Autofluorescence Intensity Research Articles

Near-Infrared Autofluorescence Signature: A New Parameter for Intraoperative Assessment of Parathyroid Glands in Primary Hyperparathyroidism.

The success of parathyroidectomy in primary hyperparathyroidism depends on the intraoperative differentiation of diseased from normal glands. Deep learning can potentially be applied to digitalize this subjective interpretation process that relies heavily on surgeon expertise. In this study, we aimed to investigate whether diseased versus normal parathyroid glands have different near-infrared autofluorescence (NIRAF) signatures and whether related deep learning models can predict normal versus diseased parathyroid glands based on intraoperative in-vivo images. This prospective study included patients who underwent parathyroidectomy for primary hyperparathyroidism or thyroidectomy using intraoperative NIRAF imaging at a single tertiary referral center from November 2019 to March 2024. Autofluorescence intensity and heterogeneity index of normal versus diseased parathyroid glands were compared, and a deep learning model was developed. NIRAF images of a total of 1,506 normal and 597 diseased parathyroid glands from 797 patients were analyzed. Normal versus diseased glands exhibited a higher median normalized NIRAF intensity [2.68 (2.19-3.23) vs 2.09 (1.68-2.56) pixels, p<.0001] and lower heterogeneity index [0.11 (0.08-0.15) vs 0.18 (0.13-0.23), p<.0001]. On receiver operating characteristics analysis, optimal thresholds to predict a diseased gland were 2.22 in pixel intensity and 0.14 in heterogeneity index. On deep learning, precision and recall of the model were 83.3% each, and area under the precision-recall curve (AUPRC) was 0.908. Normal and diseased parathyroid glands in primary hyperparathyroidism have different intraoperative NIRAF patterns that could be quantified with intensity and heterogeneity analyses. Visual deep learning models relying on these NIRAF signatures could be built to assist surgeons in differentiating normal from diseased parathyroid glands.

Surface plasmon enhanced auto-fluorescence and Raman spectroscopy for low-level detection of biological pathogens.

The current culture-based bacterial detection technique is time-consuming and requires an extended sample preparation methodology. We propose the potential of surface-enhanced Raman spectroscopy (SERS) and surface plasmon-enhanced auto-fluorescence spectroscopy (SPEAS) for the label-free identification and quantification of bacterial pathogens at low concentrations collecting its unique auto-fluorescence and Raman signatures utilising highly anisotropic three-dimensional nanostructures of silver nano dendrites (Ag-NDs). The SERS data facilitates qualitative bacterial identification using the spectral features coming from the bacterial cell wall compound, and the SPEAS data was utilised to gain unique auto-fluorescence spectra present on the bacterial cell wall with enhanced quantification. The enhancement of Raman and auto-fluorescence signals of Ag-NDs were first evaluated using rhodamine 6g (R6G) as a probe molecule that exhibits a significant enhancement of 106 and limit of detection (LOD) of 10-12 M for SERS and 15 fold intensity enhancement and LOD of 10-15 M for SPEAS measurements. Further, the SERS and SPEAS measurements of bacterial pathogens, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), using the Ag-NDs were recorded, and the results exhibit high auto-fluorescence and Raman signal intensity for both the samples up to 100 cfu/ml for both modalities. The significant photon count and distinct emission range in SPEAS measurements of bacteria enables accurate quantification. Therefore, the comprehensive investigation of plasmonic enhancement of Ag-NDs for SPEAS and SERS techniques provides complementary information about molecules to enable accurate and quick identification and quantification of pathogens.

Multimodal Optical Imaging of Ex Vivo Fallopian Tubes to Distinguish Early and Occult Tubo-Ovarian Cancers.

