Darkfield and Fluorescence Macrovision of a Series of Large Images to Assess Anatomical and Chemical Tissue Variability in Whole Cross-Sections of Maize Stems

The proportion and composition of plant tissues in maize stems vary with genotype and agroclimatic factors and may impact the final biomass use. In this manuscript, we propose a quantitative histology approach without any section labelling to estimate the proportion of different tissues in maize stem sections as well as their chemical characteristics. Macroscopic imaging was chosen to observe the entire section of a stem. Darkfield illumination was retained to visualise the whole stem cellular structure. Multispectral autofluorescence images were acquired to detect cell wall phenolic compounds after UV and visible excitations. Image analysis was implemented to extract morphological features and autofluorescence pseudospectra. By assimilating the internode to a cylinder, the relative proportions of tissues in the internode were estimated from their relative areas in the sections. The approach was applied to study a series of 14 maize inbred lines. Considerable variability was revealed among the 14 inbred lines for both anatomical and chemical traits. The most discriminant morphological descriptors were the relative amount of rind and parenchyma tissues together with the density and size of the individual bundles, the area of stem and the parenchyma cell diameter. The rind, as the most lignified tissue, showed strong visible-induced fluorescence which was line-dependant. The relative amount of para-coumaric acid was associated with the UV-induced fluorescence intensity in the rind and in the parenchyma near the rind, while ferulic acid amount was significantly correlated mainly with the parenchyma near the rind. The correlation between lignin and the tissue pseudospectra showed that a global higher amount of lignin resulted in a higher level of lignin fluorescence whatever the tissues. We demonstrated here the potential of darkfield and autofluorescence imaging coupled with image analysis to quantify histology of maize stem and highlight variability between different lines.

Label-free imaging to track reprogramming of human somatic cells

The process of reprogramming patient samples to human induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient leading to a heterogeneous population of cells. Here, we track the reprogramming status of single patient-derived cells during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. Erythroid progenitor cells (EPCs) isolated from human peripheral blood showed distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) during reprogramming. Random forest models classified starting EPCs, partially-reprogrammed intermediate cells, and iPSCs with ~95% accuracy. Reprogramming trajectories resolved at the single cell level indicated significant reprogramming heterogeneity along different branches of cell state. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique non-destructive method to assess the quality of iPSCs in real-time for various applications in regenerative medicine, cell therapy biomanufacturing, and disease modeling.

Three-dimensional Multicolor Subcellular Imaging by Fast Serial Sectioning Tomography for Centimeter-scale Specimens

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.

Ocular Characteristics of Patients with Leber Congenital Amaurosis 6 Caused by Pathogenic RPGRIP1 Gene Variation in a Chinese Cohort

Purpose. To delineate the clinical and genetic characteristics of Chinese patients with RPGRIP1-associated Leber congenital amaurosis 6 (LCA6). Methods. After screening 352 unrelated families with clinically diagnosed RP, five LCA6 patients with RPGRIP1 variations from unrelated Chinese families were identified. Full ophthalmology examinations, including decimal best-corrected visual acuity (BCVA), fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), full-field electroretinography (ffERG), multifocal electroretinography (mfERG), perimetry, and flash visual evoked potential (FVEP), were performed. Target next-generation sequencing (NGS) and Sanger sequencing were performed for the five patients to identify and to validate candidate disease-causing variants. Results. Five patients were molecularly diagnosed as the LCA6 associated with RPGRIP1 variation, with typical clinical characteristics including congenital night blindness, nystagmus, and visual defect, at an early age. Interestingly, LCA6 exhibited extensive clinical heterogeneity and the changes in the morphology and function were not completely consistent in the five LCA6 patients. Case 1 showed extensive inferior-nasal retinal atrophy with a corresponding area of hypofluorescence in fundus autofluorescence, and the fundus photograph was nearly normal in cases 2 and 3. The ERG results displayed a moderately reduced rod-system response in cases 1 and 2 and a significant reduced rod-system response in case 3. Both case 4 and case 5 showed mottled pigmentation in fundi and an unrecordable rod and cone-system response in ERG. Moreover, we identified eight compound variants and one homozygous variant in the five patients with RPGRIP1. Conclusions. This is the largest report focused on the clinical electrophysiological features of patients with associated LCA6 caused by the variation in the RPGRIP1 gene in the Chinese population with an enriched phenotypic and genotypic background of LCA6 to improve future gene therapies.

