A novel computer-assisted tool for 3D imaging of programmed death-ligand 1 expression in immunofluorescence-stained and optically cleared breast cancer specimens.

BACKGROUND: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are the two most common immune checkpoints targeted in triple-negative breast cancer (BC). Refining patient selection for immunotherapy is non-trivial and finding an appropriate digital pathology framework for spatial analysis of theranostic biomarkers for PD-1/PD-L1 inhibitors remains an unmet clinical need. METHODS: We describe a novel computer-assisted tool for three-dimensional (3D) imaging of PD-L1 expression in immunofluorescence-stained and optically cleared BC specimens (n = 20). The proposed 3D framework appeared to be feasible and showed a high overall agreement with traditional, clinical-grade two-dimensional (2D) staining techniques. Additionally, the results obtained for automated immune cell detection and analysis of PD-L1 expression were satisfactory. RESULTS: The spatial distribution of PD-L1 expression was heterogeneous across various BC tissue layers in the 3D space. Notably, there were six cases (30%) wherein PD-L1 expression levels along different layers crossed the 1% threshold for admitting patients to PD-1/PD-L1 inhibitors. The average PD-L1 expression in 3D space was different from that of traditional immunohistochemistry (IHC) in eight cases (40%). Pending further standardization and optimization, we expect that our technology will become a valuable addition for assessing PD-L1 expression in patients with BC. CONCLUSION: Via a single round of immunofluorescence imaging, our approach may provide a considerable improvement in patient stratification for cancer immunotherapy as compared with standard techniques.

Panoramic Tissue Examination That Integrates 3-Dimensional Pathology Imaging and Gene Mutation: Potential Utility in Non-Small Cell Lung Cancer.

Novel therapeutics have significantly improved the survival and quality of life of patients with malignancies in this century. Versatile precision diagnostic data were used to formulate personalized therapeutic strategies for patients. However, the cost of extensive information depends on the consumption of the specimen, raising the challenges of effective specimen utilization, particularly in small biopsies. In this study, we proposed a tissue-processing cascaded protocol that obtains 3-dimensional (3D) protein expression spatial distribution and mutation analysis from an identical specimen. In order to reuse the thick section tissue evaluated after the 3D pathology technique, we designed a novel high-flatness agarose-embedded method that could improve tissue utilization rate by 1.52 fold, whereas it reduced the tissue-processing time by 80% compared with the traditional paraffin-embedding method. In animal studies, we demonstrated that the protocol would not affect the results of DNA mutation analysis. Furthermore, we explored the utility of this approach in non-small cell lung cancer because it is a compelling application for this innovation. We used 35 cases including 7 cases of biopsy specimens of non-small cell lung cancer to simulate future clinical application. The cascaded protocol consumed 150-µm thickness of formalin-fixed, paraffin-embedded specimens, providing 3D histologic and immunohistochemical information approximately 38 times that of the current paraffin-embedding protocol, and 3 rounds of DNA mutation analysis, offering both essential guidance for routine diagnostic evaluation and advanced information for precision medicine. Our designed integrated workflow provides an alternative way for pathological examination and paves the way for multidimensional tumor tissue assessment.

Computer-assisted three-dimensional quantitation of programmed death-ligand 1 in non-small cell lung cancer using tissue clearing technology.

Immune checkpoint blockade therapy has revolutionized non-small cell lung cancer treatment. However, not all patients respond to this therapy. Assessing the tumor expression of immune checkpoint molecules, including programmed death-ligand 1 (PD-L1), is the current standard in predicting treatment response. However, the correlation between PD-L1 expression and anti-PD-1/PD-L1 treatment response is not perfect. This is partly caused by tumor heterogeneity and the common practice of assessing PD-L1 expression based on limited biopsy material. To overcome this problem, we developed a novel method that can make formalin-fixed, paraffin-embedded tissue translucent, allowing three-dimensional (3D) imaging. Our protocol can process tissues up to 150 µm in thickness, allowing anti-PD-L1 staining of the entire tissue and producing high resolution 3D images. Compared to a traditional 4 µm section, our 3D image provides 30 times more coverage of the specimen, assessing PD-L1 expression of approximately 10 times more cells. We further developed a computer-assisted PD-L1 quantitation method to analyze these images, and we found marked variation of PD-L1 expression in 3D. In 5 of 33 needle-biopsy-sized specimens (15.2%), the PD-L1 tumor proportion score (TPS) varied by greater than 10% at different depth levels. In 14 cases (42.4%), the TPS at different depth levels fell into different categories (< 1%, 1-49%, or ≥ 50%), which can potentially influence treatment decisions. Importantly, our technology permits recovery of the processed tissue for subsequent analysis, including histology examination, immunohistochemistry, and mutation analysis. In conclusion, our novel method has the potential to increase the accuracy of tumor PD-L1 expression assessment and enable precise deployment of cancer immunotherapy.