Background: There are currently no effective screening measures to detect early or occult tubo-ovarian cancers, resulting in late-stage detection and high mortality. This work explores whether an optical imaging catheter can detect early-stage tubo-ovarian cancers or precursor lesions where they originate in the fallopian tubes. Methods: This device collects co-registered optical coherence tomography (OCT) and autofluorescence imaging (AFI). OCT provides three-dimensional assessment of underlying tissue structures; autofluorescence imaging provides functional contrast of endogenous fluorophores. Ex vivo fallopian tubes (n = 28; n = 7 cancer patients) are imaged; we present methods for the calculation of and analyze eleven imaging biomarkers related to fluorescence, optical attenuation, and OCT texture for their potential to detect tubo-ovarian cancers and other lesions of interest. Results: We visualize folded plicae, vessel-like structures, tissue layering, hemosiderin deposits, and regions of fibrotic change. High-grade serous ovarian carcinoma appears as reduced autofluorescence paired with homogenous OCT and reduced mean optical attenuation. Specimens containing cancerous lesions demonstrate a significant increase in median autofluorescence intensity and decrease in Shannon entropy compared to specimens with no lesion. Non-cancerous specimens demonstrate an increase in optical attenuation in the fimbriae when compared to the isthmus or the ampulla. Conclusions: We conclude that this approach shows promise and merits further investigation of its diagnostic potential.

Near-Infrared Autofluorescence Signatures of Single- vs Multigland Disease in Primary Hyperparathyroidism

The success of parathyroidectomy depends on accurate intraoperative localization and identification of all diseased glands in parathyroid exploration based on surgeon expertise to prevent persistent hyperparathyroidism. Near-infrared autofluorescence (NIRAF) imaging has recently emerged as a promising adjunctive intraoperative tool for localizing parathyroid glands; however, its potential utility in the assessment of parathyroid glands has yet to be established. To analyze the differences in NIRAF signatures of parathyroid glands in single vs multiple glands in primary hyperparathyroidism (pHPT). This prospective diagnostic study analyzed in vivo NIRAF images of parathyroid glands obtained during parathyroidectomies between November 18, 2019, and December 31, 2023, at a single tertiary referral center. Pixel intensities of the images were measured using third-party software. Patients who underwent parathyroidectomy for sporadic pHPT using a second-generation NIRAF imaging device were included. Patients with multiple endocrine neoplasm disorders were excluded. In vivo NIRAF images obtained during the procedures were analyzed. Near-infrared autofluorescence imaging during parathyroidectomy. The primary outcomes were the autofluorescence intensity and heterogeneity of single adenomas and multigland disease (ie, double adenomas and 3- or 4-gland hyperplasia) in sporadic pHPT. Normalized autofluorescence intensity was calculated by dividing the mean pixel intensity of the parathyroid gland by the background tissue. A heterogeneity index was calculated by dividing the standard deviation by the mean pixel intensity of the gland. The secondary outcome was the visibility of each parathyroid gland on NIRAF imaging before it became apparent to the naked eye during exploration. A total of 1287 in vivo NIRAF images obtained from 377 patients (median [IQR] age, 66 [56-73] years; 299 female [79.3%]) were analyzed. Of all patients, 230 (61.0%) had a single adenoma, 91 (24.1%) had double adenomas, and 56 (14.9%) had 3- or 4-gland hyperplasia. A mean (SD) of 3.4 (1.1) parathyroid glands were identified in the procedures. A comparison of 581 diseased glands (45.1%) and 706 normal glands (54.9%) showed a lower median normalized autofluorescence intensity of 2.09 (95% CI, 1.07-4.01) vs 2.66 (95% CI, 1.43-4.20; effect size = 0.36) and higher heterogeneity index of 0.18 (95% CI, 0.07-0.41) vs 0.11 (95% CI, 0.01-0.27; effect size = 0.45), respectively. Of diseased glands, single adenomas (233 [40.1%]) vs double adenomas (187 [32.2%]) and 3- or 4-gland hyperplasia (161 [27.7%]) had a lower median autofluorescence intensity of 1.92 (95% CI, 1.02-4.44) vs 2.22 (95% CI, 1.10-3.97; effect size = 0.21), respectively. On receiver operating characteristic analysis, the optimal autofluorescence intensity threshold to differentiate between single adenomas vs multigland disease was 2.14, with a sensitivity of 64.4%, specificity of 58.1%, and area under the curve of 0.626. These findings suggest that parathyroid glands in single- vs multigland disease may exhibit different autofluorescence characteristics. Although the effect size was modest, the differences identified should be kept in mind when assessing the parathyroid glands during surgical exploration.

