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Volumetric subcellular imaging has long been essential for studying structures and dynamics in cells and tissues. However, due to limited imaging speed and depth of field, it has been challenging to perform live-cell imaging and single-particle tracking. Here we report a 2.5D fluorescence microscopy combined with highly inclined illumination beams, which significantly reduce not only the image acquisition time but also the out-of-focus background by ∼2-fold compared to epi-illumination. Instead of sequential z-scanning, our method projects a certain depth of volumetric information onto a 2D plane in a single shot using multi-layered glass for incoherent wavefront splitting, enabling high photon detection efficiency. We apply our method to multi-color immunofluorescence imaging and volumetric super-resolution imaging, covering ∼3–4 µm thickness of samples without z-scanning. Additionally, we demonstrate that our approach can substantially extend the observation time of single-particle tracking in living cells. [ABSTRACT FROM AUTHOR]

Wide‐field imaging is widely adopted due to its fast acquisition, cost‐effectiveness, and ease of use. Its extension to direct volumetric applications, however, is burdened by the trade‐off between resolution and depth of field (DOF), dictated by the numerical aperture of the system. It is demonstrated that such trade‐off is not intrinsic to wide‐field imaging, but stems from the spatial incoherence of light: images obtained through spatially coherent illumination are shown to have resolution and DOF independent of the numerical aperture. This fundamental discovery enables to demonstrate an optimal combination of coherent resolution‐DOF enhancement and incoherent tomographic sectioning for scanning‐free, wide‐field 3D microscopy on a multicolor histological section. [ABSTRACT FROM AUTHOR]

Optical brain clearing combined with immunolabeling is valuable for analyzing molecular tissue structures, including complex synaptic connectivity. However, the presence of aberrant lipid deposition due to aging and brain disorders poses a challenge for achieving antibody penetration throughout the entire brain volume. Herein, we present an efficient brain-wide immunolabeling method, the immuno-active clearing technique (iACT). The treatment of brain tissues with a zwitterionic detergent, specifically SB3-12, significantly enhanced tissue permeability by effectively mitigating lipid barriers. Notably, Quadrol treatment further refines the methodology by effectively eliminating residual detergents from cleared brain tissues, subsequently amplifying volumetric fluorescence signals. Employing iACT, we uncover disrupted axonal projections within the mesolimbic dopaminergic (DA) circuits in 5xFAD mice. Subsequent characterization of DA neural circuits in 5xFAD mice revealed proximal axonal swelling and misrouting of distal axonal compartments in proximity to amyloid-beta plaques. Importantly, these structural anomalies in DA axons correlate with a marked reduction in DA release within the nucleus accumbens. Collectively, our findings highlight the efficacy of optical volumetric imaging with iACT in resolving intricate structural alterations in deep brain neural circuits. Furthermore, we unveil the compromised integrity of DA pathways, contributing to the underlying neuropathology of Alzheimer's disease. The iACT technique thus holds significant promise as a valuable asset for advancing our understanding of complex neurodegenerative disorders and may pave the way for targeted therapeutic interventions. The axonal projection of DA neurons in the septum and the NAc showed dystrophic phenotypes such as growth cone-like enlargement of the axonal terminus and aggregated neurites. Brain-wide imaging of structural defects in the neural circuits was facilitated with brain clearing and antibody penetration assisted with SB3-12 and Quadrol pre-treatment. The whole volumetric imaging process could be completed in a week with the robust iACT method. Created with https://www.biorender.com/. [ABSTRACT FROM AUTHOR]

Abstract Background To evaluate the effectiveness of the computed tomographic (CT) volumetric analysis in postoperative lung function assessment and the predicting value for postoperative complications in patients who had segmentectomy for lung cancer. Methods CT scanning and pulmonary function examination were performed for 100 patients with lung cancer. CT volumetric analyses were performed by specific software, for the volume of the inspiratory phase (Vin), the mean inspiratory lung density (MLDin), the volume of expiratory phase (Vex), and the mean lung density at expiratory phase (MLDex). Pulmonary function examination results and CT volumetric analysis results were used to predict postoperative lung function. The concordance and correlations of these values were assessed by Bland-Altman analysis and Pearson correlation analysis, respectively. Multivariate binomial logistic regression analysis was executed to assess the associations of CT data with complication occurrence. Results Correlations between CT scanning data and pulmonary function examination results were significant in both pre- and post-operation (0.8083 ≤ r ≤ 0.9390). Forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and the ratio of FVC and FEV1 estimated by CT volumetric analyses showed high concordance with those detected by pulmonary function examination. Preoperative (Vin-Vex) and (MLDex- MLDin) values were identified as predictors for post-surgery complications, with hazard ratios of 5.378 and 6.524, respectively. Conclusions CT volumetric imaging analysis has the potential to determine the pre- and post-operative lung function, as well as to predict post-surgery complication occurrence in lung cancer patients with pulmonary lobectomy.

