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.     

High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep‐tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)‐expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP‐expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity‐dependent expression pattern of tdTomato.      

Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs

Whole‐organ and whole‐body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion‐based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.      

K‐distribution three‐dimensional mapping of biological tissues in optical coherence tomography

Probability density function (PDF) analysis with K‐distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres‐in‐water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M‐mode or two‐dimenstional slice analysis, to full three‐dimensional (3D) imaging maps of the shape parameter α of the K‐distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.      

Structured illumination imaging with quasi‐periodic patterns

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.     

Inside Cover: Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs (J. Biophotonics 5/2018)

Optical tissue clearing is a method allowing post‐mortem deep imaging of organs in three dimensions. By optimizing the CUBIC clearing protocol, the authors provide rapid and simple approach to clear the entire adult rat organism within as little as four days, which is accompanied by the variety of its staining and imaging techniques. The image was captured with polarizers and demonstrates transparent rodent heart with thread‐like crystals of clearing reagent. Further details can be found in the article by Pawe? Matryba et al. ( e201700248 ).

     

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

Acquiring information of the neural structures in the whole‐brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high‐throughput light‐sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain‐wide distribution of specific neurons or neural circuits.      

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.      

A guidebook for DISCO tissue clearing

Histological analysis of biological tissues by mechanical sectioning is significantly time‐consuming and error‐prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue‐clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands‐on implementation.     

Development by three‐dimensional approaches and four‐dimensional imaging: To the knowledge frontier and beyond

Many advances have been taken on elucidating embryonic development and tissue homeostasis and repair by the use of experimental strategies that preserve the three‐dimensional (3D) organization and allow quantitative analysis of images over time (four‐dimensional). Ranging from the understanding about the relationship between blastomeres and the events that take place during gastrulation by the use of time‐lapse imaging through 3D cultures that mimic organogenesis, the advances in this area are of critical value. The studies on embryonic development without disrupting the original architecture and the development of 3D organoid cultures pave a new avenue for unprecedented experimental advances that will positively impact the emergence of new treatments applying regenerative principles for both tissue repair and organ transplant. Birth Defects Research (Part C) 105:1–8, 2015. © 2015 Wiley Periodicals, Inc.     

Enhanced contrast acoustic‐resolution photoacoustic microscopy using double‐stage delay‐multiply‐and‐sum beamformer for vasculature imaging

In acoustic‐resolution photoacoustic microscopy (AR‐PAM) systems, the lateral resolution in the focal zone of the ultrasound (US) transducer is determined by the numerical aperture (NA) of the transducer. To have a high lateral resolution, a large NA is used. However, the larger the NA, the smaller the depth of focus [DOF]. As a result, the lateral resolution is deteriorated at depths out of the focal region. The synthetic aperture focusing technique (SAFT) along with a beamformer can be used to improve the resolution outside the focal region. In this work, for image formation in AR‐PAM, we propose the double‐stage delay‐multiply‐and‐sum (DS_DMAS) algorithm to be combined with SAFT. The proposed method is evaluated experimentally using hair targets and in vivo vasculature imaging. It is shown that DS_DMAS provides a higher resolution and contrast compared to other methods. For the B‐mode images obtained using the hair phantom, the proposed method reduces the average noise level for all the depths by about 134%, 57% and 23%, compared to the original low‐ resolution, SAFT+DAS and SAFT+DMAS methods, respectively. All the results indicate that the proposed method can be an appropriate algorithm for image formation in AR‐PAM systems.      

High resolution,label‐free photoacoustic imaging of live chicken embryo developing in bioengineered eggshell

Chicken embryos have been proven to be an attractive vertebrate model for biomedical research. They have helped in making significant contributions for advancements in various fields like developmental biology, cancer research and cardiovascular studies. However, a non‐invasive, label‐free method of imaging live chicken embryo at high resolution still needs to be developed and optimized. In this work, we have shown the potential of photoacoustic tomography (PAT) for imaging live chicken embryos cultured in bioengineered eggshells. Laser pulses at wavelengths of 532 and 740 nm were used for attaining cross‐sectional images of chicken embryos at different developmental stages. Cross‐sections along different depths were imaged to gain knowledge of the relative depth of different vessels and organs. Due to high optical absorption of vasculature and embryonic eye, images with good optical contrast could be acquired using this method. We have thus reported a label‐free method of performing cross‐sectional imaging of chicken embryos at high resolution demonstrating the capacity of PAT as a promising tool for avian embryo imaging.     

Three‐dimensional imaging technologies: a priority for the advancement of tissue engineering and a challenge for the imaging community

Tissue engineering/regenerative medicine (TERM) is an interdisciplinary field that applies the principle of engineering and life sciences to restore/replace damaged tissues/organs with in vitro artificially‐created ones. Research on TERM quickly moves forward. Today newest technologies and discoveries, such as 3D‐/bio‐printing, allow in vitro fabrication of ex‐novo made tissues/organs, opening the door to wide and probably never‐ending application possibilities, from organ transplant to drug discovery, high content screening and replacement of laboratory animals. Imaging techniques are fundamental tools for the characterization of tissue engineering (TE) products at any stage, from biomaterial/scaffold to construct/organ analysis. Indeed, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular features, allowing three‐dimensional (3D) and time‐lapse in vivo analysis, in a non‐destructive, quantitative, multidimensional analysis of TE constructs, to analyze their pre‐implantation quality assessment and their fate after implantation. This review focuses on the newest developments in imaging technologies and applications in the context of requirements of the different steps of the TERM field, describing strengths and weaknesses of the current imaging approaches.

     

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.      

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.     

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.      

Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging

Light‐sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co‐aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2‐photon LSFM (OS‐2P‐LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning‐descanning system. This system also provides improved light‐sheet confinement against scattering by using a 2‐photon Bessel beam. The OS‐2P‐LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high‐speed, translation‐free and scattering‐robust 3D imaging of large biological specimens.      

Cellular phone‐based photoplethysmographic imaging

We present study results on visible light reflection photoplethysmographic (PPG) imaging with a mobile cellular phone operated in video imaging mode. PPG signal components around 0.1 Hz attributed to the sympathetic component of the heart rate, 1 Hz as the heart rate and 2 Hz as heart rate high order harmonic were quantified on the index finger of a healthy volunteer. The green channel reported PPG signals throughout the sampled area. The blue and red channel returned plethysmographic information, but the signal strength was highly position specific. Our results obtained with a cellular phone as the data acquisition device are encouraging, especially in the broad context of personal or home‐based care and the role of cellular phone technology in medical imaging. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)