UV irradiation during fluorescence measurements affects the structures and fluorescence properties of isolated lignins in solution

There are no reports on the stability of autofluorescence, although lignins and their solutions are known to exhibit autofluorescence. An aim of this study is to investigate spectral change of lignin autofluorescence under prolonged excitation with ultraviolet (UV). Both fluorescence spectra of softwood milled wood lignin (MWL) and kraft lignin (SKL) in propylene glycol significantly changed upon an UV irradiation at 280 nm under O2 and Ar atmospheres even for 10 min. By the further irradiation, the autofluorescence intensities were remarkably changed together with the emission wavelengths. However, no tendency in the autofluorescence change was observed. The change varied between lignin preparations and gas atmosphere. These phenomena should be caused by autoxidation upon UV irradiation, and the reaction resulted in not only photodegradation but also photopolymerization.

Raman and autofluorescence spectroscopy for in situ identification of neoplastic tissue during surgical treatment of brain tumors

PurposeRaman spectroscopy (RS) is a promising method for brain tumor detection. Near-infrared autofluorescence (AF) acquired during RS provides additional useful information for tumor identification and was investigated in comparison with RS for delineating brain tumors in situ.MethodsRaman spectra were acquired together with AF in situ within the solid tumor and at the tumor border during routine brain tumor surgeries (218 spectra; glioma WHO II-III, n = 6; GBM, n = 10; metastases, n = 10; meningioma, n = 3). Tissue classification for tumor identification in situ was trained on ex vivo data (375 spectra; glioma/GBM patients, n = 20; metastases, n = 11; meningioma, n = 13; and epileptic hippocampi, n = 4).ResultsBoth in situ and ex vivo data showed that AF intensity in brain tumors was lower than that in border regions and normal brain tissue. Moreover, a positive correlation was observed between the AF intensity and the intensity of the Raman band corresponding to lipids at 1437 cm− 1, while a negative correlation was found with the intensity of the protein band at 1260 cm− 1. The classification of in situ AF and RS datasets matched the surgeon’s evaluation of tissue type, with correct rates of 0.83 and 0.84, respectively. Similar correct rates were achieved in comparison to histopathology of tissue biopsies resected in selected measurement positions (AF: 0.80, RS: 0.83).ConclusionsSpectroscopy was successfully integrated into existing neurosurgical workflows, and in situ spectroscopic data could be classified based on ex vivo data. RS confirmed its ability to detect brain tumors, while AF emerged as a competitive method for intraoperative tumor delineation.

Analysis of near-infrared autofluorescence imaging for detection of inadvertently resected parathyroid glands after endoscopic thyroidectomy

BackgroundPreserving parathyroid function during thyroidectomy is crucial, but remains challenging. Real-time near-infrared autofluorescence (NIRAF) aids surgeons in intraoperative parathyroid gland (PTG) identification. However, its role in detecting PTGs unintentionally removed during surgery is unclear. Study designThis prospective study included adult patients undergoing endoscopic thyroidectomy. Surgeons identified and documented PTGs visually. Excised specimens underwent visual inspection and NIRAF imaging (PDE-Neo II). All fluorescent tissues were dissected and pathologically evaluated (reference standard). One scanned image per lobe was chosen to quantify autofluorescence (AF) intensity. ResultsOverall, 95 patients underwent endoscopic thyroidectomies, with NIRAF imaging applied to 152 excised lobes. Of these, 19 lobes displayed a total of 23 spots with increased intensity. 175 specimens were sent for pathological evaluation, and 7 were confirmed to be parathyroid tissue. NIRAF demonstrated 100.0 % sensitivity and 90.5 % specificity for predicting parathyroid tissue, with 30.4 % positive predictive value, 100.0 % negative predictive value of and 90.9 % accuracy. Quantitatively normalized, the AF signal intensity was significantly higher in NIRAF-positive tissues than negative (4.3 vs 1.2 times, p < 0.0001). Additionally, the AF signal intensity in regions pathologically confirmed of parathyroid tissue was higher than non-parathyroid tissue (9.1 vs 2.1 times, p < 0.0001). ConclusionThis study suggests that NIRAF has high sensitivity and specificity for detecting inadvertently resected PTGs after endoscopic thyroidectomy, contributing to preservation efforts. However, NIRAF-positive tissues still require additional confirmation through multiple methods, emphasizing other examinations to verify that they are indeed parathyroid tissues. Further research is warranted to refine NIRAF imaging parameters.