Background: To evaluate the effectiveness of the computed tomographic (CT) volumetric analysis in postoperative lung function assessment and the predicting value for postoperative complications in patients who had segmentectomy for lung cancer. Methods: CT scanning and pulmonary function examination were performed for 100 patients with lung cancer. CT volumetric analyses were performed by specific software, for the volume of the inspiratory phase (Vin), the mean inspiratory lung density (MLDin), the volume of expiratory phase (Vex), and the mean lung density at expiratory phase (MLDex). Pulmonary function examination results and CT volumetric analysis results were used to predict postoperative lung function. The concordance and correlations of these values were assessed by Bland-Altman analysis and Pearson correlation analysis, respectively. Multivariate binomial logistic regression analysis was executed to assess the associations of CT data with complication occurrence. Results: Correlations between CT scanning data and pulmonary function examination results were significant in both pre- and post-operation (0.8083 ≤ r ≤ 0.9390). Forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and the ratio of FVC and FEV1 estimated by CT volumetric analyses showed high concordance with those detected by pulmonary function examination. Preoperative (Vin-Vex) and (MLDex- MLDin) values were identified as predictors for post-surgery complications, with hazard ratios of 5.378 and 6.524, respectively. Conclusions: CT volumetric imaging analysis has the potential to determine the pre- and post-operative lung function, as well as to predict post-surgery complication occurrence in lung cancer patients with pulmonary lobectomy. [ABSTRACT FROM AUTHOR]

To uncover the underlying fluid mechanisms, it is crucial to explore imaging techniques for high-resolution and large-scale three-dimensional (3D) measurements of the flow field. Plenoptic background-oriented schlieren (Plenoptic BOS), an emerging volumetric method in recent years, has demonstrated being able to resolve volumetric flow dynamics with a single plenoptic camera. The focus-stack-based plenoptic BOS system can qualitatively infer the position of the density gradient in 3D space based on the relative sharpness of the refocused BOS image. Plenoptic BOS systems based on tomography or specular enhancement techniques are realized for use in high-fidelity 3D flow measurements due to the increased number of acquisition views. Here, we first review the fundamentals of plenoptic BOS, and then discuss the system configuration and typical application of single-view and multi-view plenoptic BOS. We also discuss the related challenges and outlook on the potential development of plenoptic BOS in the future. [ABSTRACT FROM AUTHOR]

Vascular diseases may occur in the upper extremities, and the lesions can span the entire length of the blood vessel. One of the most popular methods to identify vascular disorders is ultrasound Doppler imaging. However, traditional two-dimensional (2D) ultrasound Doppler imaging cannot capture the entire length of a long vessel in one image. Medical professionals often have to painstakingly reconstruct three-dimensional (3D) data using 2D ultrasound images to locate the lesions, especially for large blood vessels. 3D ultrasound Doppler imaging can display the morphological structure of blood vessels and the distribution of lesions more directly, providing a more comprehensive view compared to 2D imaging. In this work, we propose a wide-range 3D volumetric ultrasound Doppler imaging system with dual modality, in which a high-definition camera is adopted to automatically track the movement of the ultrasound transducer, simultaneously capturing a corresponding sequence of 2D ultrasound Doppler images. We conducted experiments on human arms using our proposed system and separately with X-ray computerized tomography (X-CT). The comparison results prove the potential value of our proposed system in the diagnosis of arm vascular diseases. [ABSTRACT FROM AUTHOR]

Significance: Two-photon fluorescence microscopy (TPFM) excited by Gaussian beams requires axial tomographic scanning for three-dimensional (3D) volumetric imaging, which is a time-consuming process, and the slow imaging speed hinders its application for in vivo brain imaging. The Bessel focus, characterized by an extended depth of focus and constant resolution, facilitates the projection of a 3D volume onto a two-dimensional image, which significantly enhances the speed of volumetric imaging. Aim: We aimed to demonstrate the ability of a TPFM with a sidelobe-free Bessel beam to provide a promising tool for research in live biological specimens. Approach: Comparative in vivo imaging was conducted in live mouse brains and transgenic zebrafish to evaluate the performance of TPFM and Bessel-beam-based TPFM. Additionally, an image-difference method utilizing zeroth-order and thirdorder Bessel beams was introduced to effectively suppress background interference introduced by sidelobes. Results: In comparison with traditional TPFM, the Bessel-beams-based TPFM demonstrated a 30-fold increase in imaging throughput and speed. Furthermore, the effectiveness of the image-difference method was validated in live biological specimens, resulting in a substantial enhancement of image contrast. Importantly, our TPFM with a sidelobe-free Bessel beam exhibited robustness against axial displacements, a feature of considerable value for in vivo experiments. Conclusions: We achieved rapid, high-contrast, and robust volumetric imaging of the vasculature in live mouse brains and transgenic zebrafish using our TPFM with a sidelobe-free Bessel beam. [ABSTRACT FROM AUTHOR]

This paper introduces an innovative approach to integral volumetric imaging employing time and polarization multiplexing techniques to present volumetric three-dimensional images. Traditional integral volumetric imaging systems with a coarse lens array often face moiré pattern issues stemming from layered panel structures. In response, our proposed system utilizes a combination of time and polarization multiplexing to achieve two focal planes using a single display panel. [ABSTRACT FROM AUTHOR]

