixel imaging based on frame theory, which enables general, mathematically rigorous analysis. We apply our theoretical framework to existing single-pixel imaging techniques, as well as provide a foundation for developing more-advanced methods of image acquisition and reconstruction. The proposed frame theoretic framework for single-pixel imaging results in improved noise robustness, decrease in acquisition time, and can take advantage of special properties of the specimen under study. By building on this framework, new methods of imaging with a single element detector can be developed to realize the full potential associated with single-pixel imaging.

Previously, a wavefront encoded (WFE) imaging system implemented using a squared cubic (SQUBIC) phase mask has been verified to reduce the sensitivity of the imaging system to spherical aberration (SA). The strength of the SQUBIC phase mask and, as consequence, the performance of the WFE system are controlled by a design parameter, A. Although the higher the A-value, the more tolerant the WFE system is to SA, this is accomplished at the expense of the effective imaging resolution. In this contribution, we investigate this tradeoff in order to find an optimal A-value to balance the effect of SA and loss of resolution.

Thick samples such as embryos present particular challenges to phase imaging. Among these are scattering and phase wrapping. Nevertheless it has been shown that information can be obtained from deep inside living mouse embryos with large numbers of cells. Techniques for collection and processing of images will be discussed.

An overview of single molecule microscopy and relevant notions of resolution will be given.

Collagen organization plays an integral role in many diseases including cancer. Here we introduce a low-cost, open-access collagen imaging and image analysis platform for quantifying fibrillar collagen organization. LC-PolScope was used as the imaging modality that incorporates a precision universal compensator made of two computer controlled liquid crystal variable retarders. This imaging system can be easily implemented on standard microscopes as a cost-effective alternative to second harmonic generation (SHG) imaging and staining on a wide range of available pathology slide formats, including the most commonly used H&E stained slides. In the collagen image analysis, a two-step registration process was first used for overlaying bright-field images on polarized images of collagen: 1) Extract collagenous stroma from H&E bright-field images by image segmentation in HSV color space and performing color separation using K-means clustering algorithm to find the best collagen estimate; 2) Use an iterative intensity-based image registration algorithm to find the affine transform that registers the collagen extracted image to the SHG image at different resolutions. Then, the registered bright field H&E image was used as a guide to evaluate collagen organization near any biological structure such as blood vessels, tumors etc. These algorithms have been implemented in our open source collagen analysis software tool “CurveAlign” package that has been widely used for collagen feature extraction, including detection of tumor associated collagen signatures. As a proof of concept, we are now using this platform to investigate collagen organization in metastatic pancreatic cancer vs non-metastatic pancreatic cancer.

Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information determines the 3D location of single objects at a speed only limited by the array detector’s frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads; the 3D positions and trajectories of single fluorescence beads can be determined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were determined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is superdiffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

Fluorescence lifetime imaging (FLIM) is a technique that allows calculating the fluorescence lifetime at every pixel of an imaged fluorescent sample. The fluorescence lifetime is a property that characterizes each fluorophore and its environment, which makes FLIM a powerful quantitative analytical tool extensively used in a wide range of biomedical applications. In order to fully exploit the potentials of FLIM in the medical field, practical implementations that would enable fast and accurate in vivo FLIM imaging are needed. We present a handheld FLIM system capable of both acquiring and processing time-resolved fluorescence measurements at a pixel rate of at least 30 kHz. The handheld instrument provides a field of view of ~1 cm in diameter with an optical resolution of ~100 μm. Real-time FLIM processing is achieved by means of a bi-exponential model curve fitting algorithm based on a lookup table and pattern recognition techniques. The handheld FLIM system was validated by safely imaging fluorescence standards and the oral mucosa of healthy volunteers. The acquired fluorescence lifetime maps were in agreement with the fluorescence lifetime values estimated using the standard non-linear least square iterative reconvolution method (LSIR). These results demonstrated practical and accurate in vivo video rate FLIM imaging capabilities of this novel handheld FLIM implementation, which would facilitate practical FLIM applications, including clinical ones, such as clinical diagnosis and image guided interventions.

