Search

Osteoarthritis (OA) is a complex disease of cartilage characterised by joint pain, functional limitation, and reduced quality of life with affected joint movement leading to pain and limited mobility. Current methods to diagnose OA are predominantly limited to X-ray, MRI and invasive joint fluid analysis, all of which lack chemical or molecular specificity and are limited to detection of the disease at later stages. A rapid minimally invasive and non-destructive approach to disease diagnosis is a critical unmet need. Label-free techniques such as Raman Spectroscopy (RS), Coherent anti-Stokes Raman scattering (CARS), Second Harmonic Generation (SHG) and Two Photon Fluorescence (TPF) are increasingly being used to characterise cartilage tissue. However, current studies are based on whole tissue analysis and do not consider the different and structurally distinct layers in cartilage. In this work, we use Raman spectroscopy to obtain signatures from the superficial (top) and deep (bottom) layer of healthy and osteoarthritic cartilage samples from 64 patients (19 control and 45 OA). Spectra were acquired both in the 'fingerprint' region from 700 to 1720 cm− 1 and high-frequency stretching region from 2500 to 3300 cm− 1. Principal component and linear discriminant analysis was used to identify the peaks that contributed significantly to classification accuracy of the different samples. The most pronounced differences were observed at the proline (855 cm− 1 and 921 cm− 1) and hydroxyproline (877 cm− 1 and 938 cm− 1), sulphated glycosaminoglycan (sGAG) (1064 cm− 1 and 1380 cm− 1) frequencies for both control and OA as well as the 1245 cm− 1 and 1272 cm− 1, 1320 cm− 1 and 1345 cm− 1, 1451 cm− 1 collagen modes were altered in OA samples, consistent with expected collagen structural changes. Classification accuracy based on Raman fingerprint spectral analysis of superficial and deep layer cartilage for controls was found to be 97% and 93% on using individual/all spectra and, 100% and 95% on using mean spectra per patient, respectively. OA diseased cartilage was classified with an accuracy of 88% and 84% for individual/all spectra, and 96% and 95% for mean spectra per patient based on analysis of the superficial and the deep layers, respectively. Raman spectra from the C-H stretching region (2500–3300 cm− 1) resulted in high classification accuracy for identification of different layers and OA diseased cartilage but low accuracy for controls. Differential changes in superficial and deep layer cartilage signatures were observed with age (under 60 and over 60 years), in contrast, less significant differences were observed with gender. Prominent chemical changes in the different layers of cartilage were preliminarily imaged using CARS, SHG and TPF. Cell clustering was observed in OA together with differences in pericellular matrix and collagen structure in the superficial and the deep layers correlating with the Raman spectral analysis. The current study demonstrates the potential of Raman Spectroscopy and multimodal imaging to interrogate cartilage tissue and provides insight into the chemical and structural composition of its different layers with significant implications for OA diagnosis for an increasing aging demographic. [ABSTRACT FROM AUTHOR]

We report a novel stimulated Raman scattering (SRS) microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid, deep tissue 3D chemical imaging with subcellular resolution. To accomplish phase-controlled SRS (PC-SRS), we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams, enabling z-scanning-free optical sectioning in the sample. By incorporating Zernike polynomials into the phase patterns, we simultaneously correct the system aberrations at two separate wavelengths (~240 nm difference), achieving a ~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system. PC-SRS provides >2-fold improvement in imaging depth in various samples (e.g., polystyrene bead phantoms, porcine brain tissue) as well as achieves SRS 3D imaging speed of ~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water, superior to conventional point-scanning SRS 3D imaging. We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cell-silent region, unraveling the upregulated metabolism in liver tumor compared to normal liver. This work shows that PC-SRS provides unprecedented insights into morpho-chemistry, metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level.

