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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]

Contrast-enhanced ultrasound (CEUS) presents distinct advantages in diagnostic echography. Utilizing microbubbles (MBs) as conventional contrast agents enhances vascular visualization and organ perfusion, facilitating real-time, non-invasive procedures. There is a current tendency to replace traditional polydisperse MBs with novel monodisperse formulations in an attempt to optimize contrast enhancement and guarantee consistent behavior and reliable imaging outcomes. This study investigates the contrast enhancement achieved using various-sized monodisperse MBs and their influence on non-linear imaging artifacts observed in traditional CEUS. To explore the differences between monodisperse and polydisperse populations without excessive experimentation, numerical simulations are employed for delivering precise, objective and expeditious results. The iterative non-linear contrast source (INCS) method has previously demonstrated efficacy when simulating ultrasound propagation in large populations in which each bubble has individual properties and several orders of multiple scattering are significant. Therefore, this method is employed to realistically simulate both monodisperse and polydisperse MBs. Our findings in CEUS imaging indicate that scattering from resonant monodisperse MBs is 11.8 dB stronger than scattering from polydisperse MBs. Furthermore, the amplitude of non-linear imaging artifacts downstream of the monodisperse population is 19.4 dB stronger compared with polydisperse suspension. Investigating the impact of multiple scattering on polydisperse populations compared with various monodisperse suspensions has revealed that monodisperse MBs are more effective contrast agents, especially when at resonance. Despite the strong signal-to-noise ratio of monodisperse populations, imaging artifacts caused by non-linear wave propagation are also enhanced, resulting in further mis-classification of MBs as tissue. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

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]

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]

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]

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]

Photoacoustic microscopy (PAM) is a scalable bioimaging modality; one can choose low acoustic resolution with deep penetration depth or high optical resolution with shallow imaging depth. High spatial resolution and deep penetration depth is rather difficult to achieve using a single system. Here we report a switchable acoustic resolution and optical resolution photoacoustic microscopy (AR-OR-PAM) system in a single imaging system capable of both high resolution and low resolution on the same sample. Lateral resolution of 4.2 μm (with ~1.4 mm imaging depth) and lateral resolution of 45 μm (with ~7.6 mm imaging depth) was successfully demonstrated using a switchable system. In vivo blood vasculature imaging was also performed for its biological application. [ABSTRACT FROM AUTHOR]

Measurement of photon penetration in biological tissues is a central theme in optical imaging. A great number of endogenous tissue factors such as absorption, scattering, and anisotropy affect the path of photons in tissue, making it difficult to predict the penetration depth at different wavelengths. Traditional studies evaluating photon penetration at different wavelengths are focused on tissue spectroscopy that does not take into account the heterogeneity within the sample. This is especially critical in shortwave infrared where the individual vibration- based absorption properties of the tissue molecules are affected by nearby tissue components. We have explored the depth penetration in biological tissues from 900 to 1650 nm using Monte-Carlo simulation and a hyperspectral imaging system with Michelson spatial contrast as a metric of light penetration. Chromatic aberration-free hyperspectral images in transmission and reflection geometries were collected with a spectral resolution of 5.27 nm and a total acquisition time of 3 min. Relatively short recording time minimized artifacts from sample drying. Results from both transmission and reflection geometries consistently revealed that the highest spatial contrast in the wavelength range for deep tissue lies within 1300 to 1375 nm; however, in heavily pigmented tissue such as the liver, the range 1550 to 1600 nm is also prominent. [ABSTRACT FROM AUTHOR]

Viewing subcellular details over large tissue volumes is becoming an essential condition of the success of large-scale projects aimed at visualizing cell connections in whole organs or tissues. However, tissue opacity remains an obstacle to deep tissue imaging. This situation has brought renewed interest for techniques of tissue clearing; new protocols, such as CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel), have recently been developed. So far, most of the tests of these techniques have been applied to brain or other soft tissues. Here we show that CLARITY clears mouse hindlimb skeletal muscles and maintains the basic structural features of muscle and its fibers. However, tagging with fluorescent markers was not successful. [ABSTRACT FROM AUTHOR]

