Ultrafast cell edge detection by line‐scan time‐stretch microscopy

Ultrafast time‐stretch imaging technique recently attracts an increasing interest for applications in cell classification due to high throughput and high sensitivity. A novel imaging modality of time‐stretch imaging technique for edge detection is proposed. Edge detection based on the directional derivative is realized using differential detection. As the image processing is mainly implemented in the physical layer, the computation complexity of edge extraction is significantly reduced. An imaging system for edge detection with the scan rate of 77.76 MHz is experimentally demonstrated. Resolution target is first measured to verify the feasibility of the edge extraction. Furthermore, various cells, including red blood cells, lung cancer cells and breast cancer cells, are detected. The edges of cancerous cells present in a completely different form. The imaging system for edge detection would be a good candidate for high‐throughput cell classification.      

Volumetric hand‐held optoacoustic angiography as a tool for real‐time screening of dense breast

Existing mammographic screening solutions are generally associated with several major drawbacks, such as exposure to ionizing radiation or insufficient sensitivity in younger populations with radiographically‐dense breast. Even when combined with ultrasound or magnetic resonance imaging, X‐Ray mammography may still attain unspecific or false positive results. Thus, development of new breast imaging tools represents a timely medical challenge. We report on a new approach to high‐resolution functional and anatomical breast angiography using volumetric hand‐held optoacoustic tomography, which employs light intensities safe for human use. Experiments in young healthy volunteers with fibroglandular‐dominated dense breasts revealed the feasibility of rendering three‐dimensional images representing vascular anatomy and functional blood oxygenation parameters at video rate. Sufficient contrast was achieved at depths beyond 2 cm within dense breasts without compromising the real‐time imaging performance. The suggested solution may thus find applicability as a standalone or supplemental screening tool for early detection and follow‐up of carcinomas in radiographically‐dense breasts.

Volumetric handheld optoacoustic tomography scanner uses safe pulses of near‐infrared light to render three‐dimensional images of deep vascular anatomy, blood oxygenation and breast parenchyma at video rate.     

Effects of multispectral excitation on the sensitivity of molecular optoacoustic imaging

Molecular optoacoustic (photoacoustic) imaging typically relies on the spectral identification of absorption signatures from molecules of interest. To achieve this, two or more excitation wavelengths are employed to sequentially illuminate tissue. Due to depth‐related spectral dependencies and detection related effects, the multispectral optoacoustic tomography (MSOT) spectral unmixing problem presents a complex non‐linear inversion operation. So far, different studies have showcased the spectral capacity of optoacoustic imaging, without however relating the performance achieved to the number of wavelengths employed. Overall, the dependence of the sensitivity and accuracy of optoacoustic imaging as a function of the number of illumination wavelengths has not been so far comprehensively studied. In this paper we study the impact of the number of excitation wavelengths employed on the sensitivity and accuracy achieved by molecular optoacoustic tomography. We present a quantitative analysis, based on synthetic MSOT datasets and observe a trend of sensitivity increase for up to 20 wavelengths. Importantly we quantify this relation and demonstrate an up to an order of magnitude sensitivity increase of multi‐wavelength illumination vs. single or dual wavelength optoacoustic imaging. Examples from experimental animal studies are finally utilized to support the findings.

In vivo MSOT imaging of a mouse brain bearing a tumor that is expressing a near‐infrared fluorescent protein. ( a ) Monochromatic optoacoustic imaging at the peak excitation wavelength of the fluorescent protein. ( b ) Overlay of the detected bio‐distribution of the protein (red pseudocolor) on the monochromatic optoacoustic image. ( c ) Ex vivo validation by means of cryoslicing fluorescence imaging.     

Three‐dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo

Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three‐dimensional detection of pathophysiological parameters within skin. It was recently shown that single‐wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three‐dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15–125 MHz. We employ a per‐pulse tunable laser to inherently co‐register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label‐free visualization of physiological biomarkers in skin in vivo.

Cross‐sectional optoacoustic image of human skin in vivo. The epidermal layer is characterized by melanin absorption. A vascular network runs through the dermal layer, exhibiting blood oxygenation values of 50–90%. All scale bars: 250 µm     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

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.      

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.      