Chromatic confocal technology has enabled multispectral information detection without any axial mechanical scanning. Especially, the chromatic confocal sensor is well applied in industry, such as device defect detection and ranging. However, for 3D imaging, chromatic confocal microscopy (CCM) still suffers from insufficient speed due to the slow refresh rate of a traditional spectrometer. To address this problem, a time‐stretch chromatic confocal microscopy (TSCCM) is presented for multi‐depth imaging. A supercontinuum laser is stretched and then focused on the sample using a home‐built chromatic lens, which disperses the laser to a different depth. The time‐of‐flight signal is collected by a high‐speed photodiode and is recorded and analyzed by a 3 GHz digitizer. A novel approach is achieved to significantly improve the speed of multi‐depth imaging with an up to 1 MHz A‐scan rate and 5 Hz volumetric imaging speed, which is an order faster compared with previous work of spectrometer‐based chromatic confocal microscopy. Multi‐depth volumetric imaging of nude‐mouse skin is performed to show the potential for biomedical applications. In this method, with its high A‐scan rate and multi‐depth imaging capability, a virtual tissue "light detection and ranging (LIDAR)" may be achieved. [ABSTRACT FROM AUTHOR]

The potential of microRNA regulation in oncotherapy is limited by the lack of delivery vehicles. Herein, it is shown that fluoropolymer coated DNA nanoclews (FNCs) provide outstanding ability to deliver oligonucleotide through circulation and realize near infrared (NIR) light activated angiogenesis suppression to abrogate tumors. Oligonucleotides are loaded in DNA nanoclews through sequence specific bindings and then a fluorinated zwitterionic polymer is coated onto the surface of nanoclews. Further incorporating quantum dots in the polymer coating endows the vectors with NIR‐IIb (1500–1700 nm) fluorescence and NIR light triggered release ability. The FNC vector can deliver oligonucleotides to cancer cells systemically and realize on‐demand cytosolic release of the cargo with high transfection efficiency. Taking advantage of the NIR‐IIb emission, the whole delivery process of FNCs is visualized volumetrically in vivo with a NIR light sheet microscope. Loaded by FNCs, an oligonucleotide can effectively silence the target miRNA when activated with NIR light, and inhibit angiogenesis inside tumor, leading to complete ablation of cancer. These findings suggest FNCs can be used as an efficient oligonucleotide delivery platform to modulate the expression of endogenous microRNA in gene therapy of cancer. [ABSTRACT FROM AUTHOR]

Multispectral optoacoustic tomography (MSOT) is a beneficial technique for diagnosing and analyzing biological samples since it provides meticulous details in anatomy and physiology. However, acquiring high through‐plane resolution volumetric MSOT is time‐consuming. Here, we propose a deep learning model based on hybrid recurrent and convolutional neural networks to generate sequential cross‐sectional images for an MSOT system. This system provides three modalities (MSOT, ultrasound, and optoacoustic imaging of a specific exogenous contrast agent) in a single scan. This study used ICG‐conjugated nanoworms particles (NWs‐ICG) as the contrast agent. Instead of acquiring seven images with a step size of 0.1 mm, we can receive two images with a step size of 0.6 mm as input for the proposed deep learning model. The deep learning model can generate five other images with a step size of 0.1 mm between these two input images meaning we can reduce acquisition time by approximately 71%. [ABSTRACT FROM AUTHOR]

Optical aberration is a crucial issue in optical microscopes, fundamentally constraining the achievable imaging performance. As a commonly encountered one, spherical aberration is introduced by the refractive index mismatches between samples and their surrounding environments, leading to problems like low contrast, blurring, and distortion in imaging. Light‐field microscopy (LFM) has recently emerged as a powerful tool for rapid volumetric imaging. The presence of spherical aberration in LFM will cause substantial changes of the point spread function (PSF) and thus greatly affects the imaging performance. Here, the aberration‐modeling view‐channel‐depth (AM‐VCD) network is proposed for LFM reconstruction, effectively mitigating the influence of severe spherical aberration. By quantitatively estimating the spherical aberration in advance and incorporating it into the network training, the AM‐VCD achieves aberration‐corrected high‐speed visualization of 3D processes with uniform spatial resolution and real‐time reconstruction speed. Without necessitating hardware modifications, this method provides a convenient way for directly observing the 3D dynamics of samples in solution. The capability of AM‐VCD under a large refractive index mismatch is demonstrated through volumetric imaging of a large‐scale fishbone of a largemouth bass. Furthermore, the capability of AM‐VCD in nearly 100 Hz volumetric imaging of neutrophil migration and a beating heart in living zebrafish is investigated. [ABSTRACT FROM AUTHOR]

To uncover the underlying fluid mechanisms, it is crucial to explore imaging techniques for high-resolution and large-scale three-dimensional (3D) measurements of the flow field. Plenoptic background-oriented schlieren (Plenoptic BOS), an emerging volumetric method in recent years, has demonstrated being able to resolve volumetric flow dynamics with a single plenoptic camera. The focus-stack-based plenoptic BOS system can qualitatively infer the position of the density gradient in 3D space based on the relative sharpness of the refocused BOS image. Plenoptic BOS systems based on tomography or specular enhancement techniques are realized for use in high-fidelity 3D flow measurements due to the increased number of acquisition views. Here, we first review the fundamentals of plenoptic BOS, and then discuss the system configuration and typical application of single-view and multi-view plenoptic BOS. We also discuss the related challenges and outlook on the potential development of plenoptic BOS in the future.

