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

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

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca²⁺‐sensitive protein kinase C α fusion protein are demonstrated.     

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography.      

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

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

Organic Field‐Effect Transistor for Energy‐Related Applications: Low‐Power‐Consumption Devices,Near‐Infrared Phototransistors,and Organic Thermoelectric Devices

The organic field‐effect transistor (OFET) is the basic building block of integrated circuits. The charge carrier mobility and operating frequency of OFETs have continued to increase; therefore, the power dissipation of OFETs can no longer be ignored. Many research efforts have been made to develop low‐power‐consumption OFETs and complementary circuits. Despite the switching function, OFETs can also be utilized in emerging energy‐related applications, such as near‐infrared (NIR) photodetectors and organic thermoelectric devices. Organic phototransistors show considerably higher photo responsivity than other photodetector architectures due to field‐effect charge modulation. The photoinduced gate modulating largely suppresses the dark current while simultaneously providing gain. These characteristics may favor NIR light detection and suggest that the organic phototransistor is a promising candidate for optoelectronic applications in the NIR regime. For organic thermoelectric applications, OFETs can work as a powerful tool for examining the charge and energy transport in the organic semiconductor, thus giving insight into organic thermoelectric studies. In this review, the authors highlight recent advances in OFET‐ related energy topics, including low‐power‐consumption OFETs, NIR photodetectors, and organic thermoelectric devices. The remaining challenges in the field will also be discussed.     

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

Structured illumination imaging with quasi‐periodic patterns

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

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Functional near infrared spectroscopy using spatially resolved data to account for tissue scattering: A numerical study and arm‐cuff experiment

Functional Near‐Infrared Spectroscopy (fNIRS) aims to recover changes in tissue optical parameters relating to tissue hemodynamics, to infer functional information in biological tissue. A widely‐used application of fNIRS relies on continuous wave (CW) methodology that utilizes multiple distance measurements on human head for study of brain health. The typical method used is spatially resolved spectroscopy (SRS), which is shown to recover tissue oxygenation index (TOI) based on gradient of light intensity measured between two detectors. However, this methodology does not account for tissue scattering which is often assumed. A new parameter recovery algorithm is developed, which directly recovers both the scattering parameter and scaled chromophore concentrations and hence TOI from the measured gradient of light‐attenuation at multiple wavelengths. It is shown through simulations that in comparison to conventional SRS which estimates cerebral TOI values with an error of ±12.3%, the proposed method provides more accurate estimate of TOI exhibiting an error of ±5.7% without any prior assumptions of tissue scatter, and can be easily implemented within CW fNIRS systems. Using an arm‐cuff experiment, the obtained TOI using the proposed method is shown to provide a higher and more realistic value as compared to utilizing any prior assumptions of tissue scatter.     

Sleep‐like behavior and 24‐h rhythm disruption in the Tc1 mouse model of Down syndrome

Down syndrome is a common disorder associated with intellectual disability in humans. Among a variety of severe health problems, patients with Down syndrome exhibit disrupted sleep and abnormal 24‐h rest/activity patterns. The transchromosomic mouse model of Down syndrome, Tc1, is a trans‐species mouse model for Down syndrome, carrying most of human chromosome 21 in addition to the normal complement of mouse chromosomes and expresses many of the phenotypes characteristic of Down syndrome. To date, however, sleep and circadian rhythms have not been characterized in Tc1 mice. Using both circadian wheel‐running analysis and video‐based sleep scoring, we showed that these mice exhibited fragmented patterns of sleep‐like behaviour during the light phase of a 12:12‐h light/dark (LD) cycle with an extended period of continuous wakefulness at the beginning of the dark phase. Moreover, an acute light pulse during night‐time was less effective in inducing sleep‐like behaviour in Tc1 animals than in wild‐type controls. In wheel‐running analysis, free running in constant light (LL) or constant darkness (DD) showed no changes in the circadian period of Tc1 animals although they did express subtle behavioural differences including a reduction in total distance travelled on the wheel and differences in the acrophase of activity in LD and in DD. Our data confirm that Tc1 mice express sleep‐related phenotypes that are comparable with those seen in Down syndrome patients with moderate disruptions in rest/activity patterns and hyperactive episodes, while circadian period under constant lighting conditions is essentially unaffected.     

Three‐dimensional visualization system for ophthalmic microscopes using visible light and near‐infrared illumination

In this paper, we describe a three‐dimensional visualization system for ophthalmic microscopes that is aimed at microsurgery without the eyepieces. A three‐dimensional visualization system for ophthalmic microscopes using the mixed illumination, which consists of visible light and near‐infrared illumination, is established in order to acquire more exact information of object and reduce the amount of light irradiated to the patients, and its usage in microsurgery without eyepieces is herein described. A custom‐designed stereoscopic three‐dimensional display which is manufactured for the convenience of the surgeons during the long‐time surgery, is connected directly to the camera of the ophthalmic microscope in order to eliminate the discomfort of eyepieces to the surgeon and signal delay between the camera, mounted on the microscope, and display device for surgeon. The main features of the established system are the signal delay‐free for surgeon and the low level of illumination for patient. In particular, it could significantly reduce the amount of light irradiated on a patient's eye via NIR illumination. Upon comparison with the conventional system during clinical ophthalmology trials, this system is confirmed to require almost the same operation time and reduced discomfort and eyestrain during long periods of observation.      

