Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Improved deep two-photon calcium imaging in vivo

Two-photon laser scanning calcium imaging has emerged as a useful method for the exploration of neural function and structure at the cellular and subcellular level in vivo. The applications range from imaging of subcellular compartments such as dendrites, spines and axonal boutons up to the functional analysis of large neuronal or glial populations. However, the depth penetration is often limited to a few hundred micrometers, corresponding, for example, to the upper cortical layers of the mouse brain. Light scattering and aberrations originating from refractive index inhomogeneties of the tissue are the reasons for these limitations. The depth penetration of two-photon imaging can be enhanced through various approaches, such as the implementation of adaptive optics, the use of three-photon excitation and/or labeling cells with red-shifted genetically encoded fluorescent sensors. However, most of the approaches used so far require the implementation of new instrumentation and/or time consuming staining protocols. Here we present a simple approach that can be readily implemented in combination with standard two-photon microscopes. The method involves an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590 and benefits from the use of ultra-short laser pulses. The approach allows in vivo functional imaging of neuronal populations with single cell resolution in all six layers of the mouse cortex. We demonstrate that stable recordings in deep cortical layers are not restricted to anesthetized animals but are well feasible in awake, behaving mice. We anticipate that the improved depth penetration will be beneficial for two-photon functional imaging in larger species, such as non-human primates.     

Vibrational two-photon microscopy for tissue imaging: Short-wave infrared surface-enhanced resonance hyper-Raman scattering

Multiphoton microscopy using short-wave infrared (SWIR) radiation offers nondestructive and high-resolution imaging through tissue. Two-photon fluorescence (TPF), for example, is commonly employed to increase the penetration depth and spatial resolution of SWIR imaging, but the broad spectral peaks limit its multiplexing capabilities. Hyper-Raman scattering, the vibrational analog of TPF, yields spectral features on the order of 20 cm^?1 and reporter-functionalized noble metal nanoparticles (NPs) provide a platform for both hyper-Raman signal enhancement and selective targeting in biological media. Herein we report the first tissue imaging study employing surface-enhanced resonance hyper-Raman scattering (SERHRS), the two-photon analog of surface-enhanced resonance Raman scattering. Specifically, we employ multicore gold-silica NPs (Au@SiO₂ NPs) functionalized with a near infrared-resonant cyanine dye, 3,3′-diethylthiatricarbocyanine iodide as a SERHRS reporter. SWIR SERHRS spectra are efficiently acquired from mouse spleen tissue. SWIR SERHRS combines two-photon imaging advantages with narrow vibrational peak widths, presenting future applications of multitargeted bioimaging.     

Deep learning: step forward to high-resolution in vivo shortwave infrared imaging

Shortwave infrared window (SWIR: 1000–1700 nm) represents a major improvement compared to the NIR-I region (700–900 nm) in terms of temporal and spatial resolutions in depths down to 4 mm. SWIR is a fast and cheap alternative to more precise methods such as X-ray and opto-acoustic imaging. Main obstacles in SWIR imaging are the noise and scattering from tissues and skin that reduce the precision of the method. We demonstrate that the combination of SWIR in vivo imaging in the NIR-IIb region (1500–1700 nm) with advanced deep learning image analysis allows to overcome these obstacles and making a large step forward to high resolution imaging: it allows to precisely segment vessels from tissues and noise, provides morphological structure of the vessels network, with learned pseudo-3D shape, their relative position, dynamic information of blood vascularization in depth in small animals and distinguish the vessels types: artieries and veins. For demonstration we use neural network IterNet that exploits structural redundancy of the blood vessels, which provides a useful analysis tool for raw SWIR images.     

A near-infrared cell tracker reagent for multiscopic in vivo imaging and quantification of leukocyte immune responses

The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging.     

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.      

Comparison of high frequency ultrasound and optical coherence tomography as modalities for high resolution and non invasive skin imaging.