Millisecond two-photon optical ribbon imaging for small-animal functional connectome study.

We developed a high-speed two-photon optical ribbon imaging system, which combines galvo-mirrors for an arbitrary curve scan on a lateral plane and a tunable acoustic gradient-index lens for a 100 kHz-1 MHz axial scan. The system provides micrometer/millisecond spatiotemporal resolutions, which enable continuous readout of functional dynamics from small and densely packed neurons in a living adult Drosophila brain. Compared to sparse sampling techniques, the ribbon imaging modality avoids motion artifacts. Combined with a Drosophila anatomical connectome database, which is the most complete among all model animals, this technique paves the way toward establishing whole-brain functional connectome.

Optimizing depth-of-field extension in optical sectioning microscopy techniques using a fast focus-tunable lens.

Volume imaging based on a fast focus-tunable lens (FTL) allows three-dimensional (3D) observation within milliseconds by extending the depth-of-field (DOF) with sub-micrometer transverse resolution on optical sectioning microscopes. However, the previously published DOF extensions were neither axially uniform nor fit with theoretical prediction. In this work, complete theoretical treatments of focus extension with confocal and various multiphoton microscopes are established to correctly explain the previous results. Moreover, by correctly placing the FTL and properly adjusting incident beam diameter, a uniform DOF is achieved in which the actual extension nicely agrees with the theory. Our work not only provides a theoretical platform for volumetric imaging with FTL but also demonstrates the optimized imaging condition.

Optogenetic control of selective neural activity in multiple freely moving Drosophila adults.

We present an automated laser tracking and optogenetic manipulation system (ALTOMS) for studying social memory in fruit flies (Drosophila melanogaster). ALTOMS comprises an intelligent central control module for high-speed fly behavior analysis and feedback laser scanning (∼40 frames per second) for targeting two lasers (a 473-nm blue laser and a 593.5-nm yellow laser) independently on any specified body parts of two freely moving Drosophila adults. By using ALTOMS to monitor and compute the locations, orientations, wing postures, and relative distance between two flies in real time and using high-intensity laser irradiation as an aversive stimulus, this laser tracking system can be used for an operant conditioning assay in which a courting male quickly learns and forms a long-lasting memory to stay away from a freely moving virgin female. With the equipped lasers, channelrhodopsin-2 and/or halorhodopsin expressed in selected neurons can be triggered on the basis of interactive behaviors between two flies. Given its capacity for optogenetic manipulation to transiently and independently activate/inactivate selective neurons, ALTOMS offers opportunities to systematically map brain circuits that orchestrate specific Drosophila behaviors.

3D resolution enhancement of deep-tissue imaging based on virtual spatial overlap modulation microscopy.

During the last decades, several resolution enhancement methods for optical microscopy beyond diffraction limit have been developed. Nevertheless, those hardware-based techniques typically require strong illumination, and fail to improve resolution in deep tissue. Here we develop a high-speed computational approach, three-dimensional virtual spatial overlap modulation microscopy (3D-vSPOM), which immediately solves the strong-illumination issue. By amplifying only the spatial frequency component corresponding to the un-scattered point-spread-function at focus, plus 3D nonlinear value selection, 3D-vSPOM shows significant resolution enhancement in deep tissue. Since no iteration is required, 3D-vSPOM is much faster than iterative deconvolution. Compared to non-iterative deconvolution, 3D-vSPOM does not need a priori information of point-spread-function at deep tissue, and provides much better resolution enhancement plus greatly improved noise-immune response. This method is ready to be amalgamated with two-photon microscopy or other laser scanning microscopy to enhance deep-tissue resolution.

Optimizing immunofluorescence with high-dynamic-range imaging to enhance PD-L1 expression evaluation for 3D pathology assessment from NSCLC tumor tissue.

Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.

Long-range parametric amplification of THz wave with absorption loss exceeding parametric gain.

Optical parametric mixing is a popular scheme to generate an idler wave at THz frequencies, although the THz wave is often absorbing in the nonlinear optical material. It is widely suggested that the useful material length for co-directional parametric mixing with strong THz-wave absorption is comparable to the THz-wave absorption length in the material. Here we show that, even in the limit of the absorption loss exceeding parametric gain, the THz idler wave can grows monotonically from optical parametric amplification over a much longer distance in a nonlinear optical material until pump depletion. The coherent production of the non-absorbing signal wave can assist the growth of the highly absorbing idler wave. We also show that, for the case of an equal input pump and signal in difference frequency generation, the quick saturation of the THz idler wave predicted from a much simplified and yet popular plane-wave model fails when fast diffraction of the THz wave from the co-propagating optical mixing waves is considered.

Deep-learning based breast cancer detection for cross-staining histopathology images.

