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‐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.      

Quantitative assessment of optical clearing methods in various intact mouse organs

Various tissue optical clearing techniques have sprung up for large volume imaging. However, there are few methods showed clearing and imaging data on different organs while most of them were focused on mouse brain, and as a result, it is difficult to select the suitable method for organs in practical applications due to lack of quantitative evaluation and comprehensive comparison. Therefore, it is necessary to evaluate and compare the performances of clearing methods for different organs. In this paper, several typical optical clearing methods were applied, including 3DISCO, uDISCO, SeeDB, FRUIT, CUBIC, ScaleS and PACT to clear intact brain, heart, kidney, liver, spleen, stomach, lung, small intestine, skin and muscle. The clearing efficiency, sample deformation, fluorescence preservation and imaging depth of these methods were quantitatively evaluated. Finally, based on the systemic evaluation of various parameters described above, the appropriate clearing method for specific organ including kidney or intestine was screened out. This paper will provide important references for selection of appropriate clearing methods in related researches.      

Slide‐free imaging of hematoxylin‐eosin stained whole‐mount tissues using combined third‐harmonic generation and three‐photon fluorescence microscopy

Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time‐consuming and labor‐intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically sectioned histopathological imaging of hematoxylin‐eosin stained whole‐mount skin tissues with a subfemtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Image: Slide‐free nonlinear microscopy imaging of H&E stained whole‐mount skin tissue showing the morphology of sweat glands.      

Whole‐body clearing of beetles by successive treatment with hydrogen peroxide and CUBIC reagents

Internal tissues of multicellular organisms cannot directly be seen because they contain pigments. For this reason, whole‐body clearing methods have been developed and applied to mammals such as mice. Insects such as beetles, however, cannot be cleared by the mammalian method because of pigments such as melanin in their exoskeletons. In this study, we tried to develop a whole‐body clearing method for large beetles. We first bleached the exoskeleton using a hydrogen peroxide treatment, and applied advanced Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) reagents to make the internal tissues transparent. The combined method of hydrogen peroxide and advanced CUBIC allowed us to successfully undertake whole‐body clearing of large beetles.     

Tissue clearing‐based method for unobstructed three‐dimensional imaging of mouse penis with subcellular resolution

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous‐ (clear, unobstructed brain imaging cocktails and computational analysis) and solvent‐based (fluorescence‐preserving capability imaging of solvent‐cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three‐dimensional structure of murine penis fibrocartilage.     

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.      

Visible‐near infrared‐II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

Skull optical clearing window permits us to perform in vivo cortical imaging without craniotomy, but mainly limits to visible (vis)‐near infrared (NIR)‐I light imaging. If the skull optical clearing window is available for NIR‐II, the imaging depth will be further enhanced. Herein, we developed a vis‐NIR‐II skull optical clearing agents with deuterium oxide instead of water, which could make the skull transparent in the range of visible to NIR‐II. Using a NIR‐II excited third harmonic generation microscope, the cortical vasculature of mice could be clearly distinguished even at the depth of 650 μm through the vis‐NIR‐II skull clearing window. The imaging depth after clearing is close to that without skull, and increases by three times through turbid skull. Furthermore, the new skull optical clearing window promises to realize NIR‐II laser‐induced targeted injury of cortical single vessel. This work enhances the ability of NIR‐II excited nonlinear imaging techniques for accessing to cortical neurovasculature in deep tissue.     

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.     

Three‐dimensional reconstruction of corticospinal tract using one‐photon confocal microscopy acquisition allows detection of axonal disruption in spinal cord injury

The principal motor tract involved in mammalian locomotor activities is known as the corticospinal tract (CST), which starts in the brain motor cortex (upper motor neuron), extends its axons across the brain to brainstem and finally reaches different regions of spinal cord, contacting the lower motor neurons. Visualization of the CST is essential to carry out studies in different kinds of pathologies such as spinal cord injury or multiple sclerosis. At present, most studies of axon structure and/or integrity that involve histological tissue sectioning present the problem of finding the region where the CST is predominant. To solve this problem, one could use a novel technique to make the tissues transparent and observe them directly without histological sectioning. However, the disadvantage of this procedure is the need of costly and non‐conventional equipment, such as two‐photon fluorescence microscopy or ultramicroscopy to perform the image acquisition. Here, we show that labeling the CST with FluoroRuby in the motor cortex and then performing the clearing technique, the z‐acquisition of the entire CST in unsectioned tissue followed by three‐dimensional reconstruction can be carried out by standard one‐photon confocal microscopy, with yields similar to those obtained by two‐photon microscopy. In addition, we present an example of the application of this method in a spinal cord injury model, where the disruption of CST is shown at the lesion site.

