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Owing to reduced light scattering and tissue autofluorescence, in vivo fluorescence imaging in the 1,000–3,000-nm near-infrared II (NIR-II) spectral range can afford non-invasive imaging at depths of millimetres within biological tissue. Infrared fluorescent probes labelled with antibodies or other targeting ligands also enable NIR-II molecular imaging at the single-cell level. Here we present recent developments in the design of fluorophores and probes emitting in the NIR-II window based on organic synthesis and nanoscience approaches. We also review advances in NIR-II wide-field and microscopy imaging modalities, with a focus on preclinical imaging and promising clinical translation case studies. Finally, we outline current issues and challenges for the wider adoption of NIR-II imaging in biomedical research and clinical imaging. A review of NIR-II fluorescence imaging is presented, with a focus on fluorophores, probes and imaging techniques.

Two-photon fluorescence microscopy 1 enables scientists in various fields including neuroscience 2 , 3 , embryology 4 and oncology 5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue 3 or the insertion of optical probes 6 , 7 . Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue. Three-photon microscopy performed at the infrared wavelength of 1,700 nm makes it possible to image hard-to-reach vascular structures and labelled neurons in the hippocampus of a mouse brain.

Many polarisation techniques have been harnessed for decades in biological and clinical research, each based upon measurement of the vectorial properties of light or the vectorial transformations imposed on light by objects. Various advanced vector measurement/sensing techniques, physical interpretation methods, and approaches to analyse biomedically relevant information have been developed and harnessed. In this review, we focus mainly on summarising methodologies and applications related to tissue polarimetry, with an emphasis on the adoption of the Stokes–Mueller formalism. Several recent breakthroughs, development trends, and potential multimodal uses in conjunction with other techniques are also presented. The primary goal of the review is to give the reader a general overview in the use of vectorial information that can be obtained by polarisation optics for applications in biomedical and clinical research.The review focuses on methodologies and biomedical applications of polarisation optics. It also presents prospects on development trends, the potential multi-modal uses in conjunction with other techniques.

Histological staining is the gold standard for tissue examination in clinical pathology and life-science research, which visualizes the tissue and cellular structures using chromatic dyes or fluorescence labels to aid the microscopic assessment of tissue. However, the current histological staining workflow requires tedious sample preparation steps, specialized laboratory infrastructure, and trained histotechnologists, making it expensive, time-consuming, and not accessible in resource-limited settings. Deep learning techniques created new opportunities to revolutionize staining methods by digitally generating histological stains using trained neural networks, providing rapid, cost-effective, and accurate alternatives to standard chemical staining methods. These techniques, broadly referred to as virtual staining , were extensively explored by multiple research groups and demonstrated to be successful in generating various types of histological stains from label-free microscopic images of unstained samples; similar approaches were also used for transforming images of an already stained tissue sample into another type of stain, performing virtual stain-to-stain transformations. In this Review, we provide a comprehensive overview of the recent research advances in deep learning-enabled virtual histological staining techniques. The basic concepts and the typical workflow of virtual staining are introduced, followed by a discussion of representative works and their technical innovations. We also share our perspectives on the future of this emerging field, aiming to inspire readers from diverse scientific fields to further expand the scope of deep learning-enabled virtual histological staining techniques and their applications. Deep Learning enables virtual histological staining of biological samples.

In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing mammals. However, the definition of the NIR-II region and the mechanism accounting for the excellent performance still need to be perfected. Herein, we simulate the photon propagation in the NIR region (to 2340 nm), confirm the positive contribution of moderate light absorption by water in intravital imaging and perfect the NIR-II window as 900–1880 nm, where 1400–1500 and 1700–1880 nm are defined as NIR-IIx and NIR-IIc regions, respectively. Moreover, 2080–2340 nm is newly proposed as the third near-infrared (NIR-III) window, which is believed to provide the best imaging quality. The wide-field fluorescence microscopy in the brain is performed around the NIR-IIx region, with excellent optical sectioning strength and the largest imaging depth of intravital NIR-II fluorescence microscopy to date. We also propose 1400 nm long-pass detection in off-peak NIR-II imaging whose performance exceeds that of NIR-IIb imaging, using bright fluorophores with short emission wavelength.Moderate light absorption by bio-tissue is conducive to the imaging performance. The second near-infrared window is perfected as 900–1880 nm, and 2080–2340 nm is proposed as the third near-infrared window.

Lasers are the pillars of modern optics and sensing. Microlasers based on whispering-gallery modes (WGMs) are miniature in size and have excellent lasing characteristics suitable for biosensing. WGM lasers have been used for label-free detection of single virus particles, detection of molecular electrostatic changes at biointerfaces, and barcode-type live-cell tagging and tracking. The most recent advances in biosensing with WGM microlasers are described in this review. We cover the basic concepts of WGM resonators, the integration of gain media into various active WGM sensors and devices, and the cutting-edge advances in photonic devices for micro- and nanoprobing of biological samples that can be integrated with WGM lasers.

