Assessment of the In Vivo Response to Nanobody-Targeted PDT Through Intravital Microscopy.

Methods that allow real-time, longitudinal, intravital detection of the fluorescence distribution and the cellular and vascular responses within tumor and normal tissue are important tools to obtain valuable information when investigating new photosensitizers and photodynamic therapy (PDT) responses. Intravital confocal microscopy using the dorsal skinfold chamber model gives the opportunity to visualize and determine the distribution of photosensitizers within tumor and normal tissue. Next to that, it also allows the visualization of the effect of treatment with respect to changes in vascular diameter and blood flow, vascular leakage, and tissue necrosis, in the first days post-illumination. Here, we describe the preparation of the skinfold chamber model and the intravital microscopy techniques involved, for a strategy we recently introduced, that is, the nanobody-targeted PDT. In this particular approach, photosensitizers are conjugated to nanobodies to target these specifically to cancer cells.

Application of 3D MAPs pipeline identifies the morphological sequence chondrocytes undergo and the regulatory role of GDF5 in this process.

The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process.

Noninvasive, in vivo assessment of the cervical microcirculation using incident dark field imaging.

AIM: This study evaluates the feasibility of handheld vital microscopy for noninvasive, objective assessment of the microcirculation of the human uterine cervix. We qualitatively and quantitatively describe the microcirculation in healthy subjects in order to provide a basis for its application in cervical pathology. METHODS: Incident dark field imaging was used to image the microcirculation in four quadrants of the uterine ectocervix in ten healthy participants. If the squamocolumnar junction was visible, measurements were repeated on the endocervical columnar epithelium as well. Image acquisition time was recorded and participants scored the experienced level of discomfort. Angioarchitecture was classified according to Weber's classification. Quantitative parameters included capillary density (CD), total and perfused vessel density (TVD, PVD), proportion of perfused vessels (PPV) and microvascular flow index (MFI). RESULTS: Image acquisition was easy, fast and well tolerated. Angioarchitecture was characterized by two distinctive and organized patterns; capillary loops underneath the squamous epithelium of the ectocervix and vascular networks underneath the columnar epithelium. In the image sequences containing capillary loops, mean CD was 33.2 cpll/mm2 (95% CI 28.2-38.2 cpll/mm2). In the image sequences with vascular networks, mean TVD was 12.5 mm/mm2 (95% CI 11.2-13.77 mm/mm2), mean PVD was 12.2 (95% CI 11.0-13.5 mm/mm2), MFI was 3 and PPV was 100%. CONCLUSIONS: Incident dark field imaging allows for noninvasive, real time visualization and objective evaluation and quantification of the microcirculation of the uterine cervix. The organized vascular patterns and optimal perfusion observed in healthy subjects allow for comparison with cervical pathology, for example in patients with cervical dysplasia or cervical cancer.

Assessment of Microvessel Permeability in Murine Atherosclerotic Vein Grafts Using Two-Photon Intravital Microscopy.

Plaque angiogenesis and plaque hemorrhage are major players in the destabilization and rupture of atherosclerotic lesions. As these are dynamic processes, imaging of plaque angiogenesis, especially the integrity or leakiness of angiogenic vessels, can be an extremely useful tool in the studies on atherosclerosis pathophysiology. Visualizing plaque microvessels in 3D would enable us to study the architecture and permeability of adventitial and intimal plaque microvessels in advanced atherosclerotic lesions. We hypothesized that a comparison of the vascular permeability between healthy continuous and fenestrated as well as diseased leaky microvessels, would allow us to evaluate plaque microvessel leakiness. We developed and validated a two photon intravital microscopy (2P-IVM) method to assess the leakiness of plaque microvessels in murine atherosclerosis-prone ApoE3*Leiden vein grafts based on the quantification of fluorescent-dextrans extravasation in real-time. We describe a novel 2P-IVM set up to study vessels in the neck region of living mice. We show that microvessels in vein graft lesions are in their pathological state more permeable in comparison with healthy continuous and fenestrated microvessels. This 2P-IVM method is a promising approach to assess plaque angiogenesis and leakiness. Moreover, this method is an important advancement to validate therapeutic angiogenic interventions in preclinical atherosclerosis models.

In Vivo Assessment of Size-Selective Glomerular Sieving in Transplanted Human Induced Pluripotent Stem Cell-Derived Kidney Organoids.

BACKGROUND: The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell-derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation. METHODS: We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell-derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin. RESULTS: After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake. CONCLUSIONS: Our results demonstrate that human pluripotent stem cell-derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling.

On the in vivo assessment of goblet cells of the human bulbar conjunctiva by confocal microscopy - A review.

BACKGROUND: In vivo confocal microscopy (IVCM) has been used for over 10 years to assess the goblet cell density (GCD) within the human conjunctiva, but the reported values have been variable with no obvious indications as to why. METHODS: From publications between 2008 and 2019, representative GCD values were extracted, as well as on the image sampling strategy used. RESULTS: Average GCD values for any particular group of individuals ranged from 7 to 979 goblet cells / sq. mm, and with one notable outlier removed, an overall group-mean value for GCD (+/- SD) from single site locations was 207 +/- 143 goblet cells / sq. mm from 15 data sets for those usually designated as control subjects, with a value of 190 +/- 161 goblet cells / sq. mm calculated from 20 single site data sets from other (patient) groups. An overall analysis indicated that the reported average values for GCD from different groups of individuals increased according to the number of images assessed / individual (Spearman rho = 0.304), on the number of individuals evaluated to generate an averaged value for each group (rho = 0.367), and the total number of images assessed (rho = 0.346, multivariate analysis partial r = greater or = to 0.522). CONCLUSIONS: In the use of confocal microscopy to assess the number of goblet cells in the human bulbar conjunctiva, the substantial differences reported appear to be linked to the protocols used for image selection, and some type of standardization needs to be developed.

A mobile-phone based high-resolution microendoscope to image cervical precancer.

Nearly