In vivo monitoring the dynamic process of acute retinal hemorrhage and repair in zebrafish with spectral‐domain optical coherence tomography

Retina, the only light sensor in the human eye, is hidden and extremely fragile. Optimized animal models and efficient imaging techniques are very important for the study of retinopathy. In this work, the rapid retinal injury process and the long‐term retinal repair process were in vivo continuously evaluated with a novel imaging technology spectral‐domain optical coherence tomography (SD‐OCT) in a unique animal model zebrafish. Acute retinal injury was constructed on adult zebrafish by needle injection surgery. SD‐OCT imaging was carried out immediately after the mechanical injury. The retinal hemorrhage, which lasted only 5 seconds, could be visualized dynamically by SD‐OCT. The process of blood clearance and retinal repair was also evaluated because SD‐OCT imaging is nondestructive. Both SD‐OCT imaging results and behavioral analyzing results demonstrated that zebrafish retina could be repaired by itself within 15 days, which was confirmed by the results of pathological experiment.     

Multicontrast endomyocardial imaging by single‐channel high‐resolution cross‐polarization optical coherence tomography

A single‐channel high‐resolution cross‐polarization (CP) optical coherence tomography (OCT) system is presented for multicontrast imaging of human myocardium in one‐shot measurement. The intensity and functional contrasts, including the ratio between the cross‐ and co‐polarization channels as well as the cumulative retardation, are reconstructed from the CP‐OCT readout. By comparing the CP‐OCT results with histological analysis, it is shown that the system can successfully delineate microstructures in the myocardium and differentiate the fibrotic myocardium from normal or ablated myocardium based on the functional contrasts provided by the CP‐OCT system. The feasibility of using A‐line profiles from the 2 orthogonal polarization channels to identify fibrotic myocardium, normal myocardium and ablated lesion is also discussed.      

Measuring polarization changes in the human outer retina with polarization‐sensitive optical coherence tomography

Morphological changes in the outer retina such as drusen are established biomarkers to diagnose age‐related macular degeneration. However, earlier diagnosis might be possible by taking advantage of more subtle changes that accompany tissues that bear polarization‐altering properties. To test this hypothesis, we developed a method based on polarization‐sensitive optical coherence tomography with which volumetric data sets of the macula were obtained from 10 young (<25 years) and 10 older (>54 years) subjects. All young subjects and 5 of the older subjects had retardance values induced by the retinal pigment epithelium and Bruch's membrane (RPE‐BM) complex that were just above the noise floor measurement (5°‐13° at 840 nm). In contrast, elevated retardance, up to 180°, was observed in the other 5 older subjects. Analysis of the degree of polarization uniformity (DOPU) demonstrates that reduced DOPU (<0.4) in the RPE is associated with elevated double pass phase retardation (DPPR) below the RPE‐BM complex, suggesting that the observed elevated DPPR in older subjects is the result of increased scattering or polarization scrambling. Collectively, our measurements show that the outer retina can undergo dramatic change in its polarization properties with age, and in some cases still retain its clinically normal appearance.      

Label‐free optical projection tomography for quantitative three‐dimensional anatomy of mouse embryo

Recent progress in three‐dimensional optical imaging techniques allows visualization of many comprehensive biological specimens. Optical clearing methods provide volumetric and quantitative information by overcoming the limited depth of light due to scattering. However, current imaging technologies mostly rely on the synthetic or genetic fluorescent labels, thus limits its application to whole‐body visualization of generic mouse models. Here, we report a label‐free optical projection tomography (LF‐OPT) technique for quantitative whole mouse embryo imaging. LF‐OPT is based on the attenuation contrast of light rather than fluorescence, and it utilizes projection imaging technique similar to computed tomography for visualizing the volumetric structure. We demonstrate this with a collection of mouse embryo morphologies in different stages using LF‐OPT. Additionally, we extract quantitative organ information applicable toward high‐throughput phenotype screening. Our results indicate that LF‐OPT can provide multi‐scale morphological information in various tissues including bone, which can be difficult in conventional optical imaging technique.     

Quantitative and rapid estimations of human sub‐surface skin mass using ultra‐high‐resolution spectral domain optical coherence tomography

Non‐invasive and quantitative estimations for the delineation of sub‐surface tumor margins could greatly aid in the early detection and monitoring of the morphological appearances of tumor growth, ensure complete tumor excision without the unnecessary sacrifice of healthy tissue, and facilitate post‐operative follow‐up for recurrence. In this study, a high‐speed, non‐invasive, and ultra‐high‐resolution spectral domain optical coherence tomography (UHR‐SDOCT) imaging platform was developed for the quantitative measurement of human sub‐surface skin mass. With a proposed robust, semi‐automatic analysis, the system can rapidly quantify lesion area and shape regularity by an en‐face‐oriented algorithm. Various sizes of nylon sutures embedded in pork skin were used first as a phantom to verify the accuracy of our algorithm, and then in vivo, feasibility was proven using benign human angiomas and pigmented nevi. Clinically, this is the first step towards an automated skin lesion measurement system.