Hematoxylin and eosin (H&E) staining is the gold standard for tissue characterization in routine pathological diagnoses. However, these visible light dyes do not exclusively label the nuclei and cytoplasm, making clear-cut segmentation of staining signals challenging. Currently, fluorescent staining technology is much more common in clinical research for analyzing tissue morphology and protein distribution owing to its advantages of channel independence, multiplex labeling, and the possibility of enabling 3D tissue labeling. Although both H&E and fluorescent dyes can stain the nucleus and cytoplasm for representative tissue morphology, color variation between these two staining technologies makes cross-analysis difficult, especially with computer-assisted artificial intelligence (AI) algorithms. In this study, we applied color normalization and nucleus extraction methods to overcome the variation between staining technologies. We also developed an available workflow for using an H&E-stained segmentation AI model in the analysis of fluorescent nucleic acid staining images in breast cancer tumor recognition, resulting in 89.6% and 80.5% accuracy in recognizing specific tumor features in H&E- and fluorescent-stained pathological images, respectively. The results show that the cross-staining inference maintained the same precision level as the proposed workflow, providing an opportunity for an expansion of the application of current pathology AI models.

Rapid Histological Assessment of Prostate Specimens in the Three-dimensional Space by Hydrophilic Tissue Clearing and Confocal Microscopy.

Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method-termed Prostate Rapid Optical examination for cancer STATus (proSTAT)-for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n=15) obtained from radical prostatectomy specimens (n=5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and-subject to independent validation-can find a wide spectrum of clinical and research applications.

A domain knowledge enhanced yield based deep learning classifier identifies perineural invasion in oral cavity squamous cell carcinoma.

Background: Perineural invasion (PNI), a form of local invasion defined as the ability of cancer cells to invade in, around, and through nerves, has a negative prognostic impact in oral cavity squamous cell carcinoma (OCSCC). Unfortunately, the diagnosis of PNI suffers from a significant degree of intra- and interobserver variability. The aim of this pilot study was to develop a deep learning-based human-enhanced tool, termed domain knowledge enhanced yield (Domain-KEY) algorithm, for identifying PNI in digital slides. Methods: Hematoxylin and eosin (H&E)-stained whole-slide images (WSIs, n = 85) were obtained from 80 patients with OCSCC. The model structure consisted of two parts to simulate human decision-making skills in diagnostic pathology. To this aim, two semantic segmentation models were constructed (i.e., identification of nerve fibers followed by the diagnosis of PNI). The inferred results were subsequently subjected to post-processing of generated decision rules for diagnostic labeling. Ten H&E-stained WSIs not previously used in the study were read and labeled by the Domain-KEY algorithm. Thereafter, labeling correctness was visually inspected by two independent pathologists. Results: The Domain-KEY algorithm was found to outperform the ResnetV2_50 classifier for the detection of PNI (diagnostic accuracy: 89.01% and 61.94%, respectively). On analyzing WSIs, the algorithm achieved a mean diagnostic accuracy as high as 97.50% versus traditional pathology. The observed accuracy in a validation dataset of 25 WSIs obtained from seven patients with oropharyngeal (cancer of the tongue base, n = 1; tonsil cancer, n = 1; soft palate cancer, n = 1) and hypopharyngeal (cancer of posterior wall, n = 2; pyriform sinus cancer, n = 2) malignancies was 96%. Notably, the algorithm was successfully applied in the analysis of WSIs to shorten the time required to reach a diagnosis. The addition of the hybrid intelligence model decreased the mean time required to reach a diagnosis by 15.0% and 23.7% for the first and second pathologists, respectively. On analyzing digital slides, the tool was effective in supporting human diagnostic thinking. Conclusions: The Domain-KEY algorithm successfully mimicked human decision-making skills and supported expert pathologists in the routine diagnosis of PNI.

Forward and backward THz-wave difference frequency generations from a rectangular nonlinear waveguide.

We report forward and backward THz-wave difference frequency generations at 197 and 469 µm from a PPLN rectangular crystal rod with an aperture of 0.5 (height in z) × 0.6 (width in y) mm(2) and a length of 25 mm in x. The crystal rod appears as a waveguide for the THz waves but as a bulk material for the optical mixing waves near 1.54 µm. We measured enhancement factors of 1.6 and 1.8 for the forward and backward THz-wave output powers, respectively, from the rectangular waveguide in comparison with those from a PPLN slab waveguide of the same length, thickness, and domain period under the same pump and signal intensity of 100 MW/cm(2).

Observation of spontaneous polarization misalignments in periodically poled crystals using second-harmonic generation microscopy.