Combining Nitellopsis obtusa autofluorescence intensity and F680/F750 ratio to discriminate responses to environmental stressors

Detection of autofluorescence parameters is a useful approach to gain insight into the physiological state of plants and algae, but the effect of reabsorption hinders unambiguous interpretation of in vivo data. The exceptional morphological features of Nitellopsis obtusa made it possible to measure autofluorescence spectra along single internodal cells and estimate relative changes in autofluorescence intensity in selected spectral regions at room temperatures, avoiding the problems associated with thick or optically dense samples. The response of algal cells to controlled white light and DCMU herbicide was analyzed by monitoring changes in peak FL intensity at 680 nm and in F680/F750 ratio. Determining the association between the selected spectral FL parameters revealed an exponential relationship, which provides a quantitative description of photoinduced changes. The ability to discern the effect of DCMU not only in the autofluorescence spectra of dark-adapted cells, but also in the case of light-adapted cells, and even after certain doses of excess light, suggests that the proposed autofluorescence analysis of N. obtusa may be useful for detecting external stressors in the field.

Correlation Between Near-Infrared Autofluorescence Properties and Sestamibi Uptakes of Parathyroid Glands in Primary Hyperparathyroidism.

Near-infrared autofluorescence (NIRAF) characteristics of parathyroid glands in primary hyperparathyroidism (pHPT) vary, with unclarity regarding the underlying mechanism. Similarly, 99mTc-sestamibi uptake in diseased parathyroid glands is variable. There is a suggestion that oxyphilic cell content may influence both imaging modalities. This study aims to analyze the relationship between NIRAF imaging characteristics, 99mTc-sestamibi uptake, and cellular composition in pHPT. Retrospective analysis of an Institutional Review Board-monitored prospective database. Single tertiary referral center. NIRAF characteristics of parathyroid glands of patients with pHPT between 2019 and 2024 were compared with 99mTc-sestamibi scan findings from a prospective database. Using third-party software, brightness intensity and heterogeneity index (HI) of the glands were calculated. A subgroup of parathyroid glands obtained from consecutive patients with pHPT in 2020 to 2021 underwent histological analysis. A total of 428 patients with 638 diseased parathyroid glands were analyzed. Forty-seven percent of the glands showed an uptake on 99mTc-sestamibi scans. The brightness intensity of the NIRAF signals from parathyroid glands that were seen versus not seen on sestamibi was 2.1 versus 2.3 (P = .002) and HI 0.18 versus 0.17 (P = .35), respectively. On multivariate analysis, low autofluorescence intensity, high gland volume, and single adenoma were associated with detectability on 99mTc-sestamibi scan (P < .0001). Intraglandular adipose tissue content was lower in diseased glands that were detected on 99mTc-sestamibi scans (0% vs 5%, P < .0001). Our findings indicate an inverse relationship between autofluorescence intensity and detectability on 99mTc-sestamibi scans and a lack of correlation between different cell types and autofluorescence properties.

Relationship Between Oxidative Stress in the Rotator Cuff and Transcutaneous Advanced Glycation End-Products Measurement in Diabetic Rats.

Diabetes mellitus increases oxidative stress due to hyperglycemia, resulting in the degeneration of rotator cuff tissue. Currently, there is no established method to non-invasively assess the extent of this oxidative stress. To address this, we aimed to investigate the relationship between the accumulation of advanced glycation end-products (AGEs), a marker of oxidative stress, and transcutaneous autofluorescence intensity in rotator cuff tissue harvested from diabetic rats. Ten control Sprague-Dawley (SD) rats and streptozotocin-induced diabetic rats (n = 10 per group) were used. The rats were euthanized eight and 16 weeks after the induction of diabetes, and rotator cuff attachment sites were collected and histologically analyzed. Prior to euthanasia, autofluorescence intensity was measured transcutaneously in the rotator cuff area. The expressions of AGEs and type I collagen were evaluated immunohistochemically with specific antibodies and the stained areas were quantified. All data were statistically analyzed using the Mann-Whitney U test. Correlation analysis was performed for skin autofluorescence intensity and the percentage of AGEs staining area using Spearman's rank correlation coefficient. The immunohistochemical expression of AGEs at the rotator cuff attachment sites and transcutaneous AGEs measurements were significantly higher in diabetic rats than in the control group at 16 weeks. There was no significant difference in the level of type 1 collagen between the two groups. This study reveals that the accumulation of AGEs in rotator cuff tissue increases due to prolonged hyperglycemia in diabetes. In addition, transcutaneous skin fluorescence intensity may be related to histological oxidative stress at the rotator cuff.