As a non-invasive technique, optical imaging has become a widely used tool in both biological research and clinical diagnostics to investigate biological tissues. A key parameter to consider is the penetration depth of optical imaging in the tissues. Several techniques have been developed to enhance the penetration depth of optical imaging within scattering biological tissues, such as optical coherence microscopy (OCM) and multi-photon microscopy (MPM). Recently, focal modulation microscopy (FMM) has been developed and an imaging depth comparable to these techniques has been achieved. Here, combined with focal modulation techniques, two-photon focal modulation microscopy (TPFMM) is demonstrated theoretically and experimentally. First, TPFMM in turbid media using a novel spatiotemporal phase modulator (STPM) is theoretically investigated using the vector diffraction theory. At the destructive stage during the excitation beam modulation, this STPM is equivalent to a strip-shaped pupil filter with a sinusoidal phase distribution. Compared to the previous filter patterns with sharp phase transitions, the contribution of out-of-focus ballistic excitation to the background is largely reduced using the continuous phase filters. In addition, this new STPM has been designed and integrated into TPFMM to achieve high performance imaging of the biological tissues. It is found that TPFMM using this new STPM can significantly suppress scattered excitation and reduce out-of-focus ballistic excitation with acceptable modulation depth and resolution. Therefore, TPFMM with some new STPMs has the great potential to further extend the penetration depth in imaging the scattering biological tissues.

Microsphere-assisted imaging can be incorporated onto conventional light microscopes allowing wide-field and flourescence imaging with enhanced resolution. We demonstrated that imaging of specimens containing subdiffraction-limited features is achievable through high-index microspheres embedded in a transparent thin film placed over the specimen. We fabricated novel microsphere-embedded microscope slides composed of barium titanate glass microspheres (with diameter ~10-100 μm and refractive index~1.9-2.2) embedded in a transparent polydimethylsiloxane (PDMS) elastomer layer with controllable thickness. We characterized the imaging performance of such microsphere-embedded devices in white-light microscopies, by measuring the imaging resolution, field-of-view, and magnification as a function of microsphere size. Our results inform on the design of novel optical devices, such as microsphere-embedded microscope slides for imaging applications.

Single fluorescent particle tracking (SPT) is a technique often used in the biophysical sciences in order to explore the environment of soft matter [C Manzo et al, Rep Prog Phys 78, 124601 (2015); H Shen et al, Chem Rev 117, 7331–7376 (2017)]. SPT has typically been performed using widefield sample illumination and fluorescent spheres at low concentration, thus limiting the achievable tracking depth and the density of mapped particle trajectories within the sample (and hence the statistical accuracy with which particle motions can be analyzed). In this talk, we propose to use a light sheet fluorescence microscope with photoactivatable carriers for high density SPT in thick samples with depth selectivity. In particular, we will demonstrate the usefulness of this approach for investigating the environments of heterogeneous soft materials, such as agarose gels, with improved statistical accuracy, and for providing precise depth information on the mechanical and dynamical properties of inhomogeneous soft matter. Furthermore, a discussion on the applicability of our method to probe material rheology at the nanometer-scale will be presented.

Current gold-standard histopathology for cancerous biopsies is destructive, time consuming, and limited to 2D slices, which do not faithfully represent true 3D tumor micro-morphology. Light sheet microscopy has emerged as a powerful tool for 3D imaging of cancer biospecimens. Here, we utilize the versatile dual-view inverted selective plane illumination microscopy (diSPIM) to render digital histological images of cancer biopsies. Dual-view architecture enabled more isotropic resolution in X, Y, and Z; and different imaging modes, such as adding electronic confocal slit detection (eCSD) or structured illumination (SI), can be used to improve degraded image quality caused by background signal of large, scattering samples. To obtain traditional H&E-like images, we used DRAQ5 and eosin (D&E) staining, with 488nm and 647nm laser illumination, and multi-band filter sets. Here, phantom beads and a D&E stained buccal cell sample have been used to verify our dual-view method. We also show that via dual view imaging and deconvolution, more isotropic resolution has been achieved for optical cleared human prostate sample, providing more accurate quantitation of 3D tumor architecture than was possible with single-view SPIM methods. We demonstrate that the optimized diSPIM delivers more precise analysis of 3D cancer microarchitecture in human prostate biopsy than simpler light sheet microscopy arrangements.