Optical scattering limits the ability to image or focus beneath one millimetre of tissue in biomedical optics. This Review summarizes recently developed 'guidestar' mechanisms that provide feedback for intra-tissue focusing. In the field of biomedical optics, optical scattering has traditionally limited the range of imaging within tissue to a depth of one millimetre. A recently developed class of wavefront-shaping techniques now aims to overcome this limit and achieve diffraction-limited control of light beyond one centimetre. By manipulating the spatial profile of an optical field before it enters a scattering medium, it is possible to create a micrometre-scale focal spot deep within tissue. To successfully operate in vivo , these wavefront-shaping techniques typically require feedback from within the biological sample. This Review summarizes recently developed 'guidestar' mechanisms that provide feedback for intra-tissue focusing. Potential applications of guidestar-assisted focusing include optogenetic control over neurons, targeted photodynamic therapy and deep tissue imaging.

Holography is a powerful tool to record waves without loss of information that has benefited optical, X-ray and electronic imaging applications by quantifying phase delays induced by light–matter interactions. However, holographic imaging is technically demanding in that it generally requires an interferometric setup, a coherent source and long-term stability. Here, we present holographic imaging in which a phase image is obtained directly from a single intensity measurement in oblique illumination. Our approach is based on space-domain Kramers–Kronig relations that transform the spatial variation in intensity to the spatial variation in phase. We demonstrate two-dimensional holographic imaging and three-dimensional refractive index tomography of microscopic objects and biological specimens from intensity images measured with an optical microscope and illumination control. The proposed method does not require iterative processes nor strict constraints and opens up a new approach to non-interferometric holographic imaging in various spectral regimes.An intensity-based holographic imaging via space-domain Kramers–Kronig relations is presented, allowing the phase image of an object to be obtained directly from a single intensity measurement with oblique illumination.

Single-photon avalanche diode (SPAD) arrays are solid-state detectors that offer imaging capabilities at the level of individual photons, with unparalleled photon counting and time-resolved performance. This fascinating technology has progressed at a very fast pace in the past 15 years, since its inception in standard CMOS technology in 2003. A host of architectures have been investigated, ranging from simpler implementations, based solely on off-chip data processing, to progressively “smarter” sensors including on-chip, or even pixel level, time-stamping and processing capabilities. As the technology has matured, a range of biophotonics applications have been explored, including (endoscopic) FLIM, (multibeam multiphoton) FLIM-FRET, SPIM-FCS, super-resolution microscopy, time-resolved Raman spectroscopy, NIROT and PET. We will review some representative sensors and their corresponding applications, including the most relevant challenges faced by chip designers and end-users. Finally, we will provide an outlook on the future of this fascinating technology.Monitoring progress of single light particle detectors for biophotonics applicationsSubstantial improvements have been made in the past 15 years to imagers based on a device that acts like a 3-in-1 light particle detector, counter and stopwatch, furthering their potential use in biological imaging technologies. Claudio Bruschini of Switzerland’s École polytechnique fédérale de Lausanne and colleagues reviewed the developments in the use of single-photon avalanche diode (SPAD) arrays for biophotonics applications. They found that, while most SPAD imagers are still used in specialised research settings, significant improvements have been made to their sensitivity, reliability and reproducibility, and a host of sensor architectures have been explored. When a photon, the unit of light, hits a SPAD, it triggers an almost immediate electric current. Compact SPAD arrays implemented in standard CMOS electronic circuits can thus be used, for example, to measure in parallel the fluorescence lifetime of molecules tagged onto living cells and tissues, improving our ability to observe them. The review was completed by an outlook of this fascinating technology and an analysis of the most relevant challenges still lying ahead.

We introduce a microscopic method that determines quantitative optical properties beyond the optical diffraction limit and allows direct imaging of unstained living biological specimens. In established holographic microscopy, complex fields are measured using interferometric detection, allowing diffraction-limited phase measurements. Here, we show that non-invasive optical nanoscopy can achieve a lateral resolution of 90 nm by using a quasi-2π-holographic detection scheme and complex deconvolution. We record holograms from different illumination directions on the sample plane and observe subwavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction. The observations are shown for nanoscopic porous cell frustule (diatoms), for the direct study of bacteria ( Escherichia coli ), and for a time-lapse approach to explore the dynamics of living dendritic spines (neurones). By recording digital holograms created from different illumination directions and subsequently processing them in a complex deconvolution scheme, scientists are able to capture details of living biological samples with subwavelength resolution.