The highly toxic N-nitrosodiethylamine (NDEA) and hydrazine (N 2 H 4) caused severe environmental contamination and serious health risks. Herein, we designed the two-photon ratiometric fluorescent probe (Nap-2), emission maximum shifted from 466 nm to 571 nm, to monitor cell viability of NDEA induced acute hepatitis via esterase activity detection. Furthermore, the probe Nap-2 evaluate the hydrazine (N 2 H 4) content in the solution and gas phase. It is worth mentioning that we used NDEA induced acute hepatitis in the mice and evaluated the negative correlation of esterase activity in the tissue cells and serum with Nap-2. The probe Nap-2 exhibited that acute hepatitis induced by NDEA decreased cell viability. Furthermore, we made convenient test papers using Nap-2 to detect N 2 H 4 in solution and gas phase. After adding N 2 H 4 , the fluorescence color changed from blue to yellow and was visible to the naked eye. This work provides a convenient tool and method for evaluating the toxicity of NDEA induced acute hepatitis and detecting N 2 H 4 in the environment. [Display omitted] • NDEA-induced acute hepatitis resulted in decreased cell viability. • Nap-2 can detect liquid and gaseous N 2 H 4 with the test papers. • The dual-function probe Nap-2 was developed to detect esterase activity and N 2 H 4. • Cyclohexane ring (Nap-2) have the best substrate for esterase detection. [ABSTRACT FROM AUTHOR]

In this study, the imaging methods for evaluating the kinetics of nanoparticles as drug delivery systems in tumor tissues were improved in BxPC3 tumor-bearing mice. First, Förster resonance energy transfer (FRET) live imaging was selected to quantitatively evaluate nanoparticle kinetics in the tumor tissue of mice. Briefly, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodide (as an acceptor)–and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (as a donor)-coloaded nanoparticles were administered intravenously to the mice, and imaging was performed using a fluorescence in vivo imager. The fluorescence intensities of images were acquired in the FRET, donor, and acceptor channels, and the nanoparticle kinetics in the tumor region was quantified by compensating for bleed-through. Second, in the cleared tumor tissue of mice, the difference in evaluation properties between the two- and three-dimensional visualization of the nanoparticles was examined. In brief, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-loaded nanoparticles were intravenously administered to the mice after fluorescently labeled tomato lectin treatment to visualize tumor vessels. Excised tumor tissue was cleared and observed using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. The three-dimensional minimum distances traveled by DiI from the tumor vessels were calculated using information about the two-dimensional distance and the slicing position using the Pythagoras theorem. These imaging techniques should facilitate the development of drug delivery systems for cancer. [Display omitted] • Nanoparticle dissociation was detected by FRET imaging. • FRET live in vivo imaging clarified nanoparticle behavior in tumor tissue. • Nanoparticle three-dimensional distribution was assessed in cleared tumor tissue. • The penetration distances from tumor vessels were calculated three-dimensionally. [ABSTRACT FROM AUTHOR]

Ytterbium (Yb3+)-sensitized upconverting nanoparticles (UCNPs) are excited at 975 nm causing relatively high absorption in tissue. A new type of UCNPs with neodymium (Nd3+) and Yb3+ codoping is excitable at a 808-nm wavelength. At this wavelength, the tissue absorption is lower. Here we quantify, both experimentally and theoretically, to what extent Nd3+-doped UCNPs will provide an increased signal at larger depths in tissue compared to conventional 975-nm excited UCNPs. [ABSTRACT FROM AUTHOR]

White matter (WM) loss is a critical event after spinal cord injury (SCI). Conventionally, such loss has been measured with histological and histochemical approaches, although the procedures are complex and may cause artifact. Recently, coherent Raman microscopy has been proven to be an emerging technology to study de- and remyelination of the injured spinal cord; however, limited penetration depth and small imaging field prevent it from comprehensive assessments of large areas of damaged tissues. Here, we report the use of bond-selective photoacoustic (PA) imaging with 1730-nm excitation, where the first overtone vibration of CH2 bond is located, to assess WM loss after a contusive SCI in adult rats. By employing the first overtone vibration of CH2 bond as the contrast, the mapping of the WM in an intact spinal cord was achieved in a label-free three-dimensional manner, and the physiological change of the spinal cord before and after injury was observed. Moreover, the recovery of the spinal cord from contusive injury with the treatment of a neuroprotective nanomedicine ferulic-acid-conjugated glycol chitosan (FA-GC) was also observed. Our study suggests that bond-selective PA imaging is a valuable tool to assess the progression of WM pathology after SCI as well as neuroprotective therapeutics in a label-free manner. [ABSTRACT FROM AUTHOR]

