Deep learning-assisted low-cost autofluorescence microscopy for rapid slide-free imaging with virtual histological staining.

Slide-free imaging techniques have shown great promise in improving the histological workflow. For example, computational high-throughput autofluorescence microscopy by pattern illumination (CHAMP) has achieved high resolution with a long depth of field, which, however, requires a costly ultraviolet laser. Here, simply using a low-cost light-emitting diode (LED), we propose a deep learning-assisted framework of enhanced widefield microscopy, termed EW-LED, to generate results similar to CHAMP (the learning target). Comparing EW-LED and CHAMP, EW-LED reduces the cost by 85×, shortening the image acquisition time and computation time by 36× and 17×, respectively. This framework can be applied to other imaging modalities, enhancing widefield images for better virtual histology.

Deep learning-assisted smartphone-based quantitative microscopy for label-free peripheral blood smear analysis.

Hematologists evaluate alterations in blood cell enumeration and morphology to confirm peripheral blood smear findings through manual microscopic examination. However, routine peripheral blood smear analysis is both time-consuming and labor-intensive. Here, we propose using smartphone-based autofluorescence microscopy (Smart-AM) for imaging label-free blood smears at subcellular resolution with automatic hematological analysis. Smart-AM enables rapid and label-free visualization of morphological features of normal and abnormal blood cells (including leukocytes, erythrocytes, and thrombocytes). 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 which show clear cellular features. The proposed technique is portable, cost-effective, and user-friendly, making it significant for broad point-of-care applications.

Exceeding the limit for microscopic image translation with a deep learning-based unified framework.

Deep learning algorithms have been widely used in microscopic image translation. The corresponding data-driven models can be trained by supervised or unsupervised learning depending on the availability of paired data. However, general cases are where the data are only roughly paired such that supervised learning could be invalid due to data unalignment, and unsupervised learning would be less ideal as the roughly paired information is not utilized. In this work, we propose a unified framework (U-Frame) that unifies supervised and unsupervised learning by introducing a tolerance size that can be adjusted automatically according to the degree of data misalignment. Together with the implementation of a global sampling rule, we demonstrate that U-Frame consistently outperforms both supervised and unsupervised learning in all levels of data misalignments (even for perfectly aligned image pairs) in a myriad of image translation applications, including pseudo-optical sectioning, virtual histological staining (with clinical evaluations for cancer diagnosis), improvement of signal-to-noise ratio or resolution, and prediction of fluorescent labels, potentially serving as new standard for image translation.

Lung Cancer Diagnosis on Virtual Histologically Stained Tissue Using Weakly Supervised Learning.

Lung adenocarcinoma (LUAD) is the most common primary lung cancer and accounts for 40% of all lung cancer cases. The current gold standard for lung cancer analysis is based on the pathologists' interpretation of hematoxylin and eosin (H&E)-stained tissue slices viewed under a brightfield microscope or a digital slide scanner. Computational pathology using deep learning has been proposed to detect lung cancer on histology images. However, the histological staining workflow to acquire the H&E-stained images and the subsequent cancer diagnosis procedures are labor-intensive and time-consuming with tedious sample preparation steps and repetitive manual interpretation, respectively. In this work, we propose a weakly supervised learning method for LUAD classification on label-free tissue slices with virtual histological staining. The autofluorescence images of label-free tissue with histopathological information can be converted into virtual H&E-stained images by a weakly supervised deep generative model. For the downstream LUAD classification task, we trained the attention-based multiple-instance learning model with different settings on the open-source LUAD H&E-stained whole-slide images (WSIs) dataset from the Cancer Genome Atlas (TCGA). The model was validated on the 150 H&E-stained WSIs collected from patients in Queen Mary Hospital and Prince of Wales Hospital with an average area under the curve (AUC) of 0.961. The model also achieved an average AUC of 0.973 on 58 virtual H&E-stained WSIs, comparable to the results on 58 standard H&E-stained WSIs with an average AUC of 0.977. The attention heatmaps of virtual H&E-stained WSIs and ground-truth H&E-stained WSIs can indicate tumor regions of LUAD tissue slices. In conclusion, the proposed diagnostic workflow on virtual H&E-stained WSIs of label-free tissue is a rapid, cost effective, and interpretable approach to assist clinicians in postoperative pathological examinations. The method could serve as a blueprint for other label-free imaging modalities and disease contexts.

