Significance: Sterility and reduction of the bioburden are crucial for healing in chronic wounds such as diabetic foot ulcers. Although there are methods for measuring bioburdens, such as semiquantitative analysis of swab/biopsy samples, microbiological sampling, and molecular diagnostics, these tools are less accessible owing to costs or not being as quick as other methods. These methods are also dependent on clinical assessment by the clinician, and high bacterial burden may appear asymptomatic. Recent Advances: Autofluorescence (AF) imaging is a novel technology for identifying and quantifying chronic inhibitory bacterial load in chronic wounds. Eighty-seven percent of bacteria that frequent chronic wounds have fluorophores that fluoresce under violet light as red or cyan, depending on the type of fluorophore. Therefore, AF image-guided treatment is becoming increasingly effective for physicians to implement wound dressing changes and debridement because bacterial burdens are difficult to locate clinically. Critical Issue: Products such as the commercially available MolecuLight i:X and MolecuLight DX function as handheld cameras for physicians to use as a reference but require additional work to ensure that the photograph will be taken with adequate lighting. Future Directions: Designs for Vision Inc. introduced a device called REVEAL, an AF imaging form factor that allows the device to be worn on top of a pair of glasses, which the physician would wear intraoperatively. The benefits of this form factor include not requiring certain lighting conditions and not having to interpret the results using a handheld camera, allowing the device to be used during active surgical debridement.

UNASSIGNED: Autofluorescence-based imaging has the potential to non-destructively characterize the biochemical and physiological properties of plants regulated by genotypes using optical properties of the tissue. A comparative study of stress tolerant and stress susceptible genotypes of Brassica rapa with respect to newly introduced stress-based phenotypes using machine learning techniques will contribute to the significant advancement of autofluorescence-based plant phenotyping research.
UNASSIGNED: Autofluorescence spectral images have been used to design a stress detection classifier with two classes, stressed and non-stressed, using machine learning algorithms. The benchmark dataset consisted of time-series image sequences from three Brassica rapa genotypes (CC, R500, and VT), extreme in their morphological and physiological traits captured at the high-throughput plant phenotyping facility at the University of Nebraska-Lincoln, USA. We developed a set of machine learning-based classification models to detect the percentage of stressed tissue derived from plant images and identified the best classifier. From the analysis of the autofluorescence images, two novel stress-based image phenotypes were computed to determine the temporal variation in stressed tissue under progressive drought across different genotypes, i.e., the average percentage stress and the moving average percentage stress.
UNASSIGNED: The study demonstrated that both the computed phenotypes consistently discriminated against stressed versus non-stressed tissue, with oilseed type (R500) being less prone to drought stress relative to the other two Brassica rapa genotypes (CC and VT).
UNASSIGNED: Autofluorescence signals from the 365/400 nm excitation/emission combination were able to segregate genotypic variation during a progressive drought treatment under a controlled greenhouse environment, allowing for the exploration of other meaningful phenotypes using autofluorescence image sequences with significance in the context of plant science.

The Retinal Pigment Epithelium (RPE) plays a prominent role in diseases such as age-related macular degeneration, but imaging individual RPE cells is challenging due to their high absorption and low autofluorescence emission. The RPE lies beneath the highly reflective photoreceptor layer (PR) and contains absorptive pigments, preventing direct backscattered light detection when the PR layer is intact. Here, we used near-infrared autofluorescence adaptive optics scanning laser ophthalmoscopy (NIRAF AOSLO) and transscleral flood imaging (TFI) in the same healthy eyes to cross-validate these approaches. Both methods revealed a consistent RPE mosaic pattern and appeared to reflect a distribution of fluorophores consistent with findings from histological studies. Interestingly, even in apparently healthy RPE, we observed dynamic changes over months, suggesting ongoing cellular activity or alterations in fluorophore distribution. These findings emphasize the value of NIRAF AOSLO and TFI in understanding RPE morphology and dynamics.

UNASSIGNED: The purpose of this study was to assess how oral and maxillofacial surgeons used various diagnostic tools for oral cancer.
UNASSIGNED: A cross-sectional methodology was used, and a standardized questionnaire was given to oral and maxillofacial surgeons randomly chosen sample. The questionnaire gathered information on demographics and the use of diagnostic tools. Data analysis methods included Chi-square testing and descriptive statistics.
UNASSIGNED: The study included 200 oral and maxillofacial surgeons in total. The most often used diagnostic tool (95%) was visual inspection, followed by toluidine blue staining (48%) and brush biopsy (32%). Less frequently used were newer methods like optical coherence tomography (12.5%) and autofluorescence imaging (15%). No significant correlations between demographic factors and patterns of use of diagnostic tools were found by Chi-square tests.
UNASSIGNED: The results show that oral and maxillofacial surgeons frequently use brush biopsy, toluidine blue staining, and ocular evaluation. However, there is a need for more widespread adoption of cutting-edge technologies. By removing obstacles and offering training opportunities, one can increase the use of diagnostic tools, improving patient outcomes and the diagnosis of oral cancer.