In vivo Raman spectroscopic and fluorescence study of suspected melanocytic lesions and surrounding healthy skin.

Cutaneous melanoma is the most lethal skin cancer and noninvasively distinguishing it from benign tumor is a major challenge. Raman spectroscopic measurements were conducted on 65 suspected melanocytic lesions and surrounding healthy skin from 47 patients. Compared to the spectra of healthy skin, spectra of melanocytic lesions exhibited lower intensities in carotenoid bands and higher intensities in lipid and melanin bands, suggesting similar variations in the content of these components. Distinct variations were observed among the autofluorescence intensities of healthy skin, benign nevi and malignant melanoma. By incorporating autofluorescence information, the classification accuracy of the support vector machine for spectra of healthy skin, nevi, and melanoma reached 90.2%, surpassing the 87.9% accuracy achieved without autofluorescence, with this difference being statistically significant. These findings indicate the diagnostic value of autofluorescence intensity, which reflect differences in fluorophore content, chemical composition, and structure among healthy skin, nevi, and melanoma.

Computer-assisted discrimination of cancerous and pre-cancerous from benign oral lesions based on multispectral autofluorescence imaging endoscopy.

Diagnosis of cancerous and pre-cancerous oral lesions at early stages is critical for the improvement of patient care, to increase survival rates and minimize the invasiveness of tumor resection surgery. Unfortunately, oral precancerous and early-stage cancerous lesions are often difficult to distinguish from oral benign lesions with the existing diagnostic tools used during standard clinical oral examination. In consequence, early diagnosis of oral cancer can be achieved in only about 30% of patients. Therefore, clinical diagnostic technologies for fast, minimally invasive, and accurate oral cancer screening are urgently needed. This study investigated the use of multispectral autofluorescence imaging endoscopy for the automated and noninvasive discrimination of cancerous and precancerous from benign oral epithelial lesions. In vivo multispectral autofluorescence endoscopic images of clinically suspicious oral lesions were acquired from 67 patients undergoing tissue biopsy examination. The imaged lesions were classified as precancerous (n = 4), cancerous (n = 29), and benign (n = 34) lesions based on histopathology diagnosis. Multispectral autofluorescence intensity feature maps were generated for each oral lesion and used to train and optimize support vector machine (SVM) models for automated discrimination of cancerous and precancerous from benign oral lesions. After a leave-one-patient-out cross-validation strategy, an optimized SVM model developed with four multispectral autofluorescence features yielded levels of sensitivity and specificity of 85% and 71%, respectively and overall accuracy of 78% in the discrimination of cancerous/precancerous versus benign oral lesions. This study demonstrates the potentials of a computer-assisted detection system based on multispectral autofluorescence imaging endoscopy for the early detection of cancerous and precancerous oral lesions.

A Comparison of Near-Infrared Autofluorescence Findings in Benign Versus Malignant Adrenal Tumors

BackgroundMany adrenal tumors are deemed radiologically indeterminate and surgically removed. Adrenal tissue, like parathyroid glands, exhibits near-infrared autofluorescence (NIRAF) properties. This study was designed to investigate the potential of NIRAF to differentiate benign versus malignant adrenal tumors.MethodsPatients undergoing adrenalectomy between October 2021 and May 2023 were prospectively studied. Adrenalectomy specimens were inspected with NIRAF imaging. Specimen autofluorescence (AF) characteristics were recorded. Comparisons were made between different tumor types and a logistic regression model was constructed to differentiate benign versus malignant tumors. A receiver operating characteristic curve was used to identify an optimal AF threshold differentiating benign versus malignant tumors.ResultsA total of 108 adrenal specimens were examined: adrenocortical adenomas/other benign lesions (n = 72), pheochromocytomas (n = 18), adrenocortical neoplasms of uncertain behavior (n = 4), and malignant tumors (n = 14). A significant difference in normalized AF intensity was identified when comparing adrenocortical adenomas (3.08 times background) with pheochromocytomas (1.95, p = 0.001) and malignant tumors (1.11, p < 0.0001). The Area Under the Curve differentiating benign vs malignant tumors was 0.87, with an optimal normalized AF threshold at 1.93.ConclusionsDifferent adrenal pathologies exhibit diverse AF properties. These findings suggest a potential intraoperative utility of NIRAF in predicting benign versus malignant nature for radiologically indeterminate adrenal tumors.