Traditional deep fluorescence imaging has primarily focused on red‐shifting imaging wavelengths into the near‐infrared (NIR) windows or implementation of multi‐photon excitation approaches. Here, the advantages of NIR and multiphoton imaging are combined by developing a dual‐infrared two‐photon microscope that enables high‐resolution deep imaging in biological tissues. This study first computationally identifies that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. A NIR two‐photon microscope is constructed next with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single‐walled carbon nanotubes (SWNTs). Spatial imaging resolutions are achieved close to the Abbe resolution limit and eliminate blur and background autofluorescence of biomolecules, 300 µm deep into brain slices and through the full 120 µm thickness of a Nicotiana benthamiana leaf. NIR‐II two‐photon microscopy can also measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature this study exploits to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. These results suggest dual‐infrared two‐photon microscopy can accomplish in‐tissue structural imaging and biochemical sensing with a minimal background, and with high spatial resolution, in optically opaque or highly autofluorescent biological tissues. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana sepals are excellent models for analyzing growth of entire organs due to their relatively small size, which can be captured at a cellular resolution under a confocal microscope. To investigate how differential growth of connected cell layers generate unique organ morphologies, it is necessary to live-image deep into the tissue. However, imaging deep cell layers of the sepal (or plant tissues in general) is practically challenging. Image processing is also difficult due to the low signal-to-noise ratio of the deeper tissue layers, an issue mainly associated with live imaging datasets. Addressing some of these challenges, we provide an optimized methodology for live imaging sepals, and subsequent image processing. For live imaging early-stage sepals, we found that the use of a bright fluorescent membrane marker, coupled with increased laser intensity and an enhanced Z- resolution produces high-quality images suitable for downstream image processing. Our optimized parameters allowed us to image the bottommost cell layer of the sepal (inner epidermal layer) without compromising viability. We used a 'voxel removal' technique to visualize the inner epidermal layer in MorphoGraphX image processing software. We also describe the MorphoGraphX parameters for creating a 2.5D mesh surface for the inner epidermis. Our parameters allow for the segmentation and parent tracking of individual cells through multiple time points, despite the weak signal of the inner epidermal cells. While we have used sepals to illustrate our approach, the methodology will be useful for researchers intending to live-image and track growth of deeper cell layers in 2.5D for any plant tissue. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana sepals are excellent models for analyzing growth of entire organs due to their relatively small size, which can be captured at a cellular resolution under a confocal microscope. To investigate how differential growth of connected cell layers generate unique organ morphologies, it is necessary to live-image deep into the tissue. However, imaging deep cell layers of the sepal (or plant tissues in general) is practically challenging. Image processing is also difficult due to the low signal-to-noise ratio of the deeper tissue layers, an issue mainly associated with live imaging datasets. Addressing some of these challenges, we provide an optimized methodology for live imaging sepals, and subsequent image processing. For live imaging early-stage sepals, we found that the use of a bright fluorescent membrane marker, coupled with increased laser intensity and an enhanced Z- resolution produces high-quality images suitable for downstream image processing. Our optimized parameters allowed us to image the bottommost cell layer of the sepal (inner epidermal layer) without compromising viability. We used a ‘voxel removal’ technique to visualize the inner epidermal layer in MorphoGraphX image processing software. We also describe the MorphoGraphX parameters for creating a 2.5D mesh surface for the inner epidermis. Our parameters allow for the segmentation and parent tracking of individual cells through multiple time points, despite the weak signal of the inner epidermal cells. While we have used sepals to illustrate our approach, the methodology will be useful for researchers intending to live-image and track growth of deeper cell layers in 2.5D for any plant tissue.

The development of fluorophores with photoemission beyond 1000 nm provides the opportunity to develop novel fluorescence microscopes sensitive to those wavelengths. Imaging at wavelengths beyond the visible spectrum enables imaging depths of hundreds of microns in intact tissue, making this attractive for volumetric imaging applications. Here, a novel shortwave‐infrared line‐scan confocal microscope is presented that is capable of deep imaging of biological specimens, as demonstrated by visualization of labeled glomeruli in a fixed uncleared kidney at depths beyond 400 µm. Imaging of brain vasculature labeled with the near‐infrared organic dye indocyanine green, the shortwave‐infrared organic dye Chrom7, and rare earth‐doped nanoparticles is also shown, thus encompassing the entire spectrum detectable by a typical shortwave‐infrared sensitive InGaAs detector. [ABSTRACT FROM AUTHOR]

Abstract Background Needle-free jet injection (NFJI) systems enable a controlled and targeted delivery of drugs into skin tissue. However, a scarce understanding of their underlying mechanisms has been a major deterrent to the development of an efficient system. Primarily, the lack of a suitable visualization technique that could capture the dynamics of the injected fluid–tissue interaction with a microsecond range temporal resolution has emerged as a main limitation. A conventional needle-free injection system may inject the fluids within a few milliseconds and may need a temporal resolution in the microsecond range for obtaining the required images. However, the presently available imaging techniques for skin tissue visualization fail to achieve these required spatial and temporal resolutions. Previous studies on injected fluid–tissue interaction dynamics were conducted using in vitro media with a stiffness similar to that of skin tissue. However, these media are poor substitutes for real skin tissue, and the need for an imaging technique having ex vivo or in vivo imaging capability has been echoed in the previous reports. Methods A near-infrared imaging technique that utilizes the optical absorption and fluorescence emission of indocyanine green dye, coupled with a tissue clearing technique, was developed for visualizing a NFJI in an ex vivo porcine skin tissue. Results The optimal imaging conditions obtained by considering the optical properties of the developed system and mechanical properties of the cleared ex vivo samples are presented. Crucial information on the dynamic interaction of the injected liquid jet with the ex vivo skin tissue layers and their interfaces could be obtained. Conclusions The reported technique can be instrumental for understanding the injection mechanism and for the development of an efficient transdermal NFJI system as well.

Abstract Optical imaging has been essential for scientific observations to date, however its biomedical applications has been restricted due to its poor penetration through tissues. In living tissue, signal attenuation and limited imaging depth caused by the wave distortion occur because of scattering and absorption of light by various molecules including hemoglobin, pigments, and water. To overcome this, methodologies have been proposed in the various fields, which can be mainly categorized into two stategies: developing new imaging probes and optical techniques. For example, imaging probes with long wavelength like NIR-II region are advantageous in tissue penetration. Bioluminescence and chemiluminescence can generate light without excitation, minimizing background signals. Afterglow imaging also has high a signal-to-background ratio because excitation light is off during imaging. Methodologies of adaptive optics (AO) and studies of complex media have been established and have produced various techniques such as direct wavefront sensing to rapidly measure and correct the wave distortion and indirect wavefront sensing involving modal and zonal methods to correct complex aberrations. Matrix-based approaches have been used to correct the high-order optical modes by numerical post-processing without any hardware feedback. These newly developed imaging probes and optical techniques enable successful optical imaging through deep tissue. In this review, we discuss recent advances for multi-scale optical imaging within deep tissue, which can provide reseachers multi-disciplinary understanding and broad perspectives in diverse fields including biophotonics for the purpose of translational medicine and convergence science. Graphical Abstract Methodologies for multi-scale optical imaging within deep tissues are discussed in diverse fields including biophotonics for the purpose of translational medicine and convergence science. Recent imaging probes have tried deep tissue imaging by NIR-II imaging, bioluminescence, chemiluminescence, and afterglow imaging. Optical techniques including direct/indirect and coherence-gated wavefront sensing also can increase imaging depth.