Periodically poled crystal (PPC) is a key component for nonlinear optical applications. Its poling quality relies largely on successful domain inversion and the alignment of spontaneous polarization (SP) vectors in each domain. Here we report the unexpected observation of bulk second harmonic generation (SHG) in PPC when excitation propagating along its optical axis. Based on its tensorial nature, SHG is highly sensitive to the orientation of SP, and therefore the misalignment of SP in each domain of PPC can be revealed noninvasively by SHG microscopy. This nonlinear imaging modality provides optical sectioning capability with 3D sub-micrometer resolution, so it will be useful for in situ investigation of poling quality in PPC.

Pulse width tunable passively mode-locked Nd:YVO4 laser based on hybrid laser gain medium locations.

A pulse width tunable passively mode-locked laser with a wavelength of 1,064 nm and two Nd:YVO(4) crystals located in asymmetric positions is demonstrated. By adjusting the pump power of the crystals, the pulse width can be continuously tuned from 8.8 to 20.3 ps at a stable mode-locked repetition rate of 122 MHz. A theoretical model is proposed to describe the experiment results phenomenologically. In this system, a maximum output power of 4.44 W is achieved with a pump of 13.68 W, corresponding to an optical-to-optical efficiency of 32.5%. The beam quality factor, M(2), is found to be M(2)(x) = 1.15 and M(2)(y) = 1.13 in the orthogonal directions at an output power of 3.2 W.

Coherent anti-Stokes Raman scattering microscopy using a single-pass picosecond supercontinuum-seeded optical parametric amplifier.

We have demonstrated coherent anti-Stokes Raman scattering (CARS) microscopy with a single-pass picosecond supercontinuum-seeded optical parametric amplifier (SCOPA). The SCOPA was pumped by a frequency-doubled picosecond passively mode-locked Nd:YVO(4) laser, and was seeded by a supercontinuum light source. Compared with the conventional experimental setups of CARS microscopy, our exposition is substantially simpler because the pump and Stokes lasers are overlapped in the SCOPA automatically and thus steered into a microscope coherently. The feasibility of this novel light source to CARS imaging was illustrated by acquiring the fundamental and overtone CARS images of the aromatic C-H stretching mode of polystyrene beads and an image of the pharynx of a C. elegans of the aliphatic C-H stretching mode.

Manipulated nucleation of Fe nanostructures on Au(111) with combined growth methods.

The manipulated nucleation of Fe on hexagonal close-packed (hcp) and face centered cubic (fcc) areas of an Au(111) herringbone surface was demonstrated with the combined growth methods of Xe buffer layer, seed effect and low temperature deposition. Besides the herringbone kinks, bi-layer stacking at fcc gaps was observed. This preference resulted in various nucleation patterns, such as zigzag stripes and doublet islands, by properly choosing the growth condition. The idea of nucleation control through combined growth methods can be applied in other nanopatterned templates for the fabrication of self-assembled nanostructures.

Drosophila Brain Functional Data Analysis: A Unified Framework.

A unified framework for the analysis of fluorescence data taken by a two-photon imaging system is presented. As in the processing of blood-oxygen-level-dependent signals of functional magnetic resonance imaging, the acquired functional images have to be co-registered with a structural brain atlas before delineating the regions activated by a given stimulus. The voxels whose calcium traces are highly correlated with the predicted responses are demarcated without the need for subjective reasoning. Experimental data acquired while presenting olfactory stimuli are used to demonstrate the efficacy of the proposed schemes. The results indicate that the functional images of a Drosophila individual can be normalized into a standard stereotactic space, and the expected brain regions can be delineated adequately. This framework provides an opportunity to enable the development of a Drosophila functional connectome database.

Broadband tunable optical parametric amplification from a single 50 MHz ultrafast fiber laser.

We have demonstrated a 0.7 microm - 1.9 microm wavelength-tunable light source based on a single-pass optical parametric amplification (OPA) in a multiperiod magnesium oxide-doped periodically poled lithium niobate crystal. The OPA pump was a frequency-doubled ultrafast ytterbium-doped fiber oscillator, and the residual 1040 nm laser power after frequency doubling was recycled to generate a supercontinuum seeding source. Compared with conventional OPAs, this system is free from timing jitter between the pump laser and the seeding source. Over 50% conversion efficiency was obtained with 10 nJ pump energy. Combined with a 50 MHz repetition rate, this versatile source is ideal for biomedical and spectroscopic applications.

A compact and portable laser radioactive decontamination system using passive Q-switched fiber laser and polygon scanner.

We designed a compact and portable laser radioactive decontamination system using a passive Q-switched fiber laser and a polygon scanner. The passive Q-switched fiber laser produced average power of 15 W at a repetition rate of 60 kHz. The scanner system contained a 12-sided spinning polygon mirror driven by a DC motor. By varying the spinning speed of the polygon mirror, we achieved a processing speed of up to 88,500 mm/s. This system was demonstrated to be very efficient for radioactive decontamination on surfaces of various metals: stainless steel, brass, iron, and aluminum.