High frequency ultrasound (HFUS) and optical coherence tomography (OCT) are techniques for high resolution imaging of tissues. The penetration depth of these modalities is limited, but it is sufficiently large enough for non invasive skin imaging. HFUS and OCT are based on the same concept. Waves (ultrasonic waves, respectively light waves) propagate along a narrow beam, are backscattered at tissue inhomogeneities and analyzed over time of flight to obtain spatially resolved morphological information. The objective of this paper is to compare HFUS and OCT in terms of resolution, dynamic range and contrast and to assess their value as tools for high resolution skin imaging. Measurements on phantoms and in vivo have been performed with a 100 MHz ultrasound system and an OCT-scanner working in the near infrared spectrum at 1300 nm wave-length. From the measurements, it can be concluded that OCT delivers an almost isotropic resolution (axial resolution about 5.8 microns, lateral resolution about 4.1 microns), whereas the resolution of the investigated HFUS system is more anisotropic (axial resolution about 9.3 microns, lateral resolution about 60 microns). HFUS and OCT show different penetration depths and a different contrast. Both techniques can, therefore, be combined advantageously in a multimodality approach to account for their individual characteristics.     

Advances in nonlinear optical microscopy techniques for in vivo and in vitro neuroimaging

Understanding the mechanism of the brain via optical microscopy is one of the challenges in neuroimaging, considering the complex structures. Advanced neuroimaging techniques provide a more comprehensive insight into patho-mechanisms of brain disorders, which is useful in the early diagnosis of the pathological and physiological changes associated with various neurodegenerative diseases. Recent advances in optical microscopy techniques have evolved powerful tools to overcome scattering of light and provide improved in vivo neuroimaging with sub-cellular resolution, endogenous contrast specificity, pinhole less optical sectioning capability, high penetration depth, and so on. The following article reviews the developments in various optical imaging techniques including two-photon and three-photon fluorescence, second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, and stimulated Raman scattering in neuroimaging. We have outlined the potentials and drawbacks of these techniques and their possible applications in the investigation of neurodegenerative diseases.     

Full-field OCT

Optical coherence tomography (OCT) is an emerging technique for imaging of biological media with micrometer-scale resolution, whose most significant impact concerns ophthalmology. Since its introduction in the early 1990's, OCT has known a lot of improvements and sophistications. Full-field OCT is our original approach of OCT, based on white-light interference microscopy. Tomographic images are obtained by combination of interferometric images recorded in parallel by a detector array such as a CCD camera. Whereas conventional OCT produces B-mode (axially-oriented) images like ultrasound imaging, full-field OCT acquires tomographic images in the en face (transverse) orientation. Full-field OCT is an alternative method to conventional OCT to provide ultrahigh resolution images (approximately 1 microm), using a simple halogen lamp instead of a complex laser-based source. Various studies have been carried, demonstrating the performances of this technology for three-dimensional imaging of ex vivo specimens. Full-field OCT can be used for non-invasive histological studies without sample preparation. In vivo imaging is still difficult because of the object motions. A lot of efforts are currently devoted to overcome this limitation. Ultra-fast full-field OCT was recently demonstrated with unprecedented image acquisition speed, but the detection sensitivity has still to be improved. Other research directions include the increase of the imaging penetration depth in highly scattering biological tissues such as skin, and the exploitation of new contrasts such as optical birefringence to provide additional information on the tissue morphology and composition.     

Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle

Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

In vivo near-infrared fluorescence imaging

Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in vivo imaging with near-infrared light and exogenous contrast agents. Recent examples of in vivo near-infrared fluorescence imaging of animals and humans are presented, including imaging of normal and diseased vasculature, tissue perfusion, protease activity, hydroxyapatite and cancer.     

重组腺相关病毒小鼠骨骼肌中近红外荧光蛋白表达及活体成像

近红外荧光蛋白因激发光和发射光波长位于近红外区,在动物组织中光吸收和光散射最低,更适宜于动物活体组织的深层成像.构建了一种携带近红外荧光蛋白(near-infrared fluorescent protein,iRFP)713基因的重组表达质粒pAAV-iRFP713,将重组表达质粒与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒(recombinant adeno-associated virus,rAAV)rAAV-iRFP713.重组腺相关病毒表达载体感染体外培养的癌细胞,48h后,荧光显微镜检测显示近红外荧光蛋白在癌细胞中高效表达,荧光明亮.重组腺相关病毒表达载体注射小鼠骨骼肌,48h后,用近红外荧光活体成像系统检测证明近红外荧光蛋白在小鼠骨骼肌中表达较强, 活体组织成像清晰.实验结果表明近红外荧光蛋白在体内体外均能很好地表达并荧光成像,为动物活体组织标记和成像的研究提供新方法.     