In vivo optical coherence tomography (OCT) image of angioma (A). Thin red arrows point to a blood vessel (BV).     

Hyperspectral optical coherence tomography for in vivo visualization of melanin in the retinal pigment epithelium

Previous studies for melanin visualization in the retinal pigment epithelium (RPE) have exploited either its absorption properties (using photoacoustic tomography or photothermal optical coherence tomography [OCT]) or its depolarization properties (using polarization sensitive OCT). However, these methods are only suitable when the melanin concentration is sufficiently high. In this work, we present the concept of hyperspectral OCT for melanin visualization in the RPE when the concentration is low. Based on white light OCT, a hyperspectral stack of 27 wavelengths (440‐700 nm) was created in post‐processing for each depth‐resolved image. Owing to the size and shape of the melanin granules in the RPE, the variations in backscattering coefficient as a function of wavelength could be identified—a result which is to be expected from Mie theory. This effect was successfully identified both in eumelanin‐containing phantoms and in vivo in the low‐concentration Brown Norway rat RPE.      

Retinal pulse wave velocity measurement using spectral‐domain optical coherence tomography

The human eyes provide a natural window for noninvasive measurement of the pulse wave velocity (PWV) of small arteries. By measuring the retinal PWV, the stiffness of small arteries can be assessed, which may better detect early vascular diseases. Therefore, retinal PWV measurement has attracted increasing attention. In this study, a jump‐scanning method was proposed for noninvasive measurement of retinal PWV using spectral‐domain optical coherence tomography (SD‐OCT). The jump‐scanning method uses the phase‐resolved Doppler OCT to obtain the pulse shapes. To realize PWV measurement, the jump‐scanning method extracts the transit time of the pulse wave from an original OCT scanning site to another through a transient jump. The measured retinal arterial PWV of a young human subject with normal blood pressure was in the order of 20 to 30 mm/s, which was consistent with previous studies. As a comparison, PWV of 50 mm/s was measured for a young human subject with prehypertension, which was in accordance with the finding of strong association between retinal PWV and blood pressure. In summary, it is believed the proposed jump‐scanning method could benefit the research and diagnosis of vascular diseases through the window of human eyes.      

Dual‐polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells

Raman spectral imaging is gaining more and more attention in biological studies because of its label‐free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information.

The dual‐polarization Raman images of a HeLa cell.     

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Imaging tissue samples by polarization‐resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label‐free manner. Polarization‐resolved second harmonic generation microscopy goes beyond simple intensity‐based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization‐resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas.

SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.     

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Image‐derived arterial input function for quantitative fluorescence imaging of receptor‐drug binding in vivo

Receptor concentration imaging (RCI) with targeted‐untargeted optical dye pairs has enabled in vivo immunohistochemistry analysis in preclinical subcutaneous tumors. Successful application of RCI to fluorescence guided resection (FGR), so that quantitative molecular imaging of tumor‐specific receptors could be performed in situ, would have a high impact. However, assumptions of pharmacokinetics, permeability and retention, as well as the lack of a suitable reference region limit the potential for RCI in human neurosurgery. In this study, an arterial input graphic analysis (AIGA) method is presented which is enabled by independent component analysis (ICA). The percent difference in arterial concentration between the image‐derived arterial input function (AIF_ICA) and that obtained by an invasive method (ICA_CAR) was 2.0 ± 2.7% during the first hour of circulation of a targeted‐untargeted dye pair in mice. Estimates of distribution volume and receptor concentration in tumor bearing mice (n = 5) recovered using the AIGA technique did not differ significantly from values obtained using invasive AIF measurements (p = 0.12). The AIGA method, enabled by the subject‐specific AIF_ICA, was also applied in a rat orthotopic model of U‐251 glioblastoma to obtain the first reported receptor concentration and distribution volume maps during open craniotomy.

     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

A simple approach for non‐invasive transcranial optical vascular imaging (nTOVI)

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non‐invasive optical‐based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non‐invasive optical‐based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro‐imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models.

An example of 1 × 1.5 cm color‐coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.     