Diffuse Pneumonitis after Lutetium-177-PSMA-617 Treatment in a Patient with Metastatic Castration-Resistant Prostate Cancer.

We present the case of a patient with heavily pretreated metastatic castration-resistant prostate cancer (mCRPC) who received lutetium Lu-177 vipivotide tetraxetan (also known as 177Lu-PSMA-617) due to progressive disease despite chemotherapy, hormonal therapy and radiation, including palliative mediastinal and central nervous system radiation. He was subsequently hospitalised for worsening acute onset dyspnoea despite clinically responding to therapy. Interval imaging revealed progressive multifocal ground-glass opacities superimposed on a background of underlying peribronchovascular fibrosis. Further workup, including an extensive workup to identify a possible infectious aetiology, ruled out most aetiologies leaving radiation pneumonitis (RP), radiation recall pneumonitis (RRP) and drug-induced pneumonitis as possible diagnoses secondary to 177Lu -PSMA-617. The associated imaging findings of ground-glass opacities and consolidation can be like other aetiologies such as acute infection and subsequently may be treated incorrectly. In the use of theragnostics like 177Lu -PSMA-617, it is fundamental to apply the practices of radioprotection learnt from radiotherapy, as well as to consider prior radiotherapy treatments and their possible side effects when used in conjunction.

Co-aggregation as A Simple Strategy for Preparing Fluorogenic Tetrazine Probes with On-Demand Fluorogen Selection.

Life science has progressed with applications of fluorescent probes-fluorophores linked to functional units responding to biological events. To meet the varied demands across experiments, simple organic reactions to connect fluorophores and functional units have been developed, enabling the on-demand selection of fluorophore-functional unit combinations. However, organic synthesis requires professional equipment and skills, standing as a daunting task for life scientists. In this study, we present a simple, fast, and convenient strategy for probe preparation: co-aggregation of hydrophobic molecules. We focused on tetrazine-a difficult-to-prepare yet useful functional unit that provides effective bioorthogonal reactivity and strong fluorogenicity. Simply mixing the tetrazine molecules and aggregation-induced emission (AIE) luminogens in water, co-aggregation is induced, and the emission of AIE luminogens is quenched. Subsequent click reaction bioorthogonally turns on the emission, identifying these coaggregates as fluorogenic probes. Thanks to this bioorthogonal fluorogenicity, we established a new time-gated fluorescence bioimaging technique to distinguish overlapping emission signals, enabling multi-organelle imaging with two same-color fluorophores. Our study showcases the potential of this co-aggregation method for the on-demand preparation of fluorescent probes as well as protocols and molecular design principles in this approach, offering an effective solution to evolving needs in life science research.

Rapid Expansion of a Teledermatology Web Application for Digital Dermatology Assessment Necessitated by the COVID-19 Pandemic: Retrospective Evaluation.

BACKGROUND: The COVID-19 pandemic necessitated a change in the provision of outpatient care in dermatology. OBJECTIVE: A novel, asynchronous, digital consultation platform was codeveloped with 2 National Health Service dermatology teams to improve access and enhance choice in outpatient care. METHODS: The rollout of the platform was accelerated during the initial COVID-19 lockdown, and its wider use across 2 Scottish health boards was retrospectively evaluated. Integrated with the hospital booking system and electronic patient record, the platform provides an alternative to face-to-face consultations, using information and images submitted by the patients. RESULTS: In total, 297 new patient consultations and 108 return patient consultations were assessed, and 80% (324/405) of the images submitted were of satisfactory quality. The consultations were, on average, 3 minutes shorter than equivalent face-to-face interactions, and a total of 5758 km of patient travel was avoided. Outcomes included web-based reviews (66/405, 16.3%), face-to-face reviews (190/405, 46.9%), biopsies (46/405, 11.4%), discharge (89/405, 22%), and other treatments or investigations (14/405, 3.5%). High levels of patient satisfaction (92/112, 82.1%) were reported. CONCLUSIONS: Digital dermatology assessments are now included in the choices for consultation types that are available to patients, helping to augment service capacity during pandemic recovery.

