Search

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.

Histopathology based on formalin-fixed and paraffin-embedded tissues remains the gold standard for surgical margin assessment (SMA). However, routine pathological practice is lengthy and laborious, failing to provide immediate feedback to surgeons and pathologists for intraoperative decision-making. In this report, we propose a cost-effective and easy-to-use histological imaging method with speckle illumination microscopy (i.e., HiLo). HiLo can achieve rapid and non-destructive imaging of large and fluorescently-labelled resection tissues at an acquisition speed of 5 cm2/min with 1.3-μm lateral resolution and 5.8-μm axial resolution, demonstrating a great potential as an intraoperative SMA tool that can be used by surgeons and pathologists to detect residual tumors at surgical margins. It is experimentally validated that HiLo enables rapid diagnosis of different subtypes of human lung adenocarcinoma and hepatocellular carcinoma, producing images with remarkably recognizable cellular features comparable to the gold-standard histology. This work will facilitate the clinical translations of HiLo microscopy to improve the current standard-of-care.

We proposed a high-throughput label-free and slide-free imaging technique by ultraviolet-excited autofluorescence, assisted by speckle illumination and a deep learning algorithm, to generate subcellular-resolution histology-like images of unprocessed tissues, simplifying the workflow of standard-of-care histopathology.

By implementing microscopy with ultraviolet surface excitation using speckle illumination, we have improved the spatial resolution by 1.5 times and preserved the long depth of focus, achieving sharp imaging of thick and rough specimens.

Microscopy with ultraviolet surface excitation (MUSE) is a promising slide-free imaging technique to improve the time-consuming histopathology workflow. However, since the penetration depth of the excitation light is tissue dependent, the image contrast could be significantly degraded when the depth of field of the imaging system is shallower than the penetration depth. High-resolution cellular imaging normally comes with a shallow depth of field, which also restricts the tolerance of surface roughness in biological specimens. Here we propose the incorporation of MUSE with speckle illumination (termed MUSES), which can achieve sharp imaging on thick and rough specimens. Our experimental results demonstrate the potential of MUSES in providing histological images with ∼ 1 μm spatial resolution and improved contrast, within 10 minutes for a field of view of 1.7 mm × 1.2 mm . With the extended depth of field feature, MUSES also relieves the constraint of tissue flatness. Furthermore, with a color transformation assisted by deep learning, a virtually stained histological image can be generated without manual tuning, improving the applicability of MUSES in clinical settings.

Acquiring ultrafast and high spectral resolution optical images is key to measure transient physical or chemical processes, such as photon propagation, plasma dynamics, and femtosecond chemical reactions. At a trillion Hz frame rate, most ultrafast imaging modalities can acquire only a limited number of frames. Here, we present a compressed ultrafast spectral-temporal (CUST) photographic technique, enabling both an ultrahigh frame rate of 3.85 trillion Hz and a large frame number. We demonstrate that CUST photography records 60 frames, enabling precisely recording light propagation, reflection, and self-focusing in nonlinear media over 30 ps. CUST photography has the potential to further increase the frame number beyond hundreds of frames. Using spectral-temporal coupling, CUST photography can record multiple frames with a subnanometer spectral resolution with a single laser exposure, enabling ultrafast spectral imaging. CUST photography with high frame rate, high spectral resolution, and high frame number in a single modality offer a new tool for observing many transient phenomena with high temporal complexity and high spectral precision.

Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded (TRUE) optical focusing in reflection mode, we focused 532 nm light through a highly scattering ex vivo adult human cataractous lens of 112 mean free path thick. This work demonstrates a potential clinical application of wavefront shaping techniques.

Label-free mid-infrared (MIR) imaging provides rich chemical and structural information of biological tissues without staining. Conventionally, the long MIR wavelength severely limits the lateral resolution owing to optical diffraction; moreover, the strong MIR absorption of water ubiquitous in fresh biological samples results in high background and low contrast. Here, we present a novel approach, called ultraviolet-localized MIR photoacoustic microscopy (ULM-PAM), to achieve high-resolution and water-background–free MIR imaging of fresh biological samples. In our approach, a pulsed MIR laser thermally excites the sample at the focal spot, and a pulsed ultraviolet (UV) laser photoacoustically detects the resulting transient temperature rise owing to the Gruneisen relaxation effect, thereby reporting the intensity of the MIR absorption by the sample. The imaging resolution of our method is determined by the wavelength of the UV laser, which is one order of magnitude shorter than that of the mid-IR laser (2.5 μm to 12 μm). In addition, in the UV region from 200 nm to 230 nm, most important organic molecules in biological tissues, including proteins, lipids and nuclei acids, have strong absorption, while water is transparent. Therefore, our method can achieve high resolution and water-background free IR imaging of fresh biological samples. For cell cultures, our method achieved high-resolution and high-contrast infrared imaging of lipids, proteins. The capability of label-free histology of this method is also demonstrated in thick biological tissues, such as brain slices. Our approach provides convenient high-resolution and high-contrast MIR imaging, which can benefit diagnosis of fresh biological samples.