In vivo blind‐deconvolution photoacoustic ophthalmoscopy with total variation regularization

Photoacoustic ophthalmoscopy (PAOM) is capable of noninvasively imaging anatomic and functional information of the retina in living rodents. However, the strong ocular aberration in rodent eyes and limited ultrasonic detection sensitivity affect PAOM's spatial resolution and signal‐to‐noise ratio (SNR) in in vivo eyes. In this work, we report a computational approach to combine blind deconvolution (BD) algorithm with a regularizing constraint based on total variation (BDTV) for PAOM imaging restoration. We tested the algorithm in retinal and choroidal microvascular images in albino rat eyes. The algorithm improved PAOM's lateral resolution by around 2‐fold. Moreover, it enabled the improvement in imaging SNR for both major vessels and capillaries, and realized the well‐preserved blood vessels' edges simultaneously, which surpasses conventional Richardson‐Lucy BD algorithm. The reported results indicate that the BDTV algorithm potentially facilitate PAOM in extracting retinal pathophysiological information by enhancing in vivo imaging quality without physically modifying PAOM's optical configuration.      

Comparison of optically‐derived biomarkers in healthy and brain tumor tissue under one‐ and two‐photon excitation

The surgical outcome of brain tumor resection and needle biopsy is significantly correlated to the patient's prognosis. Brain tumor surgery is limited to resecting the solid portion of the tumor as current intraoperative imaging modalities are incapable of delineating infiltrative regions. For accurate delineation, in situ tissue interrogation at the submicron scale is warranted. Additionally, multimodal detection is required to remediate the genetically and molecularly heterogeneous nature of brain tumors, notably, that of gliomas, meningioma and brain metastasis. Multimodal detection, such as spectrally‐ and temporally‐resolved fluorescence under one‐ and two‐photon excitation, enables characterizing tissue based on several endogenous optical contrasts. In order to assign the optically‐derived parameters to different tissue types, construction of an optical database obtained from biopsied tissue is warranted. This report showcases the different quantitative and semi‐quantitative optical markers that may comprise the tissue discrimination database. These include: the optical index ratio, the optical redox ratio, the relative collagen density, spectrally‐resolved fluorescence lifetime parameters, two‐photon fluorescence imaging and second harmonic generation imaging.     

Chemico‐mechanical imaging of Barrett's oesophagus

Barrett's oesophagus is a condition characterized by a change in the lining of the oesophagus that markedly increases the risk of adenocarcinoma. We demonstrate the first site‐matched application of Brillouin microscopy, Raman microscopy and FTIR micro‐spectroscopic imaging to ex‐vivo epithelial tissue – Barrett's oesophagus. The mechanical and chemical characters of the epithelium were assessed in histological sections from a patient subjected to endoscopic oesophageal biopsy. Previous studies have shown that both these properties change within the oesophageal wall, owing to the presence of distinct cellular and extracellular constituents which are putatively affected by oesophageal cancer. Brillouin microscopy enables maps of elasticity of the epithelium to be obtained, whilst Raman and FTIR imaging provide ’chemical images' without the need for labelling or staining. This site‐matched approach provides a valuable platform for investigating the structure, biomechanics and composition of complex heterogeneous systems. A combined Brillouin‐Raman device has potential for in‐vivo diagnosis of pathology.

First application of site‐matched micro Brillouin, Raman and FTIR spectroscopic imaging to epithelial tissue in Barrett's oesophagus     

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

Two‐photon imaging of fiber‐coupled neurons

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two‐photon imaging is shown to enable a quantitative in situ analysis of such fiber–single‐neuron coupling in the live brain of transgenic mice. Fiber‐optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two‐photon imaging. These results pave the way for fiber‐optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber‐optic single‐neuron stimulation into the optical imaging framework.

     

Optofluidic single‐cell absorption flow analyzer for point‐of‐care diagnosis of malaria

In this work, an optofluidic flow analyzer, which can be used to perform malaria diagnosis at the point‐of‐care is demonstrated. The presented technique is based on quantitative optical absorption measurements carried out on a single cell level for a given population of Human Red Blood Cells (RBCs). By measuring the optical absorption of each RBC, the decrease in the Hemoglobin (Hb) concentration in the cytoplasm of the cell due to the invasion of malarial parasite is detected. Cells are assessed on a single cell basis, as they pass through a microfluidic channel. The proposed technique has been implemented with inexpensive off‐the‐shelf components like laser diode, photo‐detector and a micro‐controller. The ability of the optofluidic flow analyzer to asses about 308,049 cells within 3 minutes has been demonstrated. The presented technique is capable of detecting very low parasitemia levels with high sensitivity.

     

Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost‐effective and label‐free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single‐cell precision to define cell identities in a large and heterogeneous population of cells—hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric‐detection time‐stretch optical microscopy (multi‐ATOM) that captures and processes quantitative label‐free single‐cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi‐ATOM, based upon ultrafast phase‐gradient encoding, outperforms state‐of‐the‐art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi‐ATOM for large‐scale, label‐free, multivariate, cell‐type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi‐ATOM could empower new strategies in large‐scale biophysical single‐cell analysis with applications in biology and enriching disease diagnostics.