Abstract The potential of microRNA regulation in oncotherapy is limited by the lack of delivery vehicles. Herein, it is shown that fluoropolymer coated DNA nanoclews (FNCs) provide outstanding ability to deliver oligonucleotide through circulation and realize near infrared (NIR) light activated angiogenesis suppression to abrogate tumors. Oligonucleotides are loaded in DNA nanoclews through sequence specific bindings and then a fluorinated zwitterionic polymer is coated onto the surface of nanoclews. Further incorporating quantum dots in the polymer coating endows the vectors with NIR‐IIb (1500–1700 nm) fluorescence and NIR light triggered release ability. The FNC vector can deliver oligonucleotides to cancer cells systemically and realize on‐demand cytosolic release of the cargo with high transfection efficiency. Taking advantage of the NIR‐IIb emission, the whole delivery process of FNCs is visualized volumetrically in vivo with a NIR light sheet microscope. Loaded by FNCs, an oligonucleotide can effectively silence the target miRNA when activated with NIR light, and inhibit angiogenesis inside tumor, leading to complete ablation of cancer. These findings suggest FNCs can be used as an efficient oligonucleotide delivery platform to modulate the expression of endogenous microRNA in gene therapy of cancer.

A computational three‐dimensional (3D) microscopy technique, termed programmable aperture light‐field microscopy (PALFM), for motion‐free, high‐resolution volumetric imaging of fluorescent or self‐luminous samples is proposed. The well‐known Fourier slice theorem is extended to incoherent tomographic imaging, which states that a detected image under an ideal aperture corresponds to a central slice in the 3D object spectrum, so the spectrum coverage can be accomplished based on the motion‐free aperture modulation. When further considering frequency extension and coverage, a hybrid aperture modulation scheme is designed consisting of non‐centrosymmetric circular and annular apertures for high‐efficiency, non‐ambiguous depth discrimination. A PALFM system with an easy‐to‐build programmable aperture module attached to an off‐the‐shelf inverted fluorescence microscope is constructed, where annular apertures can manipulate Bessel‐like beams for spectrum modulation. Experimental results on near‐diffraction‐limited imaging of a resolution target across a large depth range and high‐resolution, multi‐color 3D imaging of a mouse kidney section verify the validity and effectiveness of PALFM. High‐speed, long‐term time‐lapse volumetric imaging of HeLa cells in vitro further demonstrates that PALFM is a promising tomographic imaging tool for studying dynamic cellular processes and events without requiring complicated sample rotation or beam scanning. [ABSTRACT FROM AUTHOR]

Advancements in microscopy techniques have revolutionized our ability to explore the intricacies of biological systems, with engineered human heart tissue (EHT) being a particularly challenging target. In this article, we will provide an in-depth look at the award-winning SOLIS technique (scanned oblique light-sheet instant-volume sectioning), a new twist on multifocus fluorescence microscopy. Through its unique capabilities, SOLIS offers a new approach to organoid and tissue imaging, providing unprecedented insights into the cellular architecture of these complex artificial samples. By being able to record optically sectioned volumes during single-camera exposures, SOLIS demonstrates remarkable advantages over traditional multifocus microscopy, underscoring its potential to transform our understanding of developmental biology, disease mechanisms, and potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

Three-dimensional single-pixel imaging (3D SPI) has become an attractive imaging modality for both biomedical research and optical sensing. 3D-SPI techniques generally depend on time-of-flight or stereovision principle to extract depth information from backscattered light. However, existing implementations for these two optical schemes are limited to surface mapping of 3D objects at depth resolutions, at best, at the millimeter level. Here, we report 3D light-field illumination single-pixel microscopy (3D-LFI-SPM) that enables volumetric imaging of microscopic objects with a near-diffraction-limit 3D optical resolution. Aimed at 3D space reconstruction, 3D-LFI-SPM optically samples the 3D Fourier spectrum by combining 3D structured light-field illumination with single-element intensity detection. We build a 3D-LFI-SPM prototype that provides an imaging volume of ~390 × 390 × 3,800 µm³ and achieves 2.7-µm lateral resolution and better than 37-µm axial resolution. Its capability of 3D visualization of label-free optical absorption contrast is demonstrated by imaging single algal cells in vivo. Our approach opens broad perspectives for 3D SPI with potential applications in various fields, such as biomedical functional imaging. [ABSTRACT FROM AUTHOR]

Volumetric imaging of a mouse brain in vivo with one-photon and two-photon ultralong anti-diffracting (UAD) beam illumination was performed. The three-dimensional (3D) structure of blood vessels in the mouse brain were mapped to a two-dimensional (2D) image. The speed of volumetric imaging was significantly improved due to the long focal length of the UAD beam. Comparing one-photon and two-photon UAD beam volumetric imaging, we found that the imaging depth of two-photon volumetric imaging (80 μm) is better than that of one-photon volumetric imaging (60 μm), and the signal-to-background ratio (SBR) of two-photon volumetric imaging is two times that of one-photon volumetric imaging. Therefore, we used two-photon UAD volumetric imaging to perform dynamic volumetric imaging of mouse brain blood vessels in vivo, and obtained the blood flow velocity. [ABSTRACT FROM AUTHOR]