Ultraviolet photoacoustic microscopy (UV-PAM) has recently been demonstrated as a potential imaging tool for surgical margin analysis (SMA). UV-PAM does not require staining or micrometer-thick slicing, which is inevitable in conventional histological imaging. To promote UV-PAM as a practical intraoperative diagnostic tool, the imaging speed should be improved while preserving the high-resolution imaging capability and simplistic system design. In this Letter, we developed a galvanometer mirror-based UV-PAM (GM-UV-PAM) system for high-speed histology-like imaging. By using a UV laser with a high repetition rate (55 kHz) and a one-dimensional galvanometer mirror, our GM-UV-PAM system can generate subcellular images in less than 15 min for a typical brain biopsy ( 5 m m × 5 m m ), with a lateral resolution of ∼ 1.0 µ m . The images of mouse brain slices obtained by our GM-UV-PAM system show that it can provide histological information for SMA.

In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue.

Photoacoustic microscopy (PAM) with ultraviolet (UV) laser illumination has recently been demonstrated as a promising tool that provides fast, label-free, and multilayered histologic imaging of human breast tissue. Thus far, the axial resolution has been determined ultrasonically. To enable optically defined axial resolution, we exploit the Gruneisen relaxation (GR) effect. By imaging mouse brain slices, we show that GRUV-PAM reveals detailed information about three-dimensional cell nuclear distributions and internal structures, which are important diagnostic features for cancers. Due to the nonlinear effect, GRUV-PAM also provides better contrast in images of cell nuclei.

A photoacoustic microscope system provides label-free multilayered histology-like imaging of unprocessed human breast specimens., The goal of breast-conserving surgery is to completely remove all of the cancer. Currently, no intraoperative tools can microscopically analyze the entire lumpectomy specimen, which results in 20 to 60% of patients undergoing second surgeries to achieve clear margins. To address this critical need, we have laid the foundation for the development of a device that could allow accurate intraoperative margin assessment. We demonstrate that by taking advantage of the intrinsic optical contrast of breast tissue, photoacoustic microscopy (PAM) can achieve multilayered histology-like imaging of the tissue surface. The high correlation of the PAM images to the conventional histologic images allows rapid computations of diagnostic features such as nuclear size and packing density, potentially identifying small clusters of cancer cells. Because PAM does not require tissue processing or staining, it can be performed promptly and intraoperatively, enabling immediate directed re-excision and reducing the number of second surgeries.

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population efficiently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in flow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging flow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non-imaging flow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast microfluidic flow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional fluorescence measurements widely adopted in the non-imaging flow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the "parameter space" in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design of the microfluidic chip for ultrafast fluidic flow, as well as the image acquisition and processing.

Ultraviolet photoacoustic microscopy (UV-PAM) is a promising intraoperative tool for surgical margin assessment (SMA), one that can provide label-free histology-like images with high resolution. In this study, using a microlens array and a one-dimensional (1-D) array ultrasonic transducer, we developed a high-throughput multifocal UV-PAM (MF-UV-PAM). Our new system achieved a [Formula: see text] lateral resolution and produced images 40 times faster than the previously developed point-by-point scanning UV-PAM. MF-UV-PAM provided a readily comprehensible photoacoustic image of a mouse brain slice with specific absorption contrast in [Formula: see text] , highlighting cell nuclei. Individual cell nuclei could be clearly resolved, showing its practical potential for intraoperative SMA.

Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications, which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded optical focusing in reflection mode, we focused 532-nm light through a highly scattering ex vivo adult human cataractous lens. This work demonstrates a potential clinical application of wavefront shaping techniques.

We demonstrate, by means of internal light delivery, photoacoustic imaging of the deep brain of rats in vivo . With fiber illumination via the oral cavity, we delivered light directly into the bottom of the brain, much more than can be delivered by external illumination. The study was performed using a photoacoustic computed tomography (PACT) system equipped with a 512-element full-ring transducer array, providing a full two-dimensional view aperture. Using internal illumination, the PACT system provided clear cross sectional photoacoustic images from the palate to the middle brain of live rats, revealing deep brain structures such as the hypothalamus, brain stem, and cerebral medulla.

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity- a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry- permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay., Comment: Manuscript in 28 pages, 5 figures Supplementary information in 9 pages, 9 figures

The image resolution of an aberration-corrected laser-scanning fluorescence microscopy (LSFM) system, like all other classical optical imaging modalities, is ultimately governed by diffraction limit and can be, in practice, influenced by the noise. However, consideration of only these two parameters is not adequate for LSFM with ultrafast laser-scanning, in which the dwell time of each resolvable image point becomes comparable with the fluorescence lifetime. In view of the continuing demand for faster LSFM, we here revisit the theoretical framework of LSFM and investigate the impact of the scanning speed on the resolution. In particular, we identify there are different speed regimes and excitation conditions in which the resolution is primarily limited by diffraction limit, fluorescence lifetime, or intrinsic noise. Our model also suggests that the speed of the current laser-scanning technologies is still at least an order of magnitude below the limit (∼sub-MHz to MHz), at which the diffraction-limited resolution can be preserved. We thus anticipate that the present study can provide new insight for practical designs and implementation of ultrafast LSFM, based on emerging laser-scanning techniques, e.g., ultrafast wavelength-swept sources, or optical time-stretch.