     

In vivo monitoring blood‐brain barrier permeability using spectral imaging through optical clearing skull window

The blood‐brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT‐induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low‐cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT‐induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).      

Controlling the near‐infrared transparency of costal cartilage by impregnation with clearing agents and magnetite nanoparticles

Penetration depth of near‐infrared laser radiation to costal cartilage is controlled by the tissue absorption and scattering, and it is the critical parameter to provide the relaxation of mechanical stress throughout the whole thickness of cartilage implant. To enhance the penetration for the laser radiation on 1.56 μm, the optical clearing solutions of glycerol and fructose of various concentrations are tested. The effective and reversible tissue clearance was achieved. However, the increasing absorption of radiation should be concerned: 5°C‐8°C increase of tissue temperature was detected. Laser parameters used for stress relaxation in cartilage should be optimized when applying optical clearing agents. To concentrate the absorption in the superficial tissue layers, magnetite nanoparticle (NP) dispersions with the mean size 95 ± 5 nm and concentration 3.9 ± 1.1 × 10¹¹ particles/mL are applied. The significant increase in the tissue heating rate was observed along with the decrease in its transparency. Using NPs the respective laser power can be decreased, allowing us to obtain the working temperature locally with reduced thermal effect on the surrounding tissue.      

A comparative study of ex vivo skin optical clearing using two‐photon microscopy

Multiphoton tomography (MPT) is a prospective tool for imaging the skin structure. Aiming to increase the probing depth, a comparative ex vivo study of optical clearing of porcine ear skin was performed by using two optical clearing agents (OCAs), i.e., glycerol and iohexol (Omnipaque^TM) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or Omnipaque^TM solutions onto the skin for 60 min significantly improved the depth and contrast of the MPT signals. By utilizing 40%, 60% and 100% glycerol, and 60% and 100% Omnipaque^TM it was demonstrated that both agents improve autofluorescence and SHG (second harmonic generation) signals from the skin. At the applied concentrations and agent time exposure, glycerol is more effective than Omnipaque^TM. However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. Omnipaque^TM, on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover Omnipaque^TM allows for robust multimodal optical/X‐ray imaging with automatically matched optically cleared and X‐ray contrasted tissue volumes. These findings make Omnipaque^TM more prospective than glycerol for some particular application.

     

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white‐light laser source

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti‐Stokes Raman scattering (CARS), third‐order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white‐light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study.

Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in‐plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.     

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.

     

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     

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.

     

Super‐achromatic optical coherence tomography capsule for ultrahigh‐resolution imaging of esophagus

Endoscopic optical coherence tomography (OCT) is a noninvasive technology allowing for imaging of tissue microanatomies of luminal organs in real time. Conventional endoscopic OCT operates at 1300 nm wavelength region with a suboptimal axial resolution limited to 8‐20 μm. In this paper, we present the first ultrahigh‐resolution tethered OCT capsule operating at 800 nm and offering about 3‐ to 4‐fold improvement of axial resolution (plus enhanced imaging contrast). The capsule uses diffractive optics to manage chromatic aberration over a full ~200 nm spectral bandwidth centering around 830 nm, enabling to achieve super‐achromaticity and an axial resolution of ~2.6 μm in air. The performance of the OCT capsule is demonstrated by volumetric imaging of swine esophagus ex vivo and sheep esophagus in vivo, where fine anatomic structures including the sub‐epithelial layers are clearly identified. The ultrahigh resolution and excellent imaging contrast at 800 nm of the tethered capsule suggest the potential of the technology as an enabling tool for surveillance of early esophageal diseases on awake patients without the need for sedation.      

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.

     

3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation

Background

Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining.

Methods

Using 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer.

Results

With our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier.

Conclusions

Taken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.