The volumetric imaging of two-photon microscopy expands the focal depth and improves the throughput, which has unparalleled superiority for three-dimension samples, especially in neuroscience. However, emerging in volumetric imaging is still largely customized, which limits the integration with commercial two-photon systems. Here, we analyzed the key parameters that modulate the focal depth and lateral resolution of polarized annular imaging and proposed a volumetric imaging module that can be directly integrated into commercial two-photon systems using conventional optical elements. This design incorporates the beam diameter adjustment settings of commercial two-photon systems, allowing flexibility to adjust the depth of focus while maintaining the same lateral resolution. Further, the depth range and lateral resolution of the design were verified, and the imaging throughput was demonstrated by an increase in the number of imaging neurons in the awake mouse cerebral cortex. [ABSTRACT FROM AUTHOR]

The volumetric imaging of two-photon microscopy expands the focal depth and improves the throughput, which has unparalleled superiority for three-dimension samples, especially in neuroscience. However, emerging in volumetric imaging is still largely customized, which limits the integration with commercial two-photon systems. Here, we analyzed the key parameters that modulate the focal depth and lateral resolution of polarized annular imaging and proposed a volumetric imaging module that can be directly integrated into commercial two-photon systems using conventional optical elements. This design incorporates the beam diameter adjustment settings of commercial two-photon systems, allowing flexibility to adjust the depth of focus while maintaining the same lateral resolution. Further, the depth range and lateral resolution of the design were verified, and the imaging throughput was demonstrated by an increase in the number of imaging neurons in the awake mouse cerebral cortex.

Single-cell volumetric imaging is essential for researching individual characteristics of cells. As a nonscanning imaging technique, light field microscopy (LFM) is a critical tool to achieve real-time three-dimensional imaging with the advantage of single-shot. To address the inherent limits including nonuniform resolution and block-wise artifacts, various modified LFM strategies have been developed to provide new insights into the structural and functional information of cells. This review will introduce the principle and development of LFM, discuss the improved approaches based on hardware designs and 3D reconstruction algorithms, and present the applications in single-cell imaging. [ABSTRACT FROM AUTHOR]

Optical‐resolution photoacoustic microscopy suffers from limited depth of field due to the strongly focused laser beam. Here, a novel volumetric information fusion is proposed to achieve large volumetric and high‐resolution imaging. First, three‐dimensional stationary wavelet transform was performed on the multi‐focus data to obtain eight wavelet coefficients. Differential evolution based on joint weighted evaluation was then employed to optimize the block size of division for each wavelet coefficient. The proposed fusion rule using standard deviation for focus detection was used to fuse the corresponding sub‐coefficients. Finally, photoacoustic imaging with large depth of field can be achieved by the inverse stationary wavelet transform. Performance test shows that the depth of field of photoacoustic imaging can be doubled without sacrificing lateral resolution. The proposed volumetric information fusion can further promote the capability of volumetric imaging of optical‐resolution photoacoustic microscopy and will be helpful in the acquisition of physiological and pathological process. [ABSTRACT FROM AUTHOR]

Purpose: To quantitatively evaluate the achievable performance of volumetric imaging based on lung motion modeling by principal component analysis (PCA). Methods: In volumetric imaging based on PCA, internal deformation was represented as a linear combination of the eigenvectors derived by PCA of the deformation vector fields evaluated from patient-specific four-dimensionalcomputed tomography (4DCT) datasets. The volumetric image was synthesized by warping the reference CT image with a deformation vector field which was evaluated using optimal principal component coefficients (PCs). Larger PCs were hypothesized to reproduce deformations larger than those included in the original 4DCT dataset. To evaluate the reproducibility of PCA-reconstructed volumetric images synthesized to be close to the ground truth as possible, mean absolute error (MAE), structure similarity index measure (SSIM) and discrepancy of diaphragm position were evaluated using 22 4DCT datasets of nine patients. Results: Mean MAE and SSIM values for the PCA-reconstructed volumetric images were approximately 80 HU and 0.88, respectively, regardless of the respiratory phase. In most test cases including the data of which motion range was exceeding that of the modeling data, the positional error of diaphragm was less than 5 mm. The results suggested that large deformations not included in the modeling 4DCT dataset could be reproduced. Furthermore, since the first PC correlated with the displacement of the diaphragm position, the first eigenvector became the dominant factor representing the respiration-associated deformations. However, other PCs did not necessarily change with the same trend as the first PC, and no correlation was observed between the coefficients. Hence, randomly allocating or sampling these PCs in expanded ranges may be applicable to reasonably generate an augmented dataset with various deformations. Conclusions:Reasonable accuracy of image synthesis comparable to those in the previous research were shown by using clinical data. These results indicate the potential of PCA-based volumetric imaging for clinical applications. [ABSTRACT FROM AUTHOR]