Confocal Microscopy (CM) enables 3-D high-resolution images acquisition of biological samples by confocal pinhole application. Whereas transverse scanning can be provided by implementation of scanning mirrors, axial scanning can be achieved by moving the sample, which limits the acquisition speed. On the other hand, with coherence gating Optical Coherence Microscopy (OCM) allows to generate micrometer resolution, cross-sectional images and volumetric data on the internal structure of back-scattering objects. The aim of our study was to assess imaging performance of different designs of CM and OCM with tunable lens technology. Mechanical scanning in axial direction was replaced by remote control of the objective focus position with the two types of lenses: Electrically Tunable Lens (ETL; up to 500 Hz) and Acousto-Optic Lens (AOL; 250 kHz). We compared focus tuning ability of ETL and AOL technologies by applying beam profiling and wavefront sensing. We assessed the impact of tunable lens technology on the CM and OCM system performance. We designed the confocal microscopic system with Bessel beam illumination to extend the depth-of-focus. The axial scanning of the collection point will be provided by our AOL. Combination of Bessel beam illumination and AOL allowed high-speed image acquisition from well-controlled depth positions along with better quality of PSF due to self-healing properties of the Bessel beam. We compared image quality of the proposed configurations with the standard design using biological specimens. To conclude, tunable lens technology implemented to CM and OCM instruments enables enhancement of instrument performance.

An interferometric fluorescent microscope and a novel theoretic image reconstruction approach were developed and used to obtain super-resolution images of live biological samples and to enable dynamic real time tracking. The tracking utilizes the information stored in the interference pattern of both the illuminating incoherent light and the emitted light. By periodically shifting the interferometer phase and a phase retrieval algorithm we obtain information that allow localization with sub-2 nm axial resolution at 5 Hz.

Optical scattering properties in tissue are diagnostic markers, ingredients to increasingly sophisticated models, and undergird many optical imaging modalities. Advances in all these areas hinge on obtaining high fidelity scattering measurements. A new optical scattering goniometry method is discussed which measures scattering into 4π sr from small (~100um) tissue regions in flat-mounted samples. This novel tissue scattering gonoimeter images the back focal plane of two opposing microscope objectives to collect light in the forward and backward direction and has several key advantages: (1) scanning the incident angle allows measurement of scattering over 4π steradians to determine the complete scattering phase function of tissue, (2) specificity of measuring scattering from small ~50um regions combined with obliquely sectioned tissue allows mapping of layered tissue, (3) spectral characterization through tuning illumination wavelength, (4) concurrent measurement of scattering coefficient. This opens up the prospect of a new level of detail in the characterization of optical scattering from tissue, including distinguishing properties of thin layers. A tissue system of particular interest and an excellent candidate on which to apply this new goniometry method is the retina. Existing measurements are limited to bulk retina properties or inferred from methodologies that do not have access to transmitted scattering. Scattering coefficient and anisotropy measurements are presented for the various retinal layers. These novel measurements may be used to model light transport and scattering in the retina. Examples of modeling imaging modalities based on scattered light are discussed.

Scanning laser optical tomography (SLOT) is a 3D imaging technique, based on the principle of computed tomography to visualize samples up to magnitude of several centimeters. Intrinsic contrast mechanisms as absorption, scattering and autofluorescence provide information about the 3D architecture and composition of the sample. Another valuable intrinsic contrast mechanism is second harmonic generation (SHG), which is generated in noncentrosymmetric materials and commonly used to image collagen in biological samples. The angular dependence of the SHG signal, however, produces artifacts in reconstructed optical tomography datasets (OPT, SLOT). Thus, successful use of this intrinsic contrast mechanism is impaired. We investigate these artifacts by simulation and experiment and propose an elimination procedure that enables successful reconstruction of SHG-SLOT data. Nevertheless, in many cases specific labeling of certain structures is necessary to make them visible. Using multiple dyes in one sample can lead to crosstalk between the different channels and reduce contrast of the images. Also autofluorescence of the sample itself can account for that. By using multispectral imaging in combination with spectral unmixing techniques, this loss can be compensated. Therefore either a spectrally resolved detection path, or spectrally resolved excitation is required. Therefore we integrated a white supercontinuum light source in our SLOT-setup that enables a spectral selection of the excitation beam and extended the detection path to a four channel setup. This enables the detection of three fluorescence channels and one absorption channel in parallel, and increases the contrast in the reconstructed 3D images significantly.

Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ovarian cancer. To quantify these alterations, we implemented a form of 3D texture analysis method based on textons to delineate the fibrillar morphology observed in 3D Second Harmonic Generation (SHG) microscopy image data of normal (1) and high risk (2) ovarian stroma, (3) benign ovarian tumors, low grade (4) and high grade (5) serous tumors, and endometrioid tumors (6). We developed a tailored set of 3D filters which extract textural features in the 3D image sets to build (or learn) statistical models of each tissue class. By applying k-nearest neighbor classification using these learned models, we achieved 83-91% accuracies for the six classes. The 3D method outperformed the analogous 2D classification by 10-15% on the same tissues, where we suggest this is due the increased information content available in 3D voxels. This classification based on ECM structural changes will complement conventional classification based on genetic profiles and can serve as an additional biomarker. Moreover, the texture analysis algorithm is quite general, as it does not rely on single morphological metrics such as fiber alignment, length, and width but their combined convolution with a customizable basis. We further discuss a new approach to achieve complete 3D SHG imaging, that is based on a rotating multiview platform. We show this visualizes axially oriented features missing in conventional en face imaging. The data sets are compatible with the texture analysis here and will further improve upon this approach.

A patterned microretarder array positioned in the rear conjugate plane of a microscope enables rapid polarizationdependent nonlinear optical microscopy. The pattern introduced to the array results in periodic modulation of the polarization-state of the incident light as a function of position within the field of view with no moving parts or active control. Introduction of a single stationary optical element and a fixed polarizer into the beam of a nonlinear optical microscope enabled nonlinear optical tensor recovery, which informs on local structure and orientation. Excellent agreement was observed between the measured and predicted second harmonic generation (SHG) of z-cut quartz, selected as a test system with well-established nonlinear optical properties. Subsequent studies of spatially varying samples further support the general applicability of this relatively simple strategy for detailed polarization analysis in both conventional and nonlinear optical imaging of structurally diverse samples.

Recently, it has been demonstrated that sectioning within a turbid media using structured illumination microscopy can be greatly enhanced through the use of a Hilbert transform algorithm. This technique, known as single image structured illumination microscopy (SISIM), provides high quality sectioning even within a strongly refracting/scattering medium, such as tissue. It is found that motion artifacts comprise a large portion of the error in in-vivo imaging. Thus, we expand on the SISIM technique by implementing an image registration algorithm which greatly mitigates motion induced artifacts. Multiple images and movies are developed to demonstrate SISIMs ability to provide real-time, in-vivo imaging of human tissue.

Three-dimensional imaging is affected by depth-induced spherical aberration (SA) when imaging deep into an optically thick sample. In this work, we evaluate the impact of SA on the performance of incoherent grating-projection structured illumination microscopy (SIM). In particular, we analyze the reduction of the contrast in the structured pattern and compare the reconstructed SIM images for different amounts of SA. In order to mitigate the impact of SA, we implement and evaluate in SIM a wavefront encoded imaging system using a square cubic (SQUBIC) phase mask, an approach shown previously to be successful in conventional microscopy.

Coded aperture imaging for 3D reconstructions have been applied in both photography and microscopy to successfully recover depth information from images captured with a patterned pupil plane. Generally, single-shot coded aperture microscopy methods require the 3D sample to be extremely sparse and with no overlapping features. Here, we extend coded aperture microscopy methods to multi-shot 3D fluorescent imaging with wave-optical forward models, enabling thicker and denser 3D samples to be faithfully reconstructed with efficient data capture via compressed sensing and advanced inverse algorithms.. We demonstrate successful reconstruction of a 500um thick brine shrimp sample. We further study the minimum required number of codes to capture full fluorescent information under wide-field illumination The experiments are carried out with a 4f system and a spatial light modulator in the Fourier (aperture) domain to display the codes.

Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

Publisher’s Note: This conference presentation, originally published on 3/21/18, was withdrawn per author request on 3/21/18.