Wide‐field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy

We present a new hyperspectral reflected light microscopy system with a scanned broadband supercontinuum light source. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. The presented system can be used for real‐time 3D NP tracking as spectral sensors, thus providing new avenues in the spatio‐temporal characterization and detection of bioanalytes.

3D image of the distribution of functionalized AuNPs attached to CD44‐expressing MDA‐MB‐231 human cancer cells.     

Nonlinear optoacoustic readings from diffusive media at near‐infrared wavelengths

Optoacoustic (photoacoustic) imaging assumes that the detected signal varies linearly with laser energy. However, nonlinear intensity responses as a function of light fluence have been suggested in optoacoustic microscopy, that is, within the first millimeter of tissue. In this study, we explore the presence of nonlinearity deeper in tissue (~4 mm), as it relates to optoacoustic mesoscopy, and investigate the fluence required to delineate a switch from linear to nonlinear behavior. Optoacoustic signal nonlinearity is studied for different materials, different wavelengths and as a function of changes in the scattering and absorption coefficient of the medium imaged. We observe fluence thresholds in the mJ/cm² range and preliminary find that different materials may exhibit different nonlinearity patterns. We discuss the implications of nonlinearity in relation to image accuracy and quantification in optoacoustic tomography.      

In vivo study of rat cortical hemodynamics using a stereotaxic‐apparatus‐compatible photoacoustic microscope

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic‐apparatus‐compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2‐dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm². Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.      

Convolutional neural network‐based malaria diagnosis from focus stack of blood smear images acquired using custom‐built slide scanner

The present paper introduces a focus stacking‐based approach for automated quantitative detection of Plasmodium falciparum malaria from blood smear. For the detection, a custom designed convolutional neural network (CNN) operating on focus stack of images is used. The cell counting problem is addressed as the segmentation problem and we propose a 2‐level segmentation strategy. Use of CNN operating on focus stack for the detection of malaria is first of its kind, and it not only improved the detection accuracy (both in terms of sensitivity [97.06%] and specificity [98.50%]) but also favored the processing on cell patches and avoided the need for hand‐engineered features. The slide images are acquired with a custom‐built portable slide scanner made from low‐cost, off‐the‐shelf components and is suitable for point‐of‐care diagnostics. The proposed approach of employing sophisticated algorithmic processing together with inexpensive instrumentation can potentially benefit clinicians to enable malaria diagnosis.      

Tracking mesenchymal stromal cells using an ultra‐bright TAT‐functionalized plasmonic‐active nanoplatform

High‐resolution tracking of stem cells remains a challenging task. An ultra‐bright contrast agent with extended intracellular retention is suitable for in vivo high‐resolution tracking of stem cells following the implantation. Here, a plasmonic‐active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide‐functionalized gold nanostars (TAT‐GNS) that emit ultra‐bright two‐photon photoluminescence capable of tracking MSCs under high‐resolution optical imaging. In vitro experiment showed TAT‐GNS‐labeled MSCs retained a similar differentiability to that of non‐labeled MSCs controls. Due to their star shape, TAT‐GNS exhibited greater intracellular retention than that of commercial Q‐Tracker. In vivo imaging of TAT‐GNS‐labeled MSCs five days following intra‐arterial injections in mice kidneys showed possible MSCs implantation in juxta‐glomerular (JG) regions, but non‐specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic‐active nanoplatforms may be a useful intracellular tracking tool for stem cell research.

An ultra‐bright intracellular contrast agent is developed using TAT peptide‐functionalized gold nanostars (TAT‐GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two‐photon photoluminescence and superior labeling efficiency than commercial Q‐Tracker. Following renal implantation, some TAT‐GNS‐labeled MSCs permeate blood vessels and migrate to the juxta‐glomerular region.     

Inside Cover: Label‐free multi‐parametric imaging of single cells: dual picosecond optoacoustic microscopy (J. Biophotonics 8/2019)

Sub‐picosecond light pulses are used to launch high‐frequency ultrasound in cells. The dual detection of acoustic echoes and of the time‐domain Brillouin scattering allows mapping remotely and in a single run experiment the cell adhesion, thickness, storage modulus and mass density, all with micron resolution. The dual picosecond opto‐acoustic microscope is demonstrated with the multiple imaging of a mitotic macrophage‐like cell. This novel modality is compatible with simultaneous fluorescence imaging. Further details can be found in the article by Liwang Liu, Laurent Plawinski, Marie‐Christine Durrieu, Bertrand Audoin ( e201900045 ).