A single‐shot adaptation of Optical Projection Tomography (OPT) for high‐speed volumetric snapshot imaging of dynamic mesoscopic biological samples is presented. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio, but the sequential acquisition of projection images typically requires samples to be immobilized during the acquisition. A proof‐of‐principle system capable of single‐shot tomography of a ~1 mm3 volume is presented, demonstrating camera‐limited rates of up to 62.5 volumes/s, which has been applied to 3D imaging of a freely swimming zebrafish embryo. This is achieved by recording eight projection views simultaneously on four low‐cost CMOS cameras. With no stage required to rotate the sample, this single‐shot OPT system can be implemented with a component cost of under £5000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including high throughput flow cytometry applied to model organisms and fluid dynamics studies. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the benefits of fast spin echo (FSE) imaging over rapid gradient‐echo (RAGE) for magnetization‐prepared inhomogeneous magnetization transfer (ihMT) imaging. Methods: A 3D FSE sequence was modified to include an ihMT preparation (ihMT‐FSE) with an optional CSF suppression based on an inversion‐recovery (ihMT‐FLAIR). After numeric simulations assessing SNR benefits of FSE and the potential impact of an additional inversion‐recovery, ihMT‐RAGE, ihMT‐FSE, and ihMT‐FLAIR sequences were compared in a group of six healthy volunteers, evaluating image quality, thermal, and physiological noise as well as quantification using an ihMT saturation (ihMTsat) approach. A preliminary exploration in the cervical spinal cord was also conducted in a group of three healthy volunteers. Results: Several fold improvements in thermal SNR were observed with ihMT‐FSE in agreement with numerical simulations. However, we observed significantly higher physiological noise in ihMT‐FSE compared to ihMT‐RAGE that was mitigated in ihMT‐FLAIR, which provided the best total SNR (+74% and +49% compared to ihMT‐RAGE in the white and gray matter, P ≤ 0.004). IhMTsat quantification was successful in all cases with strong correlation between all sequences (r2 > 0.75). Early experiments showed potential for spinal cord imaging. Conclusions: FSE generally offers higher SNR compared to gradient‐echo based acquisitions for magnetization‐prepared contrasts as illustrated here in the case of ihMT. However, physiological noise has a significant effect, but an inversion–recovery‐based CSF suppression was shown to be efficient in mitigating effects of CSF motion. [ABSTRACT FROM AUTHOR]

There is an increased desire for miniature ultrasound probes with small apertures to provide volumetric images at high frame rates for in-body applications. Satisfying these increased requirements makes simultaneous achievement of a good lateral resolution a challenge. As micro-beamforming is often employed to reduce data rate and cable count to acceptable levels, receive processing methods that try to improve spatial resolution will have to compensate the introduced reduction in focusing. Existing beamformers do not realize sufficient improvement and/or have a computational cost that prohibits their use. Here we propose the use of adaptive beamforming by deep learning (ABLE) in combination with training targets generated by a large aperture array, which inherently has better lateral resolution. In addition, we modify ABLE to extend its receptive field across multiple voxels. We illustrate that this method improves lateral resolution both quantitatively and qualitatively, such that image quality is improved compared with that achieved by existing delay-and-sum, coherence factor, filtered-delay-multiplication-and-sum and Eigen-based minimum variance beamformers. We found that only in silica data are required to train the network, making the method easily implementable in practice. [ABSTRACT FROM AUTHOR]

Two-photon excitation laser scanning microscopy (TPLSM) has provided many insights into the life sciences, especially for thick biological specimens, because of its superior penetration depth and less invasiveness owing to the near-infrared wavelength of its excitation laser light. This paper introduces our four kinds of studies to improve TPLSM by utilizing several optical technologies as follows: (1) A high numerical aperture objective lens significantly deteriorates the focal spot size in deeper regions of specimens. Thus, approaches to adaptive optics were proposed to compensate for optical aberrations for deeper and sharper intravital brain imaging. (2) TPLSM spatial resolution has been improved by applying super-resolution microscopic techniques. We also developed a compact stimulated emission depletion (STED) TPLSM that utilizes electrically controllable components, transmissive liquid crystal devices, and laser diode-based light sources. The spatial resolution of the developed system was five times higher than conventional TPLSM. (3) Most TPLSM systems adopt moving mirrors for single-point laser beam scanning, resulting in the temporal resolution caused by the limited physical speed of these mirrors. For high-speed TPLSM imaging, a confocal spinning-disk scanner and newly-developed high-peak-power laser light sources enabled approximately 200 foci scanning. (4) Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups based on deep expertise for microscopic technologies, resulting in a high threshold for biologists. Recently, an easy-to-use light-needle-creating device was proposed for conventional TPLSM systems to achieve one-touch volumetric imaging. [ABSTRACT FROM AUTHOR]

Fan-Slicer (https://github.com/UCL/fan-slicer) is a Python package that enables the fast sampling (slicing) of 2D ultrasound-shaped images from a 3D volume. To increase sampling speed, CUDA kernel functions are used in conjunction with the Pycuda package. The main features include functions to generate images from both 3D surface models and 3D volumes. Additionally, the package also allows for the sampling of images from curvilinear (fan shaped planes) and linear (rectangle shaped planes) ultrasound transducers. Potential uses of Fan-slicer include the generation of large datasets of 2D images from 3D volumes and the simulation of intra-operative data among others. [ABSTRACT FROM AUTHOR]

This paper introduces an innovative approach to integral volumetric imaging employing time and polarization multiplexing techniques to present volumetric three-dimensional images. Traditional integral volumetric imaging systems with a coarse lens array often face moiré pattern issues stemming from layered panel structures. In response, our proposed system utilizes a combination of time and polarization multiplexing to achieve two focal planes using a single display panel.