Fluid-structure interaction in the developing heart is an active area of research in developmental biology. However, investigation of heart dynamics is mostly limited to computational uid dynamics simulations using heart wall structure information only, or single plane blood ow information - so there is a need for 3D + time resolved data to fully understand cardiac function. We present an imaging platform combining selective plane illumination microscopy (SPIM) with micro particle image velocimetry (μPIV) to enable 3D-resolved flow mapping in a microscopic environment, free from many of the sources of error and bias present in traditional epi uorescence-based μPIV systems. By using our new system in conjunction with optical heart beat synchronization, we demonstrate the ability obtain non-invasive 3D + time resolved blood flow measurements in the heart of a living zebrafish embryo.

How do you use imaging to analyse the development of the heart, which not only changes shape but also undergoes constant, high-speed, quasi-periodic changes? We have integrated ideas from prospective and retrospective optical gating to capture long-term, phase-locked developmental time-lapse videos. In this paper we demonstrate the success of this approach over a key developmental time period: heart looping, where large changes in heart shape prevent previous prospective gating approaches from capturing phase- locked videos. We use the comparison with other approaches to in vivo heart imaging to highlight the importance of collecting the most appropriate data for the biological question.

Hemodynamics is a critical factor for healthy embryonic and fetal development, and when altered, could result in congenital heart defects (CHD), the most common birth defect in the newborns. Previous studies have shown that the fluid mechanical forces in the blood flow during early cardiac development could influence overall morphogenesis of cardiovascular system. Though near-infrared light (NIR) point-scan OCT has been used to quantitatively assess the hemodynamics in the embryo, high speed visualization of the developing chicken embryo is still lacking. Here, we developed a line-scanning NIR OCT for high speed visualization of chicken embryo hemodynamics. The line-scanning approach also lowered the threshold of maximal exposure limit for the power delivered to the samples. The supercontinuum light source, with the output filtered to harness NIR wavelengths between 600 – 950 nm, will be used in the system. The noise performance of the supercontinuum light source will be characterized across different pulse repetition rates, camera exposure time, and wavelengths. A careful design of the spectrometer employing a low noise two-dimensional CMOS camera will be performed in order to optimize the maximal sensitivity and sensitivity rolloff. The effective performance of the line-scan OCT system will be compared to a point-scan OCT counterpart in term of maximal sensitivity, imaging speed, and contrast by imaging developing chicken embryo. The structural and functional information of dynamic cardiac tissue deformation and blood flow in ultrahigh spatiotemporal resolution will further enhance our understanding of the roles of hemodynamics and in embryonic development and in CHD.

Fluorescence microscopy is an essential tool in bio-imaging, yet there are no widely adopted standards for the calibration of fluorescent microscopes. Calibration provides a wide range of information relating to microscope performance. Without calibration, images taken on two separate microscopes cannot be directly compared as they may have differing magnifications, illumination intensities or detector sensitivities. As the range of microscopy techniques capturing 3D information continues to increase, the need for standardisation becomes ever greater. Widely used methods for determining microscope performance are currently limited to basic techniques such as fluorescent beads, which don’t form a regularly spaced pattern and reflective etched gratings, which are limited to being two-dimensional and require changes to the microscope filter sets. Using ultrafast laser processing inside plastic substrates, we demonstrate the generation of bright fluorescent patterns in three dimensions offering new possibilities for calibration in fluorescence microscopy. The fabricated calibration slides can be used to quantify a range of parameters that determine microscope performance. For example, spatial distortions within the field of view can be quantified by a regular array of bright fluorescent points. Other patterns can determine factors such as detector linearity, field flatness and changes in the point spread function across the field of view and over depth. The patterns can additionally be used to calibrate spatial length-scales and for colour channel registration.

Light-sheet microscopy has evolved as an indispensable tool in imaging biological samples. It can image 3D samples at fast speed, with high-resolution optical sectioning, and with reduced photobleaching effects. These properties make light-sheet microscopy ideal for imaging fluorophores in a variety of biological samples and organisms, e.g. zebrafish, drosophila, cleared mouse brains, etc. While most commercial turnkey light-sheet systems are expensive, the existing lower cost implementations, e.g. OpenSPIM, are focused on achieving high-resolution imaging of small samples or organisms like zebrafish. In this work, we substantially reduce the cost of light-sheet microscope system while targeting to image much larger samples, i.e. cleared mouse brains, at single-cell resolution. The expensive components of a lightsheet system – excitation laser, water-immersion objectives, and translation stage – are replaced with an incoherent laser diode, dry objectives, and a custom-built Arduino-controlled translation stage. A low-cost CUBIC protocol is used to clear fixed mouse brain samples. The open-source platforms of μManager and Fiji support image acquisition, processing, and visualization. Our system can easily be extended to multi-color light-sheet microscopy.