BACKGROUND: Kabuki Syndrome is a rare and genetically heterogenous condition with both ophthalmic and systemic complications and typical facial features. We detail the macular phenotype in two unrelated patients with Kabuki syndrome due to de novo nonsense variants in KMT2D, one novel. A follow-up of 10 years is reported. Pathogenicity of both de novo nonsense variants is analyzed.
METHODS: Four eyes of two young patients were studied by full clinical examination, kinetic perimetry, short wavelength autofluorescence, full field (ff) ERGs, and spectral-domain optical coherence tomography (SD-OCT). One patient had adaptive optic (AO) imaging. Whole exome sequencing was performed in both patients.
RESULTS: Both patients had de novo nonsense variants in KMTD2. One patient had c.14843C>G; p. (Ser4948ter) novel variant and the second c.11119C>T; p. (Arg3707ter). Both had a stable Snellen visual acuity of 0.2-0.3. The retinal multimodal imaging demonstrated abnormalities at the fovea in both eyes: hyperreflectivity to blue light and a well-delimited gap-disruption of ellipsoid and interdigitation layer on OCT. The dark area on AO imaging is presumed to be absent for, or with structural change to photoreceptors. The ff ERGs and kinetic visual fields were normal. The foveal findings remained stable over several years.
CONCLUSIONS: Kabuki syndrome-related maculopathy is a distinct loss of photoreceptors at the fovea as shown by multimodal imaging including, for the first time, AO imaging. This report adds to the literature of only one case with maculopathy with two additional macular dystrophies in patients with Kabuki syndrome. Although underestimated, these cases further raise awareness of the potential impact of retinal manifestations of Kabuki syndrome not only among ophthalmologists but also other healthcare professionals involved in the care of patients with this multisystem disorder.

OBJECTIVE: This prospective observational study investigated wound area reduction (WAR) outcomes in a complex wound population composed of non-healing acute and chronic wounds. The relationship between bacterial autofluorescence signals and WAR was investigated. Area measurements were collected both manually and digitally, and both methods were compared for accuracy.
METHODS: Twenty-six participants with 27 wounds of varying etiologies were observed twice weekly for two weeks. Digital wound measurement, wound bacterial status assessment, and targeted debridement were performed through a point-of-care fluorescence imaging device (MolecuLight® i: X, MolecuLight Inc, Toronto, Canada). The wound area reduction (WAR) rate was calculated using baseline and last visit measurements. Statistical analyses, including t-tests, Fisher exact tests, the Wilcoxon signed rank test for method comparison, and ANOVA for bacterial subgroups, were applied as pertinent.
RESULTS: The overall average WAR was -3.80 cm2, or a decrease of 46.88% (manual measurement), and -2.62 cm2, or a 46.05% decrease (digital measurement via MolecuLight® device). There were no statistically significant differences between the WAR of acute and chronic wounds (p = 0.7877). A stepwise correlation between the WAR and bacterial status classification per fluorescence findings was observed, where persistent bacteria resulted in worse WAR outcomes. An overestimation of wound area by manual measurement was 23% on average.
CONCLUSIONS: Fluorescence imaging signals were linked to WAR outcome and could be considered predictive. Wounds exhibiting bacterial loads that persisted at the end of the study period had worse WAR outcomes, while those for which management was able to effectively remove them demonstrated greater WAR. Manual measurement of the wound area consistently overestimated wound size when compared to digital measurement. However, if performed by the same operator, the overestimation was uniform enough that the WAR was calculated to be close to accurate. Notwithstanding, single wound measurements are likely to result in overestimation.

BACKGROUND: Extant lineages of sea spiders (Pycnogonida) exhibit different types of development. Most commonly, pycnogonids hatch as a minute, feeding protonymphon larva with subsequent anamorphic development. However, especially in cold water habitats at higher latitudes and in the deep sea, some taxa have large, lecithotrophic larvae, or even undergo extended embryonic development with significantly advanced postlarval hatching stages. Similar biogeographic trends are observed in other marine invertebrates, often referred to as \"Thorson\'s rule\".
RESULTS: To expand our knowledge on the developmental diversity in the most speciose pycnogonid genus Nymphon, we studied the developmental stages of the two tropical representatives N. floridanum and N. micronesicum., We compared classical scanning electron microscopy with fluorescence-based approaches to determine which imaging strategy is better suited for the ethanol-fixed material available. Both species show epimorphic development and hatch as an advanced, lecithotrophic postlarval instar possessing the anlagen of all body segments. Leg pairs 1-3 show a considerable degree of differentiation at hatching, but their proximal regions remain coiled and hidden under the cuticle of the hatching instar. The adult palp and oviger are not anteceded by three-articled larval limbs, but differentiate directly from non-articulated limb buds during postembryonic development.
CONCLUSIONS: Fluorescence imaging yielded more reliable morphological data than classical scanning electron microscopy, being the method of choice for maximal information gain from rare and fragile sea spider samples fixed in high-percentage ethanol. The discovery of epimorphic development with lecithotrophic postlarval instars in two small Nymphon species from tropical shallow-water habitats challenges the notion that this developmental pathway represents an exclusive cold-water adaptation in Nymphonidae. Instead, close phylogenetic affinities to the likewise more direct-developing Callipallenidae hint at a common evolutionary origin of this trait in the clade Nymphonoidea (Callipallenidae + Nymphonidae). The lack of functional palpal and ovigeral larval limbs in callipallenids and postlarval hatchers among nymphonids may be a derived character of Nymphonoidea. To further test this hypothesis, a stable and well-resolved phylogenetic backbone for Nymphonoidea is key.