In vivo study of rat cortical hemodynamics using a stereotaxic‐apparatus‐compatible photoacoustic microscope

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic‐apparatus‐compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2‐dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm². Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.      

Design,synthesis and photocytotoxicity of upconversion nanoparticles: Potential applications for near‐infrared photodynamic and photothermal therapy

Photodynamic therapy (PDT) and photothermal therapy (PTT) are emerging modalities for the treatment of tumors and nonmalignant conditions, based on the use of photosensitizers to generate singlet oxygen or heat, respectively, upon light (laser) irradiation. They have potential advantages over conventional treatments, being minimally invasive with precise spatial‐temporal selectivity and reduced side effects. However, most clinically employed PDT agents are activated at visible (vis) wavelengths for which the tissue penetration and, hence, effective treatment depth are compromised. In addition, the lipophilicity of near‐infrared (NIR) photothermal agents limits their use and efficiency. To achieve combined PDT/PTT effects, both excitation wavelengths need to be tuned into the NIR spectral window of biological tissues. This paper reports the synthesis of neodymium‐doped upconversion nanoparticles (NaYF₄:Yb,Er,Nd@NaYF₄:Nd) that convert 800 nm light into vis wavelengths, which can then activate conventional photosensitizers on the nanoparticle surface for PDT. Covalently bonded IR‐780 dyes can readily be activated by 800 nm laser irradiation. The PEGylated nanoplatform exhibited a narrow size distribution, good stability and efficient generation of singlet oxygen under laser irradiation. The in vitro photocytotoxicity of this engineered nanoplatform as either a PDT or PTT agent in HeLa cells is demonstrated, while fluorescence microscopy in nanoplatform‐incubated cells highlights its potential for bioimaging.     

Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo

The main objective of this work is to quantify the impact of photodynamic/photothermal treatment by using visible LED and NIR laser irradiation through the skin of subcutaneous fat in vivo followed up by tissue sampling and histology. The optical method may provide reduction of regional or site‐specific accumulations of abdominal or subcutaneous adipose tissue precisely and least‐invasively by inducing cell apoptosis and controlled necrosis of fat tissue. As photodynamic/photothermal adipose tissue sensitizers Brilliant Green (BG) or Indocyanine Green (ICG) dyes were injected subcutaneously in rats. The CW LED device (625 nm) or CW diode laser (808 nm) were used as light sources, respectively. Biopsies of skin together with subcutaneous tissues were taken for histology. The combined action BG‐staining and LED‐irradiation (BG + LED) or ICG‐staining and NIR‐laser irradiation (ICG + NIR) causes pronounced signs of damage of adipose tissue characterized by a strong stretching, thinning, folding and undulating of cell membranes and appearance of necrotic areas. As a posttreatment after 14 days only connective tissue was observed at the site of necrotic areas. The data obtained are important for safe light treatment of site‐specific fat accumulations, including cellulite. This work provides a basis for the development of fat lipolysis technologies and to move them to clinical applications. Schematics of animal experiment.      

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD‐FLIM system based on a complementary metal‐oxide semiconductor camera equipped with two‐tap true correlated double sampling lock‐in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31‐harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean‐Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD‐FLIM system is a valuable tool in the biomedical field.      

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.      

Signal generation and Raman‐resonant imaging by non‐degenerate four‐wave mixing under tight focusing conditions

The authors demonstrate Raman‐resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3‐color coherent anti‐Stokes Raman scattering (CARS) experiment. The interaction of three coincident short‐pulsed laser beams simultaneously generates both 2‐color (degenerate) CARS and 3‐color (non‐degenerate) CARS signals, which are collected and characterized spectroscopically – allowing for resonant, doubly‐resonant, and non‐resonant contrast mechanisms. Images obtained from both 2‐color and 3‐color CARS signals are compared and found to provide complementary information. The 3‐color CARS microscopy scheme provides a versatile multiplexed modality for biological imaging, which may extend the capabilities of label‐free non‐linear microscopy, e.g. by probing multiple Raman resonances. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A label‐free immunoassay using eggshell membrane as matrix and poly(diallyldimethylammonium chloride) as light‐scattering enhancer

A label‐free immunoassay system using eggshell membrane as a matrix was developed. A common spectrofluorometer was used to collect light‐scattering signals. The rabbit anti‐human IgG (Ab) was first immobilized on the eggshell membrane with glutaraldehyde. Then, based on the immunoreactions and electrostatic interaction, the target human IgG antigen (Ag) and poly(diallyldimethylammonium chloride) (PDDA) were captured on the eggshell membrane. It was found that the light‐scattering signal resulting from the PDDA immunotargeted on modified eggshell membrane was related to the concentration of target antigen. Under the optimal conditions, the light scattering intensity is directly proportional to the concentration of Ag in the range of 5.00–500 ng/mL (r = 0.995) with the limit of detection of 2.31 ng/mL [signal:noise ratio (S:N) = 3]. The proposed method was successfully applied to the determination of IgG in human serum, and the results were in agreement with those obtained by a general immunonephelometric method. Copyright © 2011 John Wiley & Sons, Ltd.