Optical clearing is a useful tool for investigating large biological tissues in 3D, but it has not been widely adapted in regular biomedical research community, partially due to the high complexity and low speed of the current optical clearing methods. Therefore, we developed an optical clearing technique, termed lipid-preserving index matching for prolonged imaging depth (LIMPID), that simplifies the clearing procedure while maintaining the advantages of the state of the art clearing methods. (1) LIMPID is designed as an aqueous solution that directly diffuses into the tissue and makes the refractive indices uniform. It is capable of clearing the tissue in a single step, simply by immersing fixed and pre-labeled samples in the clearing solution. In contrast, most current clearing techniques involve multiple steps and some of steps are complicated and time consuming. (2) LIMPID clears the tissue quickly. The solution has low viscosity and rapidly diffuses into the tissue at room temperature. For samples with submillimeter thickness, it clears the tissue within an hour. Clearing times for larger samples are also impressive. (3) LIMPID preserves fluorescence and tissue morphology while maintaining high transparency. No dehydration, organic solvent exchange or lipid extraction is required. We have used LIMPID to study several animal and disease models. For instance, it revealed abnormal peripheral nerve innervation in the embryonic quail heart in a fetal alcohol syndrome model. We believe this simple, quick method with no discernable disadvantages could become the optical clearing protocol of choice for many microscopy applications.

We report a miniature head mounted two-photon fiber-coupled microscope (TP-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. Full three-dimensional two-photon imaging of GCaMP6s showing individual neuron activity in multiple focal planes was achieved in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is demonstrated. Additionally, the axial scanning of the electrowetting lens allows dynamic control of tilt to the focal plane allowing rapid scanning of different regions of interest in three dimensions. Two-photon imaging allows increased penetration depth in tissue with a field-of-view of 240 μm diameter and 200 μm variable axial focus. The TP-FCM has a light-weight design (~4 g) and excellent image stability. TP-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening up unique opportunities in neuroscience research.

We describe a quantitative fluorescence projection tomography technique which measures the three-dimensional fluorescence spectrum in biomedical samples with size up to several millimeters. This is achieved by acquiring a series of hyperspectral images, by using laser scanning scheme, at different projection angles. We demonstrate that this technique provide a quantitative measure of the fluorescence signal by comparing the spectrum and intensity profile of a fluorescent bead phantom and also demonstrate its application to differentiating the extrinsic label and the autofluorescence in a mouse embryo.

The implementation of time domain fluorescence lifetime imaging (TD-FLIM) requires high cost bandwidth electronics and large storage capacity. A cross-level sampling technique for TD-FLIM is proposed. A simulation of this sampling technique was implemented using synthetic FLIM data. FLIM images were synthetically generated at different noise levels using a wide range of lifetimes. Each of the images was resampled using a cross-level approach and the lifetime maps were computed. Simulation results displayed strong correlation between the lifetime maps of the original and resampled images, suggesting that this sampling method could be adopted to reduce bandwidth and data transfer/ storage requirements.

The estimation of the Point Spread Function (PSF) of an imaging system is important for various post acquisition processes. The PSF can be estimated by knowing the optical arrangement of the imaging system or can be obtained by using a point object. Both the techniques have their own limitations. In this paper we propose a new PSF estimation technique based on a target that can be reconfigured programmably. We will show that a target with different illumination areas can be imaged to establish a relation between the image plane and the object plane via a PSF. The relation thus allows one to estimate the PSF of the imaging system.

The high resolution applications of a laser scanning imaging system very much demand the accurate positioning of the illumination beam. The galvanometer scanner based beam scanning imaging systems, on the other hand, suffer from both short term and long term beam instability issues. Fortunately Computer generated holography based beam scanning offers extremely accurate beam steering, which can be very useful for imaging in high-resolution applications in confocal microscopy. The holographic beam scanning can be achieved by writing a sequence of holograms onto a spatial light modulator and utilizing one of the diffracted orders as the illumination beam. This paper highlights relative advantages of such a holographic beam scanning based confocal system and presents some of preliminary experimental results.