No proof for retinal amyloid after oral curcumin in amyloid confirmed AD cases

INTRODUCTIONPrevious work showed in-vivo presence of retinal amyloid in AD patients using curcumin. We aimed to replicate these findings in an amyloid biomarker confirmed cohort.METHODSTwenty-six AD patients (age 66 (±9), MMSE≥18) and 14 controls (age 71(±12)) used three curcumin formulations: Longvida®, Theracurmin® and Novasol®. Plasma levels were determined and pre- and post-curcumin retinal scans acquired using autofluorescence imaging. Images were both visually and quantitatively assessed.RESULTSVisual assessment showed no difference between AD patients and controls for pre- and post-curcumin images. This was confirmed by quantitative analyses. Mean plasma curcumin levels were 198.7 nM (Longvida®), 576.6 nM (Theracurmin®) and 1605.8 nM (Novasol®).DISCUSSIONWe found no difference in retinal focal fluorescence in an amyloid biomarker confirmed cohort of AD patients and controls, using Longvida® (previously used for this purpose) and two additional curcumin formulations yielding higher curcumin plasma levels. We therefore question the presence of retinal amyloid.

A Clinical Study to Evaluate Autofluorescence Imaging of Diabetic Foot Ulcers Using a Novel Artificial Intelligence Enabled Noninvasive Device

Diabetic foot ulcers, with worldwide prevalence ranging from 12%-25%, are an important cause of nontraumatic lower limb amputation. Evidence-based assessment of early infection can help the clinician provide the right first line treatment thus helping improve the wound closure rate. Illuminate®, a novel point of care device working on multispectral autofluorescence imaging, helps in the rapid identification and classification of bacteria. This study was aimed to evaluate the diagnostic accuracy of the device in detecting bacterial gram type against standard culture methods. A total of 178 patients from a tertiary care center for diabetes was recruited and 203 tissue samples were obtained from the wound base by the plastic surgeon. The device was handled by the trained investigator to take wound images. The tissue samples were taken from the color-coded infected region as indicated by the device's Artificial Intelligence algorithm and sent for microbial assessment. The results were compared against the Gram type inferred by the device and the device was found to have an accuracy of 89.54%, a positive predictive value of 86.27% for detecting Gram-positive bacteria, 80.77% for Gram-negative bacteria, and 91.67% for no infection. The negative predictive value corresponded to 87.25% for Gram-positive, 92% for Gram-negative, and 96.12% for no infection. The Results exhibited the accuracy of this novel autofluorescence device in identifying and classifying the gram type of bacteria and its potential in significantly aiding clinicians towards early infection assessment and treatment.

Evaluation of Autofluorescence in Identifying Parathyroid Glands by Measuring Parathyroid Hormone in Fine-Needle Biopsy Washings

Background: Near-infrared autofluorescence imaging has potentially great value for assisting endocrine surgeons in identifying parathyroid glands and may dramatically change the surgical strategy of endocrine surgeons in thyroid surgery. This study is designed to objectively evaluate the role of near-infrared autofluorescence imaging in identifying parathyroid glands during thyroid surgery by measuring intraoperative parathyroid hormone in fine-needle aspiration biopsy washings.Methods: A case series study was conducted at a tertiary referral teaching hospital in China from September 2019 to April 2020. Patients undergoing total thyroidectomy with or without neck lymph node dissection were consecutively included. The surgeon used near-infrared autofluorescence imaging to identify parathyroid glands during thyroid surgery and confirmed suspicious parathyroid tissues by measuring their intraoperative parathyroid hormone. Nanocarbon was injected into the thyroid gland if the thyroid autofluorescence intensity was too strong. The sensitivity and accuracy of near-infrared autofluorescence imaging and vision for identifying parathyroid glands, and the difference in autofluorescence intensity in various tissues were the main outcomes.RESULTS: Overall, 238 patients completed the trial. Based on the pathological and aIOPTH results, the sensitivity of near-infrared autofluorescence imaging for detecting parathyroid glands（568 of 596 parathyroid glands; 95.30%）was significantly higher than that of vision（517 of 596 parathyroid glands; 86.74%, P<.001). The accuracy of near-infrared autofluorescence imaging (764 of 841 tissues; 90.84%) was significantly higher than that of vision (567 of 841 tissues; 67.42%, P<.001) when the evaluations of certain tissues were inconsistent. There was a significant difference between the autofluorescence intensity of the parathyroid glands and that of the lymph nodes (74.19 ± 17.82 vs 33.97 ± 10.64, P<.001).CONCLUSIONS: The use of near-infrared autofluorescence imaging, along with intraoperative parathyroid hormone and nanocarbon for the identification of parathyroid glands in thyroid surgery may increase the number of confirmed parathyroid glands. Using near-infrared autofluorescence imaging can effectively distinguish lymph nodes and parathyroid glands during lymph node dissection.