Background: Needle-free jet injection (NFJI) systems enable a controlled and targeted delivery of drugs into skin tissue. However, a scarce understanding of their underlying mechanisms has been a major deterrent to the development of an efficient system. Primarily, the lack of a suitable visualization technique that could capture the dynamics of the injected fluid–tissue interaction with a microsecond range temporal resolution has emerged as a main limitation. A conventional needle-free injection system may inject the fluids within a few milliseconds and may need a temporal resolution in the microsecond range for obtaining the required images. However, the presently available imaging techniques for skin tissue visualization fail to achieve these required spatial and temporal resolutions. Previous studies on injected fluid–tissue interaction dynamics were conducted using in vitro media with a stiffness similar to that of skin tissue. However, these media are poor substitutes for real skin tissue, and the need for an imaging technique having ex vivo or in vivo imaging capability has been echoed in the previous reports. Methods: A near-infrared imaging technique that utilizes the optical absorption and fluorescence emission of indocyanine green dye, coupled with a tissue clearing technique, was developed for visualizing a NFJI in an ex vivo porcine skin tissue. Results: The optimal imaging conditions obtained by considering the optical properties of the developed system and mechanical properties of the cleared ex vivo samples are presented. Crucial information on the dynamic interaction of the injected liquid jet with the ex vivo skin tissue layers and their interfaces could be obtained. Conclusions: The reported technique can be instrumental for understanding the injection mechanism and for the development of an efficient transdermal NFJI system as well. [ABSTRACT FROM AUTHOR]

Optical microscopy is a vital tool for visualizing the cellular and sub-cellular structures of biological specimens. However, due to its limited penetration depth, its biological applicability has been hindered. The scattering and absorption of light by a wide array of biomolecules causes signal attenuation and restricted imaging depth in tissues. Researchers have put forth various approaches to address this, including designing novel probes for imaging applications and introducing adaptive optics (AO) technology. Various techniques, such as direct wavefront sensing to quickly detect and fix wavefront deformation and indirect wavefront sensing using modal and zonal methods to rectify complex aberrations, have been developed through AO paradigms. In addition, algorithmic post-processing without mechanical feedback has been utilized to correct the optical patterns using the matrix-based method. Hence, reliable optical imaging through thick biological tissue is made possible by sensorless AO. This review highlights the latest advancements in various AO-based optical microscopy techniques for depth-resolved imaging and briefly discusses their potential in various biomedical applications. [ABSTRACT FROM AUTHOR]

Optical imaging in the 1500-1700 nm region, known as near-infrared IIb (NIR-IIb), shows potential for noninvasive in vivo detection owing to its ultrahigh tissue penetration depth and spatiotemporal resolution. Rare earth-doped nanoparticles have emerged as widely used NIR-IIb probes because of their excellent optical properties. However, their downshifting emissions rarely exhibit sufficient brightness beyond 1600 nm. This study presents tetragonal-phase thulium-doped nanoparticles (Tm 3+ -NPs) with core-shell-shell structures (CSS, LiYbF 4 :3%Tm@LiYbF 4 @LiYF 4 ) that exhibit bright downshifting luminescence at 1680 nm. Enhanced luminescence is attributed to (1) the promoted nonradiative relaxation between the doping ions and (2) the maximized sensitization process. Additionally, this strategy was validated for NIR-IIb luminescence enhancement of erbium (Er 3+ )-doped NPs. After surface modification with PEGylated liposomes, tetragonal-phase Tm 3+ -NPs exhibited a prolonged blood cycle time, high colloidal stability, and good biocompatibility. Owing to the advantages of Tm 3+ -based probes in NIR-IIb imaging, in vivo thrombus detection and monitoring of angiogenesis and arteriogenesis were successfully performed in a mouse model of ischemic hind limbs.

Optical clearing technology offers a prospective solution to improve the imaging depth and quality of optical microscopy, but there is still a lack of quantitative standards to accurately evaluate transparency effects so the composition and concentration of most reagents are not optimal. Here, we propose a transparency quantitative analysis method (TQAM) based on the tissue area recognition technique to achieve the high-throughput reagent concentration gradient screening. After optimizations of reagent composition, concentration, operation time and other parameters of the optical clearing, we develop a new ultrafast optical clearing method with quantified analysis (FOCMS) with excellent transparency effect, simple operation, improved imaging depth and quality, minor morphological change and outstanding fluorescence retention. Applied the FOCMS to an application of human brain tissue, significant differences are observed between glioma and normal human brain tissue, while these differences are difficult to be found without the assistance of FOCMS. Therefore, FOCMS shows great application potential in clinical diagnosis and treatment, pathological analysis and so on.