     

Perfusion‐based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near‐infrared molecular probes

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near‐infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor‐selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor‐to‐background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real‐time intraoperative procedures.      

Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks

A critical link exists between pathological changes of cerebral vasculature and diseases affecting brain function. Microscopic techniques have played an indispensable role in the study of neurovascular anatomy and functions. Yet, investigations are often hindered by suboptimal trade‐offs between the spatiotemporal resolution, field‐of‐view (FOV) and type of contrast offered by the existing optical microscopy techniques. We present a hybrid dual‐wavelength optoacoustic (OA) biomicroscope capable of rapid transcranial visualization of large‐scale cerebral vascular networks. The system offers 3‐dimensional views of the morphology and oxygenation status of the cerebral vasculature with single capillary resolution and a FOV exceeding 6 × 8 mm², thus covering the entire cortical vasculature in mice. The large‐scale OA imaging capacity is complemented by simultaneously acquired pulse‐echo ultrasound (US) biomicroscopy scans of the mouse skull. The new approach holds great potential to provide better insights into cerebrovascular function and facilitate efficient studies into neurological and vascular abnormalities of the brain.      

Non‐invasive sentinel lymph node mapping and needle guidance using clinical handheld photoacoustic imaging system in small animal

Translating photoacoustic imaging (PAI) into clinical setup is a challenge. Handheld clinical real‐time PAI systems are not common. In this work, we report an integrated photoacoustic (PA) and clinical ultrasound imaging system by combining light delivery with the ultrasound probe for sentinel lymph node imaging and needle guidance in small animal. The open access clinical ultrasound platform allows seamless integration of PAI resulting in the development of handheld real‐time PAI probe. Both methylene blue and indocyanine green were used for mapping the sentinel lymph node using 675 and 690 nm wavelength illuminations, respectively. Additionally, needle guidance with combined ultrasound and PAI was demonstrated using this imaging system. Up to 1.5 cm imaging depth was observed with a 10 Hz laser at an imaging frame rate of 5 frames per second, which is sufficient for future translation into human sentinel lymph node imaging and needle guidance for fine needle aspiration biopsy.      

Synthetic data framework to estimate the minimum detectable concentration of contrast agents for multispectral optoacoustic imaging of small animals

The concentrations of contrast agents for optoacoustic imaging of small animals must usually be optimized through extensive pilot experiments on a case‐by‐case basis. The present work describes a streamlined approach for determining the minimum detectable concentration (MDC) of a contrast agent given experimental conditions and imaging system parameters. The developed Synthetic Data Framework (SDF) allows estimation of MDCs of various contrast agents under different tissue conditions without extensive animal experiments. The SDF combines simulated optoacoustic signals from exogenously administered contrast agents with in vivo experimental signals from background tissue to generate realistic synthetic multispectral optoacoustic images. In this paper, the SDF is validated with in vivo measurements and demonstrates close agreement between SDF synthetic data and experimental data in terms of both image intensity and MDCs. Use of the SDF to estimate MDCs for fluorescent dyes and nanoparticles at different tissue depths and for imaging lesions of different sizes is illustrated.     

Single molecule localization deep within thick cells; a novel super‐resolution microscope

A novel 3D imaging system based on single‐molecule localization microscopy is presented to allow high‐accuracy drift‐free (<0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within the cell, distortions of the point‐spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each blink, a series of depth‐dependent calibrations are required. The system and its allied methodology are applied to image the ryanodine receptor in the cardiac myocyte. Using the depth‐dependent calibration, the receptors deep within the cell are spread over a Z range that is many hundreds of nanometers greater than implied by conventional analysis. We implemented a time domain filter to detect overlapping blinks that were not filtered by a stringent goodness of fit criterion. This filter enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography.

High‐accuracy deep imaging of the ryanodine receptor in the cardiac myocyte, using single‐molecule localization microscopy. Depth‐dependent calibrations are performed for accurate depth localization. The optical design featuring two independent and variable focal planes allows real‐time feedback for drift‐free deep imaging.     

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.