To capture the emergent properties of neural circuits, high-speed volumetric imaging of neural activity at cellular resolution is needed. Here, we introduce wavelength multiplexing to perform fast volumetric two-photon imaging of cortical columns (>2,000 neurons in 10 planes at 10 vol/s), using two different calcium indicators, an electrically tunable lens and a spatial light modulator. We image the activity of neuronal populations from layers 2/3 to 5 of primary visual cortex from awake mice, finding a lack of columnar structures in orientation responses and revealing correlations between layers which differ from trial to trial. We also simultaneously image functional correlations between presynaptic layer 1 axons and postsynaptic layer 2/3 neurons. Wavelength multiplexing enhances high-speed volumetric microscopy and can be combined with other optical multiplexing methods to easily boost imaging throughput.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Optical imaging has become a powerful tool for studying brains in vivo. The opacity of adult brains makes microendoscopy, with an optical probe such as a gradient index (GRIN) lens embedded into brain tissue to provide optical relay, the method of choice for imaging neurons and neural activity in deeply buried brain structures. Incorporating a Bessel focus scanning module into two-photon fluorescence microendoscopy, we extended the excitation focus axially and improved its lateral resolution. Scanning the Bessel focus in 2D, we imaged volumes of neurons at high-throughput while resolving fine structures such as synaptic terminals. We applied this approach to the volumetric anatomical imaging of dendritic spines and axonal boutons in the mouse hippocampus, and functional imaging of GABAergic neurons in the mouse lateral hypothalamus in vivo.

Single-cell volumetric imaging is essential for researching individual characteristics of cells. As a nonscanning imaging technique, light field microscopy (LFM) is a critical tool to achieve real-time three-dimensional imaging with the advantage of single-shot. To address the inherent limits including nonuniform resolution and block-wise artifacts, various modified LFM strategies have been developed to provide new insights into the structural and functional information of cells. This review will introduce the principle and development of LFM, discuss the improved approaches based on hardware designs and 3D reconstruction algorithms, and present the applications in single-cell imaging.

Recording the highly diverse and dynamic activities in large populations of neurons in behaving animals is crucial for a better understanding of how the brain works. To meet this challenge, extensive efforts have been devoted to developing functional fluorescent indicators and optical imaging techniques to optically monitor neural activity. Indeed, optical imaging potentially has extremely high throughput due to its non-invasive access to large brain regions and capability to sample neurons at high density, but the readout speed, such as the scanning speed in two-photon scanning microscopy, is often limited by various practical considerations. Among different imaging methods, light field microscopy features a highly parallelized 3D fluorescence imaging scheme and therefore promises a novel and faster strategy for functional imaging of neural activity. Here, we briefly review the working principles of various types of light field microscopes and their recent developments and applications in neuroscience studies. We also discuss strategies and considerations of optimizing light field microscopy for different experimental purposes, with illustrative examples in imaging zebrafish and mouse brains. [ABSTRACT FROM AUTHOR]

In this paper, we present a novel design framework of synthetic radial aperture focusing for three-dimensional (3D) transrectal ultrasound imaging (TRUS-rSAF), in which multiple transmittance/reception events at different scanning angles are synthesized to reconstruct a radial plane in the target volume, securing high spatial resolution and texture uniformity. A theory-based design approach has not been available to push the envelope of the 3D rSAF technique. Herein, a closed-form analytical description of the TRUS-rSAF method is presented for the first time, effectively delineating spatial resolution and grating lobe positions in the radial dimension of a TRUS transducer. We demonstrate a solid optimization workflow based on the theoretical foundation to improve its spatiotemporal resolution, grating lobe artifacts, and signal-to-noise ratio. A specific design criterion was considered to outperform a clinical 3D TRUS imaging as a reference (TRUS-REF), where each radial plane is reconstructed with a single transmittance/reception event using a motorized actuator. The optimized TRUS-rSAF method significantly enhanced spatial resolution up to 50% over the TRUS-REF method while providing clinically effective temporal resolution (2-8 volume/sec) with negligible grating lobe artifacts. The results indicate that the proposed design approach would enable a novel TRUS imaging solution in clinics. [ABSTRACT FROM AUTHOR]

Light-sheet fluorescence microscopy (LSFM) is a kind of volumetric imaging methodology suited for long term living specimens at high temporal-spatial resolution. A single Airy beam (SAB) light-sheet can extend the field of view of Light-sheet fluorescence microscopy benefiting from its non-diffracting nature, but at the cost of out-of-focus background and low imaging contrast caused by side lobes illumination. Here, we propose a method to generate a sort of multiple Airy beams (MAB), which are linearly superimposed of multiple single Airy beams with different scale factors. Compared to the SAB light-sheet, the energy of the multiple Airy beams light-sheet is more concentrated on the focal plane of the detection objective, which can improve the imaging contrast and decrease the photodamage effect. Furthermore, we combined the complementary beam subtraction (CBS) strategy to increase the axial resolution, termed as multiple Airy beams-complementary beam subtraction method, which enables the axial resolution of 1.2 μm while keeping the field of view of 450 μm × 450 μm. The effectiveness of the method is demonstrated by imaging of fluorescent beads and aspergillus conidiophores.

