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Hematologists evaluate alterations in blood cell enumeration and morphology to confirm the peripheral blood smear findings through manual microscopic examination. However, routine peripheral blood smear analysis is both time-consuming and labor-intensive. Here, we propose a smartphone-based autofluorescence microscopy (Smart-AM) system for imaging label-free blood smears at sub-cellular resolution and performing hematological analysis. Smart-AM enables rapid, high-quality, and label-free visualization of morphological features of different blood cells (leukocytes, erythrocytes, and thrombocytes) and abnormal variations in blood cells. Moreover, assisted with deep learning algorithms, this technique can automatically detect and classify different leukocytes with high accuracy, and transform the autofluorescence images into virtual Giemsa-stained images maintaining significant cellular features. The proposed technique is portable, cost-effective, and user-friendly, making it significant for broad point-of-care applications.

Lung cancer is one of the leading causes of cancer death worldwide. The diagnosis of lung cancer based on the analysis of formalin-fixed and paraffin-embedded (FFPE) tissues is laborious and time-consuming, failing to guide surgeons intraoperatively. Here we proposed a rapid histological imaging method, termed microscopy with ultraviolet single-plane illumination (MUSI), to enable rapidex-orin-vivoimaging of fresh and unprocessed tissues in a label-free and non-destructive manner. The MUSI system allows surgical specimens with large irregular surfaces to be screened at a speed of 0.5 mm2/s with a subcellular resolution, which is sufficient to provide immediate feedback to surgeons and pathologists for intraoperative decision-making. We demonstrate that MUSI can differentiate between different subtypes of human lung adenocarcinomas, revealing diagnostically important features that are comparable to the gold standard FFPE histology. As an assistive imaging platform, MUSI could facilitate the development of precise image-guided surgery and revolutionize the current practice in surgical pathology.

Optical-resolution photoacoustic microscopy (OR-PAM) has been widely used for imaging blood vessel and oxygen saturation of hemoglobin (sO2), providing high-resolution functional images of living animals in vivo. However, most of them require one or multiple bulky and costly pulsed lasers, hindering their applicability in preclinical and clinical settings. In this paper, we demonstrate a reflection-mode low-cost high-resolution OR-PAM system by using two cost-effective and compact laser diodes (LDs), achieving microvasculature and sO2 imaging with a high lateral resolution of ∼6 µm. The cost of the excitation sources has dramatically reduced by ∼20–40 times compared to that of the pulsed lasers used in state-of-the-art OR-PAM systems. A blood phantom study was performed to show a determination coefficient R2 of 0.96 in linear regression analysis. Experimental results of in vivo mouse ear imaging show that the proposed dual-wavelength LD-based PAM system can provide high-resolution functional images at a low cost.

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires intraoperative diagnostic tools to avoid repeated surgeries due to a positive surgical margin. Recently, by taking the advantage of the high intrinsic optical absorption of DNA/RNA at 266 nm wavelength, ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images without labeling, showing great promise as an intraoperative tool for SMA. To enable the development of UV-PAM for SMA, here, a high-speed and open-top UV-PAM system is presented, which can be operated similarly to conventional optical microscopies. The UV-PAM system provides a high lateral resolution of 1.2 µm, and a high imaging speed of 55 kHz A-line rate with one-axis galvanometer mirror scanning. Moreover, to ensure UV-PAM images can be easily interpreted by pathologists without additional training, the original grayscale UV-PAM images are virtually stained by a deep-learning algorithm to mimic the standard hematoxylin- and eosin-stained images, enabling training-free histological analysis. Mouse brain slice imaging is performed to demonstrate the high performance of the open-top UV-PAM system, illustrating its great potential for SMA applications.

Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.

Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, automated, cost-effective, and versatile multicolor 3D imaging method with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). TRUST enables exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously with the help of a UV light-emitting diode and a color camera. Commonly applied tissue preparation procedures (e.g., staining or clearing) are laborious, time-consuming, and may induce detrimental effects on processed samples. In TRUST, formalin-fixed specimens are stained with real-time double labeling layer by layer along with serial widefield optical illumination with raster scanning and mechanical sectioning to improve the staining speed and reveal rich biological information. All vital organs in mice have been imaged by TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging ability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (taking ∼2 days for imaging the embryo at the embryonic day of 15). TRUST offers a way for multicontrast and multicolor whole-organ 3D imaging with high resolution and high speed while relieving researchers from heavy sample preparation workload.

Laser diodes (LDs) have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy (PAM). However, the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously. In this paper, we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD, operating at a pulsed mode, with a repetition rate of 30 kHz, as an excitation source. A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio. By optimizing the optical system, a high lateral resolution of 4.8 μm has been achieved. In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.

Histopathology based on formalin-fixed and paraffin-embedded tissues has long been the gold standard for surgical margin assessment (SMA). However, routine pathological practice is lengthy and laborious, failing to guide surgeons intraoperatively. In this report, we propose a practical and low-cost histological imaging method with wide-field optical-sectioning microscopy (i.e., High-and-Low-frequency (HiLo) microscopy). HiLo can achieve rapid and non-destructive imaging of freshly-excised tissues at an extremely high acquisition speed of 5 cm2/min with a spatial resolution of 1.3 µm (lateral) and 5.8 µm (axial), showing great potential as an SMA tool that can provide immediate feedback to surgeons and pathologists for intraoperative decision-making. We demonstrate that HiLo enables rapid extraction of diagnostic features for different subtypes of human lung adenocarcinoma and hepatocellular carcinoma, producing surface images of rough specimens with large field-of-views and cellular features that are comparable to the clinical standard. Our results show promising clinical translations of HiLo microscopy to improve the current standard of care.

Summary Three-dimensional (3D) histology is vitally important to characterize disease-induced tissue heterogeneity at the individual cell level. However, it remains challenging for both high-throughput 3D imaging and volumetric reconstruction. Here we propose a label-free, cost-effective, and ready-to-use 3D histological imaging technique, termed microtomy-assisted autofluorescence tomography with ultraviolet excitation (MATE). With the combination of block-face imaging and serial microtome sectioning, MATE can achieve rapid and label-free imaging of paraffin-embedded whole organs at an acquisition speed of 1 cm3 per 4 h with a voxel resolution of 1.2 × 1.2 × 10 μm3. We demonstrate that MATE enables simultaneous visualization of cell nuclei, fiber tracts, and blood vessels in mouse/human brains without tissue staining or clearing. Moreover, diagnostic features, including nuclear size and packing density, can be quantitatively extracted with high accuracy. MATE is augmented to the current slide-based 2D histology, holding great promise to facilitate histopathological interpretation at the organelle level., Graphical abstract, Highlights • MATE enables label-free 3D imaging of whole organs without tissue staining or clearing • Various anatomical structures can be simultaneously visualized in a label-free manner • MATE reveals diagnostic features comparable with the gold standard histology • MATE facilitates comprehensive histopathological analysis at the subcellular level, Biological sciences; Biophysics; Histology; Optical imaging