Light field technique at a single shot can get the whole volume image of observed sample. Therefore, the original frame rate of the optical system can be taken as the volumetric image rate. For dynamically imaging whole micron-scale biosample, a light field microscope with temporal focusing illumination has been developed. In the light field microscope, the f-number of the microlens array (MLA) is adopted to match that of the objective; hence, the subimages via adjacent lenslets do not overlay each other. A three-dimensional (3D) deconvolution algorithm is utilized to deblur the out-of-focusing part. Conventional light field microscopy (LFM) illuminates whole volume sample even noninteresting parts; nevertheless, whole volume excitation causes even more damage on bio-sample and also increase the background noise from the out of range. Therefore, temporal focusing is integrated into the light field microscope for selecting the illumination volume. Herein, a slit on the back focal plane of the objective is utilized to control the axial excitation confinement for selecting the illumination volume. As a result, the developed light field microscope with the temporal focusing multiphoton illumination (TFMPI) can reconstruct 3D images within the selected volume, and the lateral resolution approaches to the theoretical value. Furthermore, the 3D Brownian motion of two-micron fluorescent beads is observed as the criterion of dynamic sample. With superior signal-to-noise ratio and less damage to tissue, the microscope is potential to provide volumetric imaging for vivo sample.

Non-invasive imaging technologies, such as magnetic resonance imaging (MRI) and optical multimodality imaging methods, are commonly used for diagnosing and supervising the development of inflammatory bowel disease (IBD). These in vivo imaging methods can provide morphology changes information of IBD in macro-scale. However, it is difficult to investigate the intestinal wall in molecular and cellular level. State-of-art light-sheet and two-photon microscopy have the ability to acquire the changes for IBD in micro-scale. The aim of this work is to evaluate the size of the enterocoel and the thickness of colon wall using both MRI for in vivo imaging, and light-sheet and two-photon microscope for in vitro imaging. C57BL/6 mice were received 3.5% Dextran sodium sulfate (DSS) in the drinking water for 5 days to build IBD model. Mice were imaged with MRI on days 0, 6 to observe colitis progression. After MRI imaging, the mice were sacrificed to take colons for tissue clearing. Then, light-sheet and two-photon microscopies are used for in vitro imaging of the cleared samples. The experimental group showed symptoms of bloody stools, sluggishness and weight loss. It showed that the colon wall was thicker while the enterocoel was narrower compare to control group. The more details are observed using light-sheet and two-photon microscope. It is demonstrated that hybrid of MRI in macro-scale and light-sheet and two-photon microscopy in micro-scale imaging is feasible for colon inflammation diagnosing and supervising.

Whole-brain imaging is challenging because it demands microscopes with high temporal and spatial resolution, which are often at odds, especially in the context of large fields of view. We have designed and built a light-sheet microscope with digital micromirror illumination and light-field detection. On the one hand, light sheets provide high resolution optical sectioning on live samples without compromising their viability. On the other hand, light field imaging makes it possible to reconstruct full volumes of relatively large fields of view from a single camera exposure; however, its enhanced temporal resolution comes at the expense of spatial resolution, limiting its applicability. We present an approach to increase the resolution of light field images using DMD-based light sheet illumination. To that end, we develop a method to produce synthetic resolution targets for light field microscopy and a procedure to correct the depth at which planes are refocused with rendering software. We measured the axial resolution as a function of depth and show a three-fold potential improvement with structured illumination, albeit by sacrificing some temporal resolution, also three-fold. This results in an imaging system that may be adjusted to specific needs without having to reassemble and realign it. This approach could be used to image relatively large samples at high rates.

In digital holography, it is critical to know the distance in order to reconstruct the multi-sectional object. This autofocusing is traditionally solved by reconstructing a stack of in-focus and out-of-focus images and using some focus metric, such as entropy or variance, to calculate the sharpness of each reconstructed image. Then the distance corresponding to the sharpest image is determined as the focal position. This method is effective but computationally demanding and time-consuming. To get an accurate estimation, one has to reconstruct many images. Sometimes after a coarse search, a refinement is needed. To overcome this problem in autofocusing, we propose to use deep learning, i.e., a convolutional neural network (CNN), to solve this problem. Autofocusing is viewed as a classification problem, in which the true distance is transferred as a label. To estimate the distance is equated to labeling a hologram correctly. To train such an algorithm, totally 1000 holograms are captured under the same environment, i.e., exposure time, incident angle, object, except the distance. There are 5 labels corresponding to 5 distances. These data are randomly split into three datasets to train, validate and test a CNN network. Experimental results show that the trained network is capable of predicting the distance without reconstructing or knowing any physical parameters about the setup. The prediction time using this method is far less than traditional autofocusing methods.