Autonomous and Self-sustained Circadian Oscillators Displayed in Human Primary Parathyroid Cell Culture Drive Parathormone Secretion

Abstract Background Most cells in our body possess circadian oscillators controlling diurnal rhythmicity of body metabolism. The role of molecular clocks in regulating the function and disfunction of human parathyroid gland (PG), responsible for calcium homeostasis, has not been unraveled. Hyperparathyroidism is a common endocrine pathology, characterized by an unadapted PTH secretion to calcium levels. Aims Here we aimed at characterizing molecular makeup of circadian clocks in human primary parathyroid cell culture (HPPCC), and at establishing differential transcriptional patterns of normal and pathological PGs. Methods RNA extracted from normal, adenomatous, and hyperplastic PG tissues was subjected to RNA sequencing (RNAseq) analysis. The rim of normal PG tissue was dissected from parathyroid adenoma specimens based on the higher autofluorescence intensity assessed by the near infra-red imaging. HPPCCs were established from adenomatous and hyperplastic PG biopsies. Efficient circadian clock disruption was achieved in HPPCCs by small interfering RNA-mediated knockdown of CLOCK. The functionality of circadian clock machinery was assessed by continuous recording of circadian bioluminescence introduced via lentivectors and paralleled with around-the-clock measurement of PTH secretion using perfusion system. Results The RNAseq analyses revealed transcriptional signatures that distinguished between pathological and normal PGs, and among adenomatous and hyperplastic PGs. We report, for the first time, robust anti-phasic circadian rhythmicity of Per2-luciferase and Bmal1-luciferase reporters in HPPCCs synchronized in vitro. Strikingly, we observed circadian rhythmicity of PTH secretion by parathyroid adenoma cells. Moreover, clock disruption in PG adenoma cells has an impact on transcription of functional genes. Conclusion Our data indicate presence of cell-autonomous molecular clock in human PG cells paralleled with circadian rhythmic PTH secretion and provide large-scale transcriptional pattern of parathyroid adenoma and hyperplastic tissues.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensitiesand fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Comparative analysis of the integration of autologous and allogeneic cryopreserved tendons in the femoral canal on a model of laboratory animals

Allogeneic tendon grafts are seriously demand in knee joint plastic surgery. The novel method of tendon cryopreservation, including sterilization with supercritical carbon dioxide, was developed in N.V. Sklifosovsky Research Institute for Emergency Cryopreserved tendons retain their normal fiber structure without significant loss of mechanical properties. At the next stage it was necessary to evaluate cryopreserved tendons′ integration inside bone canal in experimental animals.The aim of study. To evaluate morphologic changes of autologous and allogeneic tendons inside the femur in rats and to determine the effect of tendon transplantation on the physical activity.Material and methods. The study was conducted on white inbreed male rats. Three groups of animals were formed: the control group (animals without tendon transplantation), the 1st experimental group – animals with autologous tendon transplantation, the 2nd experimental group – animals with allogeneic tendon transplantation. In animals of the experimental groups the through channel was formed in the distal metaepiphysis of the femur and a tail tendon graft 0.5 x 0.1 cm was placed there. To assess the physical activity of the animals, we studied maximum distance that the animals could run 3 and 6 weeks after transplantation was determined, using treadmill test. The graft structure was evaluated on histological preparations in transmitted light, stained with hematoxylin-eosin and Van Gieson′s stain. To assess the preservation of collagen fibers we checked the autofluorescence intensity of collagen.Results. According to the treadmill test, the distance run by the animals of both experimental groups did not significantly differ from the values in the control group. Histological analysis after 3 weeks in both experimental groups revealed signs of fibers′ decomposition in the absence of inflammatory infiltration and maintaining close contact with bone trabeculae. The autofluorescence intensity of the collagen fibers in grafts corresponded to normal or was close to normal. After 6 weeks, the animals of both experimental groups revealed areas of graft fusion with their own bone, Sharpe fibers were actively formed. In both groups, numerous small vessels with diameters up to 10 microns were detected in the area of tendon-bone contact. Infiltration of grafts by inflammatory cells was absent or very insignificant, active migration of fibroblasts to the tendon area was also not observed. In both groups, tendon grafts had areas where fiber decompactization was observed. In the area of contact with the bone, the autofluorescence of tendon fibers was sharply increased, which indicates the chemical cleavage of collagen. At 3 and 6 weeks after transplantation the effect of fixation (integration) of the tendon with bone tissue was observed in both experimental groups.Conclusions. Allogeneic tendon grafts did not cause a pronounced inflammatory or immune reaction in experimental animals. 6 weeks after transplantation of autologous and allogeneic tendons, the integration of grafts inside the femoral canal was observed. Cryopreserved allogeneic tendons were able to integrate into the body's own tissues without pronounced structural and functional disorders. According to the treadmill test, the distance covered by the animals of both experimental groups did not differ statistically significantly from the values in the control group (without tendon transplantation) after 3 and 6 weeks