综述——新型纳米生物材料的研发：稀土上转换荧光纳米材料的制备与生物应用

近几年,稀土上转换荧光纳米材料作为新型的荧光探针受到研究者的广泛关注,其优势在于光化学稳定性好、发射谱带窄、荧光寿命长、Stokes位移大等．同时,它利用近红外激光器作为激发光源,组织穿透能力好、对生物组织的损伤小、几乎没有背景荧光,使其应用于生物活体荧光成像成为可能．本文主要综述了最近稀土上转换荧光纳米材料在制备与生物应用方面的研究进展．     

The efficiency of blood photomodification with therapeutic doses of optical radiation of different wavelengths

Biophysics - Spectra of radiation penetration depth into the blood and skin tissues were calculated for the wavelength range of 405 to 950 nm with the optical properties of biological tissues taken...     

金纳米棒-聚苯胺核壳纳米材料的光学相干层析成像能力研究

对比试剂的使用能够显著提升光学相干层析(OCT)的成像效果。聚苯胺(PANI)是一种有机导电聚合物,在近红外(NIR)区有着很强的光吸收。本文采用PANI对常见的OCT成像对比试剂--金纳米棒(GNRs)进行修饰,合成了PANI/GNRs核壳粒子,并对其OCT成像对比能力进行了研究。PANI/GNRs展现出良好的NIR光吸收特性;同时,PANI对GNRs的包裹也显著提升了金纳米结构的稳定性、降低了GNRs原有的毒性。选用离体猪肝组织作为检测样本,发现纳米材料使用能够显著提升OCT的成像效果。与未修饰的GNRs及PANI粒子相比,PANI/GNRs的OCT成像对比效果明显更好。因此,PANI包裹的GNRs核壳纳米材料有望成为一种低毒性且效果良好的OCT对比试剂用于生物组织成像。     

Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy

Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.      

Imaging Depths of Near-infrared Quantum Dots in First and Second Optical Windows

AbstractPotential advantages of quantum dot (QD) imaging in the second optical window (SOW) at 1,000 to 1,400 nm over the first optical window (FOW) at 700 to 900 nm have attracted much interest. QDs that emit at 800 nm (800QDs) and QDs that emit at 1,300 nm (1,300QDs) are used to investigate the imaging depths at the FOW and SOW. QD images in biologic tissues are processed binarized via global thresholding method, and the imaging depths are determined using the criteria of contrast to noise ratio and relative apparent size. Owing to the reduced scattering in the SOW, imaging depth in skin can be extended by approximately three times for 1,300QD/SOW over 800QD/FOW. In liver, excitation of 1,300QD/SOW can be shifted to longer wavelengths; thus, the imaging depth can be extended by 1.4 times. Effects of quantum yield (QY), concentration, incidence angle, polarization, and fluence rate F on imaging depth are comprehensively studied. Under F approved by the Food and Drug Administration, 1,300QDs with 50% QY can reach imaging depths of 29.7 mm in liver and 17.5 mm in skin. A time-gated excitation using 1,000 times higher F pulses can obtain the imaging depth of ≈ 5 cm. To validate our estimates, in vivo whole-body imaging experiments are performed using small-animal models.     

Optical fluorescence imaging with shortwave infrared light emitter nanomaterials for in vivo cell tracking in regenerative medicine

In vivo tracking and monitoring of adoptive cell transfer has a distinct importance in cell‐based therapy. There are many imaging modalities for in vivo monitoring of biodistribution, viability and effectiveness of transferred cells. Some of these procedures are not applicable in the human body because of low sensitivity and high possibility of tissue damages. Shortwave infrared region (SWIR) imaging is a relatively new technique by which deep biological tissues can be potentially visualized with high resolution at cellular level. Indeed, scanning of the electromagnetic spectrum (beyond 1000 nm) of SWIR has a great potential to increase sensitivity and resolution of in vivo imaging for various human tissues. In this review, molecular imaging modalities used for monitoring of biodistribution and fate of administered cells with focusing on the application of non‐invasive optical imaging at shortwave infrared region are discussed in detail.     

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.