Increased tryptophan, but not increased glucose metabolism, predict resistance of pembrolizumab in stage III/IV melanoma.

Clinical trials of combined IDO/PD1 blockade in metastatic melanoma (MM) failed to show additional clinical benefit compared to PD1-alone inhibition. We reasoned that a tryptophan-metabolizing pathway other than the kynurenine one is essential. We immunohistochemically stained tissues along the nevus-to-MM progression pathway for tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1. We assessed tryptophan and glucose metabolism by performing baseline C11-labeled α-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imaging of tumor lesions in a prospective clinical trial of pembrolizumab in MM (clinicaltrials.gov, NCT03089606). We found higher protein expression of all TMEs and LAT1 in melanoma cells than tumor-infiltrating lymphocytes (TILs) within MM tumors (n = 68). Melanoma cell-specific TPH1 and LAT1 expressions were significantly anti-correlated with TIL presence in MM. High melanoma cell-specific LAT1 and low IDO1 expression were associated with worse overall survival (OS) in MM. Exploratory optimal cutpoint survival analysis of pretreatment 'high' vs. 'low' C11-AMT SUVmax of the hottest tumor lesion per patient revealed that the 'low' C11-AMT SUVmax was associated with longer progression-free survival in our clinical trial (n = 26). We saw no such trends with pretreatment FDG PET SUVmax. Treatment of melanoma cell lines with telotristat, a TPH1 inhibitor, increased IDO expression and kynurenine production in addition to suppression of serotonin production. High melanoma tryptophan metabolism is a poor predictor of pembrolizumab response and an adverse prognostic factor. Serotoninergic but not kynurenine pathway activation may be significant. Melanoma cells outcompete adjacent TILs, eventually depriving the latter of an essential amino acid.

The genomic landscape of familial glioma.

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

Label-free differential imaging of cellular components in mouse brain tissue by wide-band photoacoustic microscopy.

Mapping diverse cellular components with high spatial resolution is important to interrogate biological systems and study disease pathogenesis. Conventional optical imaging techniques for mapping biomolecular profiles with differential staining and labeling methods are cumbersome. Different types of cellular components exhibit distinctive characteristic absorption spectra across a wide wavelength range. By virtue of this property, a lab-made wide-band optical-resolution photoacoustic microscopy (wbOR-PAM) system, which covers wavelengths from the ultraviolet and visible to the shortwave infrared regions, was designed and developed to capture multiple cellular components in 300-µm-thick brain slices at nine different wavelengths without repetitive staining and complicated processing. This wbOR-PAM system provides abundant spectral information. A reflective objective lens with an infinite conjugate design was applied to focus laser beams with different wavelengths, avoiding chromatic aberration. The molecular components of complex brain slices were probed without labeling. The findings of the present study demonstrated a distinctive absorption of phospholipids, a major component of the cell membrane, brain, and nervous system, at 1690 nm and revealed their precise distribution with microscopic resolution in a mouse brain, for the first time. This novel imaging modality provides a new opportunity to investigate important biomolecular components without either labeling or lengthy specimen processing, thus, laying the groundwork for revealing cellular mechanisms involved in disease pathogenesis.

The GIANT trial (ECOG-ACRIN EA2186) methods paper: A randomized phase II study of gemcitabine and nab-paclitaxel compared with 5-fluorouracil, leucovorin, and liposomal irinotecan in older patients with treatment-naïve metastatic pancreatic cancer - defining a new treatment option for older vulnerable patients.