Featured Application: The NIRF-TGI-CT presented in this study is expected to be applied to the acquisition of information, that is, temperature, on the deep interior of samples, especially biological tissues possessing refractive index interfaces in inside that cause light scattering. We report a computed tomography (CT) technique for mapping near-infrared fluorescence (NIRF) lifetime as a multiplex three-dimensional (3D) imaging method, using a conventional NIR camera. This method is achieved by using a time-gated system composed of a pulsed laser and an NIR camera synchronized with a rotatable sample stage for NIRF-CT imaging. The fluorescence lifetimes in microsecond-order of lanthanides were mapped on reconstructed cross-sectional and 3D images, via back-projection of two-dimensional projected images acquired from multiple angles at each time point showing fluorescence decay. A method to select slopes (the observed decay rates in time-gated imaging) used for the lifetime calculation, termed as the slope comparison method, was developed for the accurate calculation of each pixel, resulting in reduction of image acquisition time. Time-gated NIRF-CT provides a novel choice for multiplex 3D observation of deep tissues in biology. [ABSTRACT FROM AUTHOR]

High-throughput deep tissue imaging and chemical tissue clearing protocols have brought out great promotion in biological research. However, due to uneven transparency introduced by tissue anisotropy in imperfectly cleared tissues, fluorescence imaging based on direct chemical tissue clearing still encounters great challenges, such as image blurring, low contrast, artifacts and so on. Here we reported a three-dimensional virtual optical clearing method based on unsupervised cycle-consistent generative adversarial network, termed 3D-VoCycleGAN, to digitally improve image quality and tissue transparency of biological samples. We demonstrated the good image deblurring and denoising capability of our method on imperfectly cleared mouse brain and kidney tissues. With 3D-VoCycleGAN prediction, the signal-to-background ratio (SBR) of images in imperfectly cleared brain tissue areas also showed above 40% improvement. Compared to other deconvolution methods, our method could evidently eliminate the tissue opaqueness and restore the image quality of the larger 3D images deep inside the imperfect cleared biological tissues with higher efficiency. And after virtually cleared, the transparency and clearing depth of mouse kidney tissues were increased by up to 30%. To our knowledge, it is the first interdisciplinary application of the CycleGAN deep learning model in the 3D fluorescence imaging and tissue clearing fields, promoting the development of high-throughput volumetric fluorescence imaging and deep learning techniques.

Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.

Intracellular probing at single cell resolution is key to revealing the heterogeneity of cells, learning new cell subtypes and functions, understanding the pathophysiology of disease, and ensuring precise diagnosis and treatment. Despite the best efforts, an enormous challenge remains due to the very small size, extremely low content, and dynamic microenvironment of a single cell. Whispering gallery mode (WGM) micro/nanolasers (active WGM) offer unique advantages of small mode volume, high quality factors, bright and low threshold laser emission, and narrow line width, particularly suitable for integration within a single cell. In this review, we provide a focused overview of WGM micro/nanolasers for intracellular probing. We deliver information on WGM micro/nanolaser concepts, sensing mechanism, and biocompatibility, as well as recent progress in intracellular probing applications mainly covering cellular-level sensing, molecular-level detection, and feasibility for cellular imaging. At the end, challenges and prospects of WGM micro/nanolasers for intracellular applications are discussed.

Optical microscopy is a vital tool for visualizing the cellular and sub-cellular structures of biological specimens. However, due to its limited penetration depth, its biological applicability has been hindered. The scattering and absorption of light by a wide array of biomolecules causes signal attenuation and restricted imaging depth in tissues. Researchers have put forth various approaches to address this, including designing novel probes for imaging applications and introducing adaptive optics (AO) technology. Various techniques, such as direct wavefront sensing to quickly detect and fix wavefront deformation and indirect wavefront sensing using modal and zonal methods to rectify complex aberrations, have been developed through AO paradigms. In addition, algorithmic post-processing without mechanical feedback has been utilized to correct the optical patterns using the matrix-based method. Hence, reliable optical imaging through thick biological tissue is made possible by sensorless AO. This review highlights the latest advancements in various AO-based optical microscopy techniques for depth-resolved imaging and briefly discusses their potential in various biomedical applications.

We describe a novel two-photon fluorescence microscopy system capable of producing high-quality second harmonic generation (SHG) images in thick turbid media by using an innovative detection system. This novel detection system is capable of detecting photons from a very large surface area. This system has proven effective in providing images of thick turbid samples, both biological and artificial. Due to its transmission detection geometry, the system is particularly suitable for detecting SHG signals, which are generally forward directed. In this article, we present comparative data acquired simultaneously on the same sample with the forward and epidetection schemes.

We report a computed tomography (CT) technique for mapping near-infrared fluorescence (NIRF) lifetime as a multiplex three-dimensional (3D) imaging method, using a conventional NIR camera. This method is achieved by using a time-gated system composed of a pulsed laser and an NIR camera synchronized with a rotatable sample stage for NIRF-CT imaging. The fluorescence lifetimes in microsecond-order of lanthanides were mapped on reconstructed cross-sectional and 3D images, via back-projection of two-dimensional projected images acquired from multiple angles at each time point showing fluorescence decay. A method to select slopes (the observed decay rates in time-gated imaging) used for the lifetime calculation, termed as the slope comparison method, was developed for the accurate calculation of each pixel, resulting in reduction of image acquisition time. Time-gated NIRF-CT provides a novel choice for multiplex 3D observation of deep tissues in biology.

Near-infrared fluorescence (NIRF) thermometry is an emerging method for the noncontact measurement of in vivo deep temperatures. Fluorescence-lifetime-based methods are effective because they are unaffected by optical loss due to excitation or detection paths. Moreover, the physiological changes in body temperature in deep tissues and their pharmacological effects are yet to be fully explored. In this study, we investigated the potential application of the NIRF lifetime-based method for temperature measurement of in vivo deep tissues in the abdomen using rare-earth-based particle materials. β-NaYF 4 particles codoped with Nd 3+ and Yb 3+ (excitation: 808 nm, emission: 980 nm) were used as NIRF thermometers, and their fluorescence decay curves were exponential. Slope linearity analysis (SLA), a screening method, was proposed to extract pixels with valid data. This method involves performing a linearity evaluation of the semilogarithmic plot of the decay curve collected at three delay times after cutting off the pulsed laser irradiation. After intragastric administration of the thermometer, the stomach temperature was monitored by using an NIRF time-gated imaging setup. Concurrently, a heater was attached to the lower abdomens of the mice under anesthesia. A decrease in the stomach temperature under anesthesia and its recovery via the heater indicated changes in the fluorescence lifetime of the thermometer placed inside the body. Thus, NaYF 4 :Nd 3+ /Yb 3+ functions as a fluorescence thermometer that can measure in vivo temperature based on the temperature dependence of the fluorescence lifetime at 980 nm under 808 nm excitation. This study demonstrated the ability of a rare-earth-based NIRF thermometer to measure deep tissues in live mice, with the proposed SLA method for excluding the noisy deviations from the analysis for measuring temperature using the NIRF lifetime of a rare-earth-based thermometer.

The development of drug delivery systems using nanocarriers requires intraorgan imaging techniques for evaluating the distribution of nanocarriers. In this study, we evaluated the tissue-clearing techniques for the imaging of polymeric nanoparticles, a nanocarrier, in the liver used as a model of pigment-rich organ in mice. The intraorgan imaging method of polymeric nanoparticles was examined without sectioning of organ samples for evaluating the delivery efficiency in preclinical studies. DiI-loaded polymeric nanoparticles and fluorescence-tagged tomato lectin for fluorescence labeling of liver general structures were intravenously administered to mice. Tissue-clearing treatment of the mouse liver was performed using ClearT2, ScaleSQ(0), clearing agent comprising fructose, urea, and glycerol for imaging (FUnGI), clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and modified CUBIC techniques. Intraorgan fluorescence imaging in the liver was performed by confocal laser microscopy. ClearT2 treatment exhibited insufficient clearing capability in the mouse liver. Although CUBIC treatment exhibited the best clearing capability, the CUBIC caused DiI leakage. ScaleSQ(0), FUnGI, and modified CUBIC treatments exhibited better clearing capability than ClearT2 technique while preserving the DiI. In the fluorescence imaging, the CUBIC and modified CUBIC exhibited deeper visualization than with the ScaleSQ(0) and FUnGI; however, the CUBIC led to a change in DiI distribution. The modified CUBIC enabled the deepest visualization while preserving the distribution of DiI. The intraorgan imaging method was established using modified CUBIC technique by the intravenous administration of fluorescence-tagged tomato lectin for evaluating the distribution of polymeric nanoparticles in mouse pigment-rich organs. [ABSTRACT FROM AUTHOR]

One of the thorny problems currently impeding the applications of the fluorescence imaging technique is the poor spatial resolution in deep tissue. Ultrasound-switchable fluorescence (USF) imaging is a novel imaging tool that has recently been explored to possibly surmount the above-mentioned bottleneck. Herein, a β-cyclodextrin/indocyanine green (ICG) complex-encapsulated poly(N-isopropylacrylamide) (PNIPAM) nanogel was synthesized and studied for ex vivo/in vivo deep tissue/high-resolution near infrared USF (NIR-USF) imaging. To be specific, our results revealed that the average diameter of the as-prepared nanogels was significantly decreased to ~ 32 nm from ~ 335 nm compared to the reported ICG-PNIPAM nanoparticles. Additionally, the excitation/ emission characteristics of the ICG itself in present nanogels were almost completely retained, and the resultant nanogel exhibited high physiological stability and positive biocompatibility. In particular, the signal-to-noise ratio of the USF image for the PNIPAM/ β-cyclodextrin/ICG nanogel (33.01 ± 2.42 dB) was prominently higher than that of the ICG-PNIPAM nanoparticles (18.73 ± 0.33 dB) in 1.5-cm-thick chicken breast tissues. The NIR-USF imaging in 3.5-cm-thick chicken breast tissues was achieved using this new probe. The ex vivo NIR-USF imaging of the mouse liver was also successfully obtained. Animal experiments showed that the present nanogels were able to be effectively accumulated into U87 tumor-bearing mice via enhanced permeability and retention effects, and the high-resolution NIR-USF imaging of in vivo tumor was efficiently acquired. The metabolism and in vivo biodistribution of the nanogels were evaluated. Overall, the results suggest that the current nanogel is a highly promising NIR-USF probe for deep tissue and high-resolution USF imaging. [ABSTRACT FROM AUTHOR]

This paper reports on microcrystals with deep tissue penetration depth, strong red light emission, and thermometric function. The red light emission of NaLuF 4 :20 % Er3+/1 % Tm3+/10 % Yb3+ microcrystals is exceptionally strong, thanks to the introduction of Tm3+ and Yb3+ ions energy capture centers. In bioimaging, the 1532 nm laser exhibits deeper imaging depth due to weak photon scattering and autofluorescence. Furthermore, red light emission has the greatest tissue penetration depth in the visible region. The luminescence intensity and tissue penetration depth of both the excitation and emission light make them suitable for deep tissue imaging. We investigated temperature sensing performance of NaLuF 4 :20 % Er3+/1 % Tm3+/10 % Yb3+ microcrystals, and found that the maximum relative sensitivity of LIR (I 521 /I 540) reaches 0.92 %K−1. The microcrystals NaLuF 4 :20 % Er3+/1 % Tm3+/10 % Yb3+ are anticipated to have application in deep tissue imaging and temperature sensing. • The red light of NaLuF 4 : Er3+ microcrystals is enhanced by the introduction of Tm3+ and Yb3+ ions. • The microcrystals NaLuF 4 :Er3+/Tm3+/Yb3+ exhibit weak photon scattering and deeper imaging depth. • The microcrystals NaLuF 4 :Er3+/Tm3+/Yb3+ exhibit favourable temperature sensing properties. • The microcrystals NaLuF 4 :Er3+/Tm3+/Yb3+ exhibit favourable properties for red light imaging. [ABSTRACT FROM AUTHOR]

High-speed, optical-sectioning imaging is highly desired in biomedical studies, as most bio-structures and bio-dynamics are in three-dimensions. Compared to point-scanning techniques, line scanning temporal focusing microscopy (LSTFM) is a promising method that can achieve high temporal resolution while maintaining a deep penetration depth. However, the contrast and axial confinement would still be deteriorated in scattering tissue imaging. Here, we propose a HiLo-based LSTFM, utilizing structured illumination to inhibit the fluorescence background and, thus, enhance the image contrast and axial confinement in deep imaging. We demonstrate the superiority of our method by performing volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo.

Targeted photoacoustic imaging using exogenous contrast agents can potentially improve early detection of breast cancer, even at significant depths inside the breast. In this study, computer simulations were performed to compare the photoacoustic performance of 11 different near-infrared (NIR) dyes for detecting tumours deep inside the breast tissue. It was observed that the three high performing NIR dyes produced at least two-fold contrast enhancement of a spherical breast tumour embedded at 4 cm depth inside the breast than those of the corresponding endogenous contrast agents. These three selected dyes were employed to visualize small blood vessels deep inside the breast tissue. Although methylene blue provided the best contrast in visualizing tumour blood vessels at depths beyond 3 cm, considering other factors such as availability of suitable targeting agent, indocyanine green at 800 nm may be preferred over all other dyes for deep breast imaging applications. [ABSTRACT FROM AUTHOR]

Surface‐enhanced Raman spectroscopy (SERS) is rapidly emerging as a bioanalytical tool for cancer diagnosis and therapy. The SERS‐based molecular diagnostics have progressed from proof‐of‐concept studies towards analysis in animal models as well as for in vitro clinical diagnostics in the last decade. Recently, SERS has also been implemented in screening, diagnosis, and staging of clinical cancer samples. Moreover, in vivo SERS imaging has been implemented for mapping the extent of tumor growth and metastasis; SERS nanoparticles have also enabled image‐guided therapies strongly indicating SERS technology can significantly complement the practice of oncology. Despite the progress, widespread clinical translation of SERS nanoparticles is still challenging. Current SERS strategies in diagnostic oncology require further development and standardization to progress from bench‐top to point‐of‐care applications. The present review critically analyzes the current state of the art about various strategies for SERS‐based cancer detection and staging from cellular metabolites, exosomes, circulating tumor cells, extracellular fluids, and cancer cells. [ABSTRACT FROM AUTHOR]

Abstract Confocal microscopy is now widely used in biological science for its high resolution and optical sectioning capability. Here we introduce the divided cosine-shaped apertures to further improve its spatial resolution and signal-to-background ratio (SBR). The results show that by adjusting the distribution frequency of cosine-shaped apertures, the axial half width at half maximum (HWHM) can be improved up to 13.5% compared with conventional circular apertures and 11.0% compared with D-shaped apertures. The transverse HWHM can be also improved. Besides, the SBR can be improved up to 12.2% compared with conventional circular apertures and 9.9% compared with D-shaped apertures. With the better performance and simple configuration, this technique shows great potential for imaging in scattering media. [ABSTRACT FROM AUTHOR]

Optical clearing is a versatile approach to improve imaging quality and depth of optical microscopy by reducing scattered light. However, conventional optical clearing methods are restricted in the efficiency-first applications due to unsatisfied time consumption, irreversible tissue deformation, and fluorescence quenching. Here, we developed an ultrafast optical clearing method (FOCM) with simple protocols and common reagents to overcome these limitations. The results show that FOCM can rapidly clarify 300-µmthick brain slices within 2 min. Besides, the tissue linear expansion can be well controlled by only a 2.12% increase, meanwhile the fluorescence signals of GFP can be preserved up to 86% even after 11 d. By using FOCM, we successfully built the detailed 3D nerve cells model and showed the connection between neuron, astrocyte, and blood vessel. When applied to 3D imaging analysis, we found that the foot shock and morphine stimulation induced distinct c-fos pattern in the paraventricular nucleus of the hypothalamus (PVH). Therefore, FOCM has the potential to be a widely used sample mounting media for biological optical imaging. [ABSTRACT FROM AUTHOR]

Measurements of flow velocities at the level of individual arterial vessels and sinusoidal capillaries are crucial for understanding the dynamics of hematopoietic stem and progenitor cell homing in the bone marrow vasculature. We have developed two complementary intravital two-photon imaging approaches to determine blood flow dynamics and velocities in multiple vessel segments by capturing the motion of red blood cells. High-resolution spatiotemporal measurements through a cranial window to determine short-time dynamics of flowing blood cells and repetitive centerline scans were used to obtain a detailed flow-profile map with hemodynamic parameters. In addition, we observed the homing of individual hematopoietic stem and progenitor cells and obtained detailed information on their homing behavior. With our imaging setup, we determined flow patterns at cellular resolution, blood flow velocities and wall shear stress in small arterial vessels and highly branched sinusoidal capillaries, and the cellular dynamics of hematopoietic stem and progenitor cell homing.

Vascular diseases are becoming an epidemic with an increasing aging population and increases in obesity and type II diabetes. Point-of-care (POC) diagnosis and monitoring of vascular diseases is an unmet medical need. Photoacoustic imaging (PAI) provides label-free multiparametric information of deep vasculature based on strong absorption of light photons by hemoglobin molecules. However, conventional PAI systems use bulky nanosecond lasers which hinders POC applications. Recently, light-emitting diodes (LEDs) have emerged as cost-effective and portable optical sources for the PAI of living subjects. However, state-of-art LED arrays carry significantly lower optical energy (

Abstract: Propagation loss is a key impeding parameter affecting accuracy, range, and applicability of fluorescent microscopy techniques. In this work, a new nanophotonic platform for propagation loss mitigated fluorescent imaging using lifetime ratiometric measurement is proposed. By applying this methodology to local parameter sensing, a method for high sensitivity local refractive index measurement via lifetime/energy monitoring of fluorescent crystal color centers is numerically demonstrated. As the base of this platform, biocompatible diamond nanodisk structures are investigated that enable sensitive, propagation loss‐immune monitoring of refractive index changes both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Deep tissue imaging in the multiple scattering regime remains at the frontier of fluorescence microscopy. Speckle correlation imaging (SCI) can computationally uncover objects hidden behind a scattering layer, but has only been demonstrated with scattered laser illumination and in geometries where the scatterer is in the far field of the target object. Here, SCI is extended to imaging a planar fluorescent signal at the back surface of a 500‐μm‐thick slice of mouse brain. The object is reconstructed from a single snapshot through phase retrieval using a proximal algorithm that easily incorporates image priors. Simulations and experiments demonstrate improved image recovery with this approach compared to the conventional SCI algorithm. [ABSTRACT FROM AUTHOR]

Preclinical fluorescence imaging with NIR-I (700 to 900 nm) illumination and short-wave infrared or NIR-II (1000 to 1700 nm) emission increases tissue penetration depth and improves resolution through decreased scattering. Background autofluorescence decreases signal-to-background ratios (SBR) in fluorescence imaging; maximizing SBR will further improve the impact of deep tissue imaging. The impact of rodent diet, illumination wavelength, and emission range on the background fluorescence and contrast agent SBR were determined to assist with the experimental design of future imaging studies. Following illumination with 670, 760, or 808 nm, autofluorescence in the NIR-I (<975 nm), NIR-II (>1000 nm), and NIR-II LP (>1250 nm) regions was assessed in mice fed chow or a purified diet using an IR VIVO preclinical imager (Photon, Etc.). Comparison of the SBR of liver-localized indocyanine green in the various imaging conditions indicated when gut autofluorescence was a problematic confounder. Mice fed chow exhibit high levels of background autofluorescence in the gastrointestinal tract and, to a lesser extent, skin when illuminated with 670 nm light for NIR-I imaging (700 to 975 nm), interfering with the identification of fluorescently labeled tissue. Background autofluorescence was reduced by more than two orders of magnitude by any of the following changes: (1) purified diet; (2) excitation with 760 or 808 nm illumination; or (3) emission in the NIR-II (1000 to 1600 or 1250 to 1600 nm). Although the SBR was generally sufficient for feature identification except when imaging of chow-fed mice with 670 nm excitation and NIR-I emission, switching to a purified diet, using longer excitation wavelengths, or using longer emission wavelengths improved SBR significantly. Systematic comparison of imaging conditions and diet highlights the reduction in autofluorescence and increase in SBR enabled by intentional choices in the experimental parameters including diet, excitation wavelength, and emission wavelength range. [ABSTRACT FROM AUTHOR]

Development of nanoparticles with multi-functionalities is of great importance. In this study, praseodymium sulfide (Pr _2 S _3 ) and molybdenum sulfide (MoS _2 ) nanoparticles were synthesized. The structural, morphological and optical properties of the as-obtained products were investigated by XRD, XPS, TEM, UV–vis-NIR spectroscopy, and photoluminescence spectroscopy. Pr _2 S _3 is found to be used in selective photodegradation of fluorescein sodium salt. MoS _2 can be utilized for selective photodegradation of rhodamine B. In the mixture of rhodamine B, fluorescein sodium salt and rhodamine 6 G, most of rhodamine B and part of fluorescein sodium salt are optically degraded by Pr _2 S _3 . In the mixture of rhodamine B, fluorescein sodium salt and rhodamine 6 G, part of fluorescein sodium salt and most of rhodamine B is degraded by MoS _2 . Moreover, they emit near-infrared fluorescence (800–1100 nm) when excited by the 785 nm light. Deep tissues imaging with high-contrast is shown, utilizing a nanoparticle-filled centrifuge tube covered with animal tissues (pig Bacon meat). Maximum imaging depth below the tissue surface of 1 cm is achieved. Our work provides a rapid yet efficient procedure to make nanoparticles for dual-application-potential in dye-photodegradation and near-infrared deep tissue imaging.

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep. [ABSTRACT FROM AUTHOR]

A novel mitochondria-targeted ratiometric two-photon fluorescent probe ( Mito-MQ ) for detecting intracellular cysteine (Cys) and homocysteine (Hcy) has been designed. Mito-MQ showed the ratiometric fluorescent detection signal (the green-to-blue emissionfrom 517 nm to 460 nm) to cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH), along with the fast response rate (10 min). The detection mechanism was illustrated by 1 H NMR, ESI-MS and theoretical calculation. The co-localization coefficient of 0.87 between Mito-MQ and MitoTracker Red revealed that the probe was predominantly present in mitochondria, therefore, Mito-MQ was successfully applied to detect mitochondrial oxidative stress by detecting the change of Cys/Hcy. Moreover, imaging in fresh tissue slices indicated that Mito-MQ could work in deep tissue (ca. 130 µm) under two-photon excitation. Furthermore, the measurement of Cys/Hcy detection in zebrafish showed that probe can be used in determination of biothiols in vivo. [ABSTRACT FROM AUTHOR]

While lasers have been commonly used as illumination sources in photoacoustic (PA) imaging, their high purchase and maintenance costs, as well as their bulkiness, have hindered the rapid clinical dissemination of PA imaging. With this in mind, we explore an alternative illumination source for PA tomography-a xenon flash lamp with high pulse energy and a microsecond pulse width. We demonstrate that, by using a single xenon flash lamp, we can image both a black latex cord placed in chicken breast tissue at a depth of up to 3.5 cm ex vivo and an entire mouse body in vivo. Our findings indicate that the xenon flash lamp, producing optical illumination that is safe for humans, can be potentially applied to human tissue imaging. [ABSTRACT FROM AUTHOR]

[Display omitted] • Efficacious Förster-type energy transfer occurs from DOTA-Eu to ZSCE PLNPs. • ZDE PLNPs enable 3.9-fold luminous brightness and 1.4-fold lifetime over bare PLNPs. • PLNPs perform high contrast imaging and accurate tumor boundary depicting ability. • Breast tumor excision is conducted under the guidance of persistent luminescence. • Lanthanide-chelate strategy could serve as an extensible approach to optimize PLNPs. Persistent luminescence (PersL) nanoparticles (PLNPs) with excitation-afterglow emission separation character, have brought luciferous prospect for ultrasensitive optical detection to circumvent the weakness of clinical fluorescence modality, benefiting from the strengths of autofluorescence-free, low phototoxicity and superior photostability. Nevertheless, small dimensions of PLNPs (<200 nm, suitable for in vivo diagnosis) synthesized via current procedures are inevitably accompanied with PersL brightness sacrifice. Whereas the past few years have witnessed advances to promote the performance of nanophosphor, proposing a versatile and sustainable approach to boost the excitation absorption capacity, thus amplifying the PersL is of industrywide urgency. Here, we first report a "lanthanide-chelate sensitization" strategy involving Eu-DOTA as strong photon harvester and Zn 2 SnO 4 : Cr3+, Eu3+ (ZSCE) PLNPs as matched acceptor to steadily augment the intensity of obtained ZDE PLNPs via efficacious Förster-type energy transfer. Photobleaching resistant ZDE PLNPs with different DOTA-Eu substitutions enable maximal 3.9-fold luminous brightness and 1.4-fold decay lifetime over bare PLNPs. Cladded with hyaluronic acid (HA), ZDE@HA PLNPs performed high signal-to-noise ratio imaging and accurate tumors boundary depicting which facilitates PersL-guided breast tumors excision. Strikingly, the "Ln-chelate sensitization" concept could serve as an extensible approach to optimize existing PLNPs, which lightens up new perspectives for ultra-contrastive malignancy navigation. [ABSTRACT FROM AUTHOR]