Optical microscopes allow us to study highly dynamic events from the molecular scale up to the whole animal level. However, conventional three-dimensional microscopy architectures face an inherent tradeoff between spatial resolution, imaging volume, light exposure and time required to record a single frame. Many biological processes, such as calcium signalling in the brain or transient enzymatic events, occur in temporal and spatial dimensions that cannot be captured by the iterative scanning of multiple focal planes. Snapshot volumetric imaging maintains the spatio-temporal context of such processes during image acquisition by mapping axial information to one or multiple cameras. This review introduces major methods of camera-based single frame volumetric imaging: so-called multiplane, multifocus, and light field microscopy. For each method, we discuss, amongst other topics, the theoretical framework; tendency towards optical aberrations; light efficiency; applicable wavelength range; robustness/complexity of hardware and analysis; and compatibility with different imaging modalities, and provide an overview of applications in biological research.

The increasing attentions paid to magnetofluidic devices with their applications in cell manipulation, drug delivery, immunoassay, etc., call for higher requirements for precise detection of the magnetic field parameters at micro-scale for more flexible manipulations in precision. Nevertheless, conventional magnetometers can only measure magnetic field strength or flux off the device, while it still remains a challenge to determine the real field distribution within the microchips with high precision. Herein, a digital holography-based technique capable of on- chip measurement and visualization of magnetic field distribution is presented to solve this problem. The interference pattern of the ferromagnetic chain is firstly recorded by an in-line holographic microscopy system with the assistance of the magnetic particles which can be aligned and form a chain along the field direction. Each part digitally reconstructed in different depths is then auto-focalized in an extended focus image with corresponding colors. The detection angle of the ferromagnetic chain is presented in three dimensions, covering a range from -40 to 40 degrees and allowing for an automatic recording and visualization of the magnetic field direction. Our on- chip measuring approach can pronouncedly enhance the magnetic control for magnetofluidic devices with complex 3-D architectures, facilitating their effective applications in the near future. [ABSTRACT FROM AUTHOR]

Significance: The technique of remote focusing (RF) has attracted considerable attention among microscopists due to its ability to quickly adjust focus across different planes, thus facilitating quicker volumetric imaging. However, the difficulty in changing objectives to align with a matching objective in a remote setting while upholding key requirements remains a challenge., Aim: We aim to propose a customized yet straightforward technique to align multiple objectives with a remote objective, employing an identical set of optical elements to ensure meeting the criteria of remote focusing., Approach: We propose a simple optical approach for aligning multiple objectives with a singular remote objective to achieve a perfect imaging system. This method utilizes readily accessible, commercial optical components to meet the fundamental requirements of remote focusing., Results: Our experimental observations indicate that the proposed RF technique offers at least comparable, if not superior, performance over a significant axial depth compared with the conventional RF technique based on commercial lenses while offering the flexibility to switch the objective for multi-scale imaging., Conclusions: The proposed technique addresses various microscopy challenges, particularly in the realm of multi-resolution imaging. We have experimentally demonstrated the efficacy of this technique by capturing images of focal volumes generated by two distinct objectives in a water medium., (© 2024 The Authors.)

Significance: Light-sheet microscopy is a powerful imaging technique that achieves optical sectioning via selective illumination of individual sample planes. However, when the sample contains opaque or scattering tissues, the incident light sheet may not be able to uniformly excite the entire sample. For example, in the context of larval zebrafish whole-brain imaging, occlusion by the eyes prevents access to a significant portion of the brain during common implementations using unidirectional illumination., Aim: We introduce mirror-assisted light-sheet microscopy (mLSM), a simple inexpensive method that can be implemented on existing digitally scanned light-sheet setups by adding a single optical element-a mirrored micro-prism. The prism is placed near the sample to generate a second excitation path for accessing previously obstructed regions., Approach: Scanning the laser beam onto the mirror generates a second, reflected illumination path that circumvents the occlusion. Online tuning of the scanning and laser power waveforms enables near uniform sample excitation with dual illumination., Results: mLSM produces cellular-resolution images of zebrafish brain regions inaccessible to unidirectional illumination. The imaging quality in regions illuminated by the direct or reflected sheet is adjustable by translating the excitation objective. The prism does not interfere with visually guided behavior., Conclusions: mLSM presents an easy-to-implement, cost-effective way to upgrade an existing light-sheet system to obtain more imaging data from a biological sample., (© 2024 The Authors.)