INTRODUCTION: Pancreatic cancer is the fourth leading cause of cancer-related death in the US with an increasing incidence in older adults (OA) over age 70. There are currently no treatment guidelines for OA with metastatic pancreatic cancer (mPCA) and selecting a chemotherapy regimen for these patients is subjective, based largely on chronologic age and performance status (PS). Geriatric screening tools provide a more objective and accurate evaluation of a patient's overall health but have not yet been validated in patient selection for mPCA treatment. This study aims to elucidate the optimal chemotherapy treatment of vulnerable OA with mPCA and understand the geriatric factors that affect outcomes in this population. METHODS/DESIGN: The GIANT (ECOG-ACRIN EA2186) study is multicenter, randomized phase II trial enrolling patients over age 70 with newly diagnosed mPCA. This study utilizes a screening geriatric assessment (GA) which characterizes patients as fit, vulnerable, or frail. Patients with mild abnormalities in functional status and/or cognition, moderate comorbidities, or over age 80 are considered vulnerable. Enrolled patients are randomized to one of two dose-reduced treatment regimens (gemcitabine/nab-paclitaxel every other week, or dose-reduced 5-fluoruracil (5FU)/ liposomal irinotecan (nal-IRI) every other week). GA and quality of life (QoL) evaluations are completed prior to treatment initiation and at each disease evaluation. Overall survival (OS) is the primary endpoint, with secondary endpoints including progression free survival (PFS) and objective response rate (ORR). Enrolled patients will be stratified by age (70-74 vs ≥75) and ECOG PS (0-1 vs 2). Additional endpoints of interest for OA include evaluation of risk factors identified through GA, QoL evaluation, and toxicities of interest for older adults. Correlative studies include assessment of pro-inflammatory biomarkers of aging in the blood (IL-6, CRP) and imaging evaluation of sarcopenia as predictors of treatment tolerance. DISCUSSION: The GIANT study is the first randomized, prospective national trial evaluating vulnerable OA with mPCA aimed at developing a tailored treatment approach for this patient population. This trial has the potential to establish a new way of objectively selecting vulnerable OA with mPCA for modified treatment and to establish a new standard of care in this growing patient population. TRIAL REGISTRATION: This trial is registered with ClinicalTrial.gov Identifier NCT04233866.

Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining.

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, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.

Low-cost high-resolution photoacoustic microscopy of blood oxygenation with two laser diodes.

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 R 2 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.

Rapid slide-free and non-destructive histological imaging using wide-field optical-sectioning microscopy.

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.

High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning.

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.

Three-dimensional label-free histological imaging of whole organs by microtomy-assisted autofluorescence tomography.

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.

Expanding the phenotypic and molecular spectrum of NFS1-related disorders that cause functional deficiencies in mitochondrial and cytosolic iron-sulfur cluster containing enzymes.

Iron-sulfur cluster proteins are involved in critical functions for gene expression regulation and mitochondrial bioenergetics including the oxidative phosphorylation system. The c.215G>A p.(Arg72Gln) variant in NFS1 has been previously reported to cause infantile mitochondrial complex II and III deficiency. We describe three additional unrelated patients with the same missense variant. Two infants with the same homozygous variant presented with hypotonia, weakness and lactic acidosis, and one patient with compound heterozygous p.(Arg72Gln) and p.(Arg412His) variants presented as a young adult with gastrointestinal symptoms and fatigue. Skeletal muscle biopsy from patients 1 and 3 showed abnormal mitochondrial morphology, and functional analyses demonstrated decreased activity in respiratory chain complex II and variably in complexes I and III. We found decreased mitochondrial and cytosolic aconitase activities but only mildly affected lipoylation of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase enzymes. Our studies expand the phenotypic spectrum and provide further evidence for the pathogenicity and functional sequelae of NFS1-related disorders with disturbances in both mitochondrial and cytosolic iron-sulfur cluster containing enzymes.