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca2+ (BCa-1 , BCa-2 mito , and BCa-3 mem), with absorption maxima (λ max) at 350–358 nm, emission maxima (λ fl) at 464–466 nm, and TP action cross-section (Φδ max) values of 55–70 × 10−50 cm4s/photon, in the presence of excess Ca2+ at 750 nm. Moreover, the probes had dissociation constants of 0.18, 2.7, and 100 μM, respectively, which are appropriate values for sensing Ca2+ in the cytoplasm, mitochondria, and plasma membrane, respectively. The measurements were conducted using a calcium calibration buffer (10 mM 3-[ N -morpholino]propanesulfonic acid and 100 mM KCl) at pH 7.2. The TP microscopy results revealed that the probes could facilitate the real-time detection of Ca2+ in the cytoplasm, mitochondria, and plasma membranes of live cells and tissues. Additionally, we developed a green-emitting TP probe for H+ (FHEt-1 lyso) with λ max = 359 nm, λ fl = 571 nm, and Φδ max = 70 × 10−50 cm4s/photon at pH 4.3 in a universal buffer (0.1 M citric acid, 0.1 M KH 2 PO 4 , 0.1 M Na 2 B 4 O 7 , 0.1 M tris[hydroxymethyl]aminomethane, and 0.1 M KCl); this probe could detect H+ in the lysosomes. Using BCa-1 and FHEt-1 lyso , it was possible to simultaneously monitor the changes in cytosolic Ca2+ and lysosomal H+ concentrations in live cells and tissues using dual-color TP microscopy in real time. When used with TP probes emitting wavelengths of green light or longer, these blue-emitting Ca2+ probes can be used to investigate the physiological role of Ca2+ in cellular organelles as well as the crosstalk between Ca2+ and other metal ions in specific organelles. [Display omitted] • We developed organelle-specific blue-emitting two-photon Ca2+ probes. • They can detect Ca2+ in organelles of living cells and tissues in real-time. • They have appropriate K d values for Ca2+ for each organelle. • They show high sensitivity, low pH dependency, and negligible cytotoxicity. • They can monitor the crosstalk among metal ions using multicolor TPM imaging. [ABSTRACT FROM AUTHOR]

Advances in bioimaging have revolutionized our ability to study life phenomena at a microscopic scale. In particular, the stimulated emission process, a universal mechanism that competes with spontaneous emission, has emerged as a powerful driving force for advancing light microscopy. The present review summarizes and compares three related techniques that each measure a different physical quantity involved in the stimulated emission process in order to tackle various challenges in light microscopy. Stimulated emission depletion microscopy, which detects the residual fluorescence after quenching, can break the diffraction-limited resolution barrier in fluorescence microscopy. Stimulated emission microscopy is capable of imaging nonfluorescent but absorbing chromophores by detecting the intensity gain of the stimulated emission beam. Very recently, stimulated emission reduced fluorescence microscopy has been proposed, in which the reduced fluorescence due to focal stimulation is measured to extend the fundamental imaging-depth limit of two-photon microscopy. Thus, through ingenious spectroscopy design in distinct microscopy contexts, stimulated emission has opened up several new territories for bioimaging, allowing examination of biological structures that are ever smaller, darker, and deeper. [ABSTRACT FROM AUTHOR]