BACKGROUND: Macrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates.
METHODS: Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation.
RESULTS: Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population.
CONCLUSIONS: Overall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.

Actinic cheilitis (AC) is recognized as the most common precursor lesion of squamous cell carcinoma (SCC) of the lip, with a higher risk of invasiveness and metastasis. Early accurate diagnosis and appropriate therapy are essential to prevent carcinogenesis and progression of AC. Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT), a non-surgical and minimally invasive modality, has been proposed as an effective treatment for oral potentially malignant diseases (OPMDs) and oral cancers. Herein, we report a 64-year-old female patient with AC on the lower lip who received 3 sessions of ALA-PDT with an interval of 1 week. Multiple noninvasive auxiliary tests including autofluorescence imaging, toluidine blue staining, and aneuploidy with DNA image cytometry (DNA-ICM) using brushing from screening through diagnosis, treatment, and follow-up. The patient successfully showed a complete response with no adverse effects and no evidence of recurrence at the 20-month follow-up. Noninvasive auxiliary tests assisted PDT is attractive and well-tolerated and may have synergistic effects against AC.

Rationale: Mesoscopic visualization of the main anatomical structures of the whole kidney in vivo plays an important role in the pathological diagnosis and exploration of the etiology of hydronephrosis. However, traditional imaging methods cannot achieve whole-kidney imaging with micron resolution under conditions representing in vivo perfusion. Methods: We used in vivo cryofixation (IVCF) to fix acute obstructive hydronephrosis (unilateral ureteral obstruction, UUO), chronic spontaneous hydronephrosis (db/db mice), and their control mouse kidneys for cryo-micro-optical sectioning tomography (cryo-MOST) autofluorescence imaging. We quantitatively assessed the kidney-wide pathological changes in the main anatomical structures, including hydronephrosis, renal subregions, arteries, veins, glomeruli, renal tubules, and peritubular functional capillaries. Results: By comparison with microcomputed tomography imaging, we confirmed that IVCF can maintain the status of the kidney in vivo. Cryo-MOST autofluorescence imaging can display the main renal anatomical structures with a cellular resolution without contrast agents. The hydronephrosis volume reached 26.11 ± 6.00 mm3 and 13.01 ± 3.74 mm3 in 3 days after UUO and in 15-week-old db/db mouse kidneys, respectively. The volume of the cortex and inner stripe of the outer medulla (ISOM) increased while that of the inner medulla (IM) decreased in UUO mouse kidneys. Db/db mice also showed an increase in the volume of the cortex and ISOM volume but no atrophy in the IM. The diameter of the proximal convoluted tubule and proximal straight tubule increased in both UUO and db/db mouse kidneys, indicating that proximal tubules were damaged. However, some renal tubules showed abnormal central bulge highlighting in the UUO mice, but the morphology of renal tubules was normal in the db/db mice, suggesting differences in the pathology and severity of hydronephrosis between the two models. UUO mouse kidneys also showed vascular damage, including segmental artery and vein atrophy and arcuate vein dilation, and the density of peritubular functional capillaries in the cortex and IM was reduced by 37.2% and 49.5%, respectively, suggesting renal hypoxia. In contrast, db/db mouse kidneys showed a normal vascular morphology and peritubular functional capillary density. Finally, we found that the db/db mice displayed vesicoureteral reflux and bladder overactivity, which may be the cause of hydronephrosis formation. Conclusions: We observed and compared main renal structural changes in hydronephrosis under conditions representing in vivo perfusion in UUO, db/db, and control mice through cryo-MOST autofluorescence imaging. The results indicate that cryo-MOST with IVCF can serve as a simple and powerful tool to quantitatively evaluate the in vivo pathological changes in three dimensions, especially the distribution of body fluids in the whole kidney. This method is potentially applicable to the three-dimensional visualization of other tissues, organs, and even the whole body, which may provide new insights into pathological changes in diseases.