Discrimination of vocal folds lesions by multiclass classification using autofluorescence spectroscopy: An ex vivo study.

Autofluorescence spectroscopy is effective for noninvasive detection but underutilized in tissue with various pathology analyses. This study evaluates whether AFS can be used to discriminate between different types of laryngeal lesions in view of assisting in vocal fold surgery and preoperative investigations. A total of 1308 spectra were recorded from 29 vocal fold samples obtained from 23 patients. Multiclass analysis was performed on the spectral data, categorizing lesions into normal, benign, dysplastic, or carcinoma. Through an appropriate selection of spectral components and a cascading classification approach based on artificial neural networks, a classification rate of 97% was achieved for each lesion class, compared to 52% using autofluorescence intensity. The ex vivo study demonstrates the effectiveness of AFS combined with multivariate analysis for accurate classification of vocal fold lesions. Comprehensive analysis of spectral data significantly improves classification accuracy, such as distinguishing malignant from precancerous or benign lesions.

Impact of knockout of dual-specificity protein phosphatase 5 on structural and mechanical properties of rat middle cerebral arteries: implications for vascular aging.

Vascular aging influences hemodynamics, elevating risks for vascular diseases and dementia. We recently demonstrated that knockout (KO) of Dusp5 enhances cerebral and renal hemodynamics and cognitive function. This improvement correlates with elevated pPKC and pERK1/2 levels in the brain and kidneys. Additionally, we observed that Dusp5 KO modulates the passive mechanical properties of cerebral and renal arterioles, associated with increased myogenic tone at low pressure, enhanced distensibility, greater compliance, and reduced stiffness. The present study evaluates the structural and mechanical properties of the middle cerebral artery (MCA) in Dusp5 KO rats. We found that vascular smooth muscle cell layers and the collagen content in the MCA wall are comparable between Dusp5 KO and control rats. The internal elastic lamina in the MCA of Dusp5 KO rats exhibits increased thickness, higher autofluorescence intensity, smaller fenestrae areas, and fewer fenestrations. Despite an enhanced myogenic response and tone of the MCA in Dusp5 KO rats, other passive mechanical properties, such as wall thickness, cross-sectional area, wall-to-lumen ratio, distensibility, incremental elasticity, circumferential wall stress, and elastic modulus, do not significantly differ between strains. These findings suggest that while Dusp5 KO has a limited impact on altering the structural and mechanical properties of MCA, its primary role in ameliorating hemodynamics and cognitive functions is likely attributable to its enzymatic activity on cerebral arterioles. Further research is needed to elucidate the specific enzymatic mechanisms and explore potential clinical applications in the context of vascular aging.

EARLY STAGES OF COLORECTAL CANCER CHARACTERIZATION BY AUTOFLUORESCENCE 3D MICROSCOPY: A PRELIMINARY STUDY.