Large field of view multispectral imaging through scattering medium is a fundamental quest in optics community. It has gained special attention from researchers in recent years for its wide range of potential applications. However, the main bottlenecks of the current imaging systems are the requirements on specific illumination, poor image quality and limited field of view. In this work, we demonstrated a single-shot high-resolution colour-imaging through scattering media using a monochromatic camera. This novel imaging technique is enabled by the spatial, spectral decorrelation property and the optical memory effect of the scattering media. Moreover the use of deconvolution image processing further annihilate above-mentioned drawbacks arise due iterative refocusing, scanning or phase retrieval procedures.

The restored images in structured illumination microscopy (SIM) can be affected by residual fringes due to a mismatch between the illumination pattern and the sinusoidal model assumed by the restoration method. When a Fresnel biprism is used to generate a structured pattern, this pattern cannot be described by a pure sinusoidal function since it is distorted by an envelope due to the biprism’s edge. In this contribution, we have investigated the effect of the envelope on the restored SIM images and propose a computational method in order to address it. The proposed approach to reduce the effect of the envelope consists of two parts. First, the envelope of the structured pattern, determined through calibration data, is removed from the raw SIM data via a preprocessing step. In the second step, a notch filter is applied to the images, which are restored using the well-known generalized Wiener filter, to filter any residual undesired fringes. The performance of our approach has been evaluated numerically by simulating the effect of the envelope on synthetic forward images of a 6-μm spherical bead generated using the real pattern and then restored using the SIM approach that is based on an ideal pure sinusoidal function before and after our proposed correction method. The simulation result shows 74% reduction in the contrast of the residual pattern when the proposed method is applied. Experimental results from a pollen grain sample also validate the proposed approach.

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.

The Bessel beam belongs to a typical class of non-diffractive optical fields that are characterized by their invariant focal profiles along the propagation direction. However, ideal Bessel beams only rigorously exist in theory; Bessel beams generated in the lab are quasi-Bessel beams with finite focal extensions and varying intensity profiles along the propagation axis. The ability to engineer the on-axis intensity profile to the desired shape is essential for many applications. Here we demonstrate an iterative optimization-based approach to engineering the on-axis intensity of Bessel beams. The genetic algorithm is used to demonstrate this approach. Starting with a traditional axicon phase mask, in the design process, the computed on-axis beam profile is fed into a feedback tuning loop of an iterative optimization process, which searches for an optimal radial phase distribution that can generate a generalized Bessel beam with the desired onaxis intensity profile. The experimental implementation involves a fine-tuning process that adjusts the originally targeted profile so that the optimization process can optimize the phase mask to yield an improved on-axis profile. Our proposed method has been demonstrated in engineering several zeroth-order Bessel beams with customized on-axis profiles. High accuracy and high energy throughput merit its use in many applications.

A V-shape Bio-Transfer-Standard (V-BTS), developed and produced at the University of Helsinki (UH), was measured in two laboratories. In comparison to Siemens Star calibration specimens, the V-BTS performs better at high lateral frequencies close to the diffraction limit of the optical instrument. This permits determining of the Instrument Transfer Function (ITF). The V-BTS features two lipid bilayer steps that partly overlap each other at an angle of 20°, with an average height of 4.6 ± 0.1 nm. The Round Robin (RR) test aims to determine whether the V-BTS and the developed application protocol work with different optical profilers in different laboratories. First the artefact was measured at Sensofar-Tech, S.L. using an S-neox profiler working in Phase Shifting Interferometry mode. Then V-BTS was measured at UH using a custom-built Scanning White Light Interferometer. All measurements done by four different operators at the two laboratories have a range or standard deviation of ±0.1 nm which agrees with the theoretical estimates and with measurements done using an atomic force microscope and with a surface plasmon resonance based instrument. The RR results show the applicability of the V-BTS for calibration and for ITF characterization of 3D optical profilers.