Gold nanoparticles can be used as molecular contrast agents binding specifically to cancer sites and thus delineating tumor regions. Imaging gold nanoparticles deeply embedded in tissues with optical techniques possesses significant challenges due to multiple scattering of optical photons that blur the obtained images. Both diag- nostic and therapeutic applications can benefit from a minimally invasive technique that can identify, localize, and quantify the payloads of gold nanoparticles deeply embedded in biological tissues. An optical radiance technique is applied to map localized inclusions of gold nanorods in 650- to 900-nm spectral range in a porcine phantom that mimics prostate geometry. Optical radiance defines a variation in the angular density of photons impinging on a selected point in the tissue from various directions. The inclusions are formed by immersing a capillary filled with gold nanorods in the phantom at increasing distances from the detecting fiber. The technique allows the isolation of the spectroscopic signatures of the inclusions from the background and identification of inclusion locations in the angular domain. Detection of ~4 x 1010 gold nanoparticles or 0.04 mg Au/mL (detector-inclusion separation 10 mm, source-detector separation 15 mm) in the porcine tissue is demonstrated. The encouraging results indicate a promising potential of radiance spectroscopy in early prostate cancer diagnostics with gold nanoparticles [ABSTRACT FROM AUTHOR]

In this paper, we demonstrate 3-D photoacoustic imaging (PAI) of light absorbing objects embedded as deep as 5 cm inside strong optically scattering phantoms using a miniaturized (4 mm × 4 mm × 500 μm), 2-D capacitive micromachined ultrasonic transducer (CMUT) array of 16 × 16 elements with a center frequency of 5.5 MHz. Two-dimensional tomographic images and 3-D volumetric images of the objects placed at different depths are presented. In addition, we studied the sensitivity of CMUT-based PAI to the concentration of indocyanine green dye at 5 cm depth inside the phantom. Under optimized experimental conditions, the objects at 5 cm depth can be imaged with SNR of about 35 dB and a spatial resolution of approximately 500 μm. Results demonstrate that CMUTs with integrated front-end amplifier circuits are an attractive choice for achieving relatively high depth sensitivity for PAI. [ABSTRACT FROM PUBLISHER]

Developments in nanotechnology have paved the way for the early detection, treatment, and prevention of several tumors which affect mankind. In the past few years, near-infrared (NIR) fluorescence imaging techniques have emerged that enable the in vivo imaging of physiological, metabolic, and molecular function. The NIR window, also known as the diagnostic window (700–900 nm), can be explored for sensitive detection techniques. Nanoparticles, particularly semiconductor quantum dots (QDs), can be utilized for the purpose of optical imaging. These semiconductor QDs possess novel electronic, optical, magnetic, and structural properties which are quite different from those of bulk materials. NIR QDs with these unique properties can be utilized as contrast agents for optical imaging, particularly for deep tissue imaging. Deep tissue imaging provides more information about the pathological status of the disease, which makes the treatment more effective and efficient. In this review we highlight the importance of NIR QDs as probes for optical imaging. We describe the different types of NIR QDs, their synthesis, and their application for deep tissue imaging along with recently developed self-illuminating NIR QDs. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 μm within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of ≤10–15 μm into the sample, further compounding the ability to image at high-resolution deep within tissue. We show that manipulating the refractive index of the mounting media and decreasing sample opacity greatly improves image quality such that the limiting factor for a standard, inverted multi-photon microscope is determined by the working distance of the objective as opposed to detectable fluorescence. This method negates the need for mechanical sectioning of tissue and enables the routine generation of high-quality, quantitative image data that can significantly advance our understanding of tissue architecture and physiology. [ABSTRACT FROM AUTHOR]

Abstract: Intravital microscopy using two-photon excitation is proven to be a valuable tool for studying the kidney and associated disease processes. However, routine performance of intravital kidney imaging is limited by the fact that fluorescence signal is attenuated by the tissue and at certain tissue depth lost its strength completely. For most of the animal tissues, this finite imaging depth is limited to a few hundred microns. Currently it is not possible to non-invasively image the kidney beyond the superficial tissue layers of the cortex. This has imposed significant limitations on the animal models one can use for imaging since structure such the glomerulus is typically located below the superficial layer of the cortex that cannot be imaged using a conventional fluorescence microscope. Here we report the use of a needle-like lens system based on gradient-index (GRIN) microlenses capable of transferring high quality fluorescence images of the tissue through a regular microscope objective for deep tissue imaging of the kidney. By combining this GRIN lens system with a Zeiss LSM 510 NLO microscope, we are able to extend the depth for imaging kidney tissues far beyond the few hundred microns limit. This GRIN lens imaging system provides an alternative microendoscopic imaging tool that will enhance current intravital kidney imaging techniques for studying structural and functional properties of local tissues at locations below the superficial layers of the kidney. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Bi 2 Ga 4 O 9 :Cr3+(BGOC) is first reported as a kind of persistent luminescence and CT multimodel imaging nanoprobes. Chemical molecules and folic acid (FA) biological molecules were modified on the surface, and the X ray was used as the re-activated light source replacement of traditional visible light, it could be realized for a long time and high sensitivity of persistent luminescence and CT dual mode imaging in the depth-independent tumor in vivo. • Non-coreshell multimodel imaging BGOC nanoparticles were synthesized by two steps method. • X-rays re-activated simultaneously realize persistent luminescence and CT imaging in vivo. • BGOC-FA nanoprobes realize persistent luminescence and CT tumor imaging in vivo. Synthesis of excellent imaging nanoparticles plays a critical role in disease detection. Here, novel Bi 2 Ga 4 O 9 :Cr (BGOC) nanoparticles were synthesized for persistent luminescence and computed tomography dual-mode imaging. The Cr3+ ions in the BGOC luminescent nanoparticles were coped as the luminescent center, and the afterglow emission was located in the first biological imaging window. Bi3+ ions served as the computed tomography (CT) imaging contrast agent. The NH 2 groups were modified on the surface of the nanoparticles to make them highly biocompatible, and folic acid molecules were modified to facilitate the specificity. This modification facilitated simultaneous persistent luminescence and CT imaging in tumor-bearing mice due to the overexpression of the folic acid molecules' receptors. Furthermore, instead of visible light, x-rays were used as the excitation light source to activate the BGOC-folic acid nanoprobes and realize long time and high sensitivity imaging of tumors in vivo because of their capacity for deep tissue penetration. Therefore, such nanoprobes can provide a promising depth-independent detection mode for high sensitivity diagnosis and long-term monitoring of diseases. [ABSTRACT FROM AUTHOR]

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 (<0.5 mJ/pulse) and high pulse repetition frequencies (PRFs) (4 KHz) compared to the high-power laser sources (100 mJ/pulse) with low PRFs of 10 Hz. Given these tradeoffs between portability, cost, optical energy and frame rate, this work systematically studies the deep tissue PAI performance of LED and laser illuminations to help select a suitable source for a given biomedical application. To draw a fair comparison, we developed a fiberoptic array that delivers laser illumination similar to the LED array and uses the same ultrasound transducer and data acquisition platform for PAI with these two illuminations. Several controlled studies on tissue phantoms demonstrated that portable LED arrays with high frame averaging show higher signal-to-noise ratios (SNRs) of up to 30 mm depth, and the high-energy laser source was found to be more effective for imaging depths greater than 30 mm at similar frame rates. Label-free in vivo imaging of human hand vasculature studies further confirmed that the vascular contrast from LED-PAI is similar to laser-PAI for up to 2 cm depths. Therefore, LED-PAI systems have strong potential to be a mobile health care technology for diagnosing vascular diseases such as peripheral arterial disease and stroke in POC and resource poor settings. [ABSTRACT FROM AUTHOR]

We present three-dimensional (3-D) high-resolution spectral-domain optical coherence microscopy (SD-OCM) by using a supercontinuum (SC) fiber laser source with 300-nm spectral bandwidth (full-width at half-maximum) in the 1700-nm spectral band. By using low-coherence interferometry with SC light and a confocal detection scheme, we realized lateral and axial resolutions of 3.4 and 3.8 μm in tissue (n = 1.38), respectively. This is, to the best of our knowledge, the highest 3-D spatial resolution reported among those of Fourier-domain optical coherence imaging techniques in the 1700-nm spectral band. In our SD-OCM, to enhance the imaging depth, a full-range method was implemented, which suppressed the formation of a coherent ghost image and allowed us to set the zero-delay position inside the samples. We demonstrated the 3-D high-resolution imaging capability of 1700-nm SD-OCM through the measurement of an interference signal from a mirror surface and imaging of a single 200-nm polystyrene bead and a pig thyroid gland. Deep tissue imaging at a depth of up to 1.8 mm was also demonstrated. This is the first demonstration of 3-D high-resolution SD-OCM in the 1700-nm spectral band. [ABSTRACT FROM AUTHOR]