Colorectal cancer is one of the most prevalent pathologies worldwide whose prognosis is linked to early detection. Colonoscopy is the gold standard for screening, and diagnosis is usually made histologically from biopsies. Aiming to reduce the inspection and diagnostic time as well as the biopsies and resources involved, other techniques are being promoted to conduct accurate in vivo colonoscopy assessments. Optical biopsy aims to detect normal and neoplastic tissues analysing the autofluorescence spectrum based on the changes in the distribution and concentration of autofluorescent molecules caused by colorectal cancer. Therefore, the autofluorescence contribution analysed by image processing techniques could be an approach to a faster characterization of the target tissue. Quantify intensity parameters through digital processing of two data sets of three-dimensional widefield autofluorescence microscopy images, acquired by fresh colon tissue samples from a colorectal cancer murine model. Additionally, analyse the autofluorescence data to provide a characterization over a volume of approximately 50 µm of the colon mucosa for each image, at second (2nd), fourth (4th) and eighth (8th) weeks after colorectal cancer induction. Development of a colorectal cancer murine model using azoxymethane/dextran sodium sulphate induction, and data sets acquisition of Z-stack images by widefield autofluorescence microscopy, from control and colorectal cancer induced animals. Pre-processing steps of intensity value adjustments followed by quantification and characterization procedures using image processing workflow automation by Fiji's macros, and statistical data analysis. The effectiveness of the colorectal cancer induction model was corroborated by a histological assessment to correlate and validate the link between histological and autofluorescence changes. The image digital processing methodology proposed was then performed on the three-dimensional images from control mice and from the 2nd, 4th, and 8th weeks after colorectal cancer chemical induction, for each data set. Statistical analyses found significant differences in the mean, standard deviation, and minimum parameters between control samples and those of the 2nd week after induction with respect to the 4th week of the first experimental study. This suggests that the characteristics of colorectal cancer can be detected after the 2nd week post-induction. The use of autofluorescence still exhibits levels of variability that prevent greater systematization of the data obtained during the progression of colorectal cancer. However, these preliminary outcomes could be considered an approach to the three-dimensional characterization of the autofluorescence of colorectal tissue, describing the autofluorescence features of samples coming from dysplasia to colorectal cancer. • A new digital image processing method was developed to measure intensity in 3D autofluorescence images of colorectal samples using a CRC mouse model. • This method showed that autofluorescence intensity in colon mucosa is similar in healthy tissue but changes significantly in tumor development. • Statistical analysis revealed CRC traits detectable from the second week post-induction, aiding in early CRC detection. • The study provides a basis for 3D autofluorescence characterization in colorectal tissue from dysplasia to cancer, although variability in autofluorescence limits data systematization during cancer progression.

Characterization of a new fluorescence lifetime imaging ophthalmoscope

Aims/Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) allows in vivo measurement of autofluorescence intensity decays of endogenous fluorophores in the ocular fundus. So far, only devices from Heidelberg Engineering based on the Spectralis system have been used in FLIO research. Here, we present and characterize a new FLIO device based on the RETImap system from Roland Consult.Methods: The device is based on a confocal scanning laser ophthalmoscope (35° field, 512 × 512 px). A ps diode laser (BDL‐SMN 473 nm, Becker &amp; Hickl GmbH, Berlin, Germany) excites autofluorescence. The fluorescence photons are split into a short (498–560 nm, SSC) and a long (560–720 nm, LSC) spectral channel (one HPM‐100‐40 detector [Becker &amp; Hickl GmbH] each) and are detected by time‐correlated single photon counting (SPC‐160, Becker &amp; Hickl GmbH). We determined the maximum laser power (ILT2400, International Light Technologies, Inc. Peabody, MA, USA) and analysed the instrument's behaviour at three different laser power levels (150 μW, 200 μW and max.) in terms of laser spectrum (CAS140CT, Instrument Systems GmbH, Munich, Germany) and instrument response function (IRF). The IRF was determined using a 25 μM Eosin Y solution, mixed with a 5 M solution of potassium iodide, placed in a flat cuvette (110‐OS, Hellma GmbH &amp; Co. KG, Müllheim, Germany) in front of the objective lens of the FLIO device. Fluorescence measurements of approximately 1‐min duration were performed three times for all three laser powers. The IRF and the full width at half maximum (FWHM) were calculated using FLIMX software (www.flimx.de).Results: The max. laser power was 280 μW. The peak wavelengths of the laser spectra were 467.6 (150 μW), 467.9 (200 μW) and 468.0 nm (280 μW). IRF FWHM in SSC were 298.6 ± 1.1 ps (150 μW), 341.0 ± 2.5 ps (200 μW) and 347.5 ± 6.0 ps (280 μW). In LSC, the IRF FWHM were 290.4 ± 3.8 ps (150 μW), 344.0 ± 3.4 ps (200 μW) and 358.8 ± 1.3 ps (280 μW). Results are mean ± standard deviation.Conclusions: A new fluorescence lifetime imaging ophthalmoscope has been characterized. The device offers a high laser power for fluorescence excitation, a large field of view, a high spatial resolution, and a sufficiently high time resolution. Thus, it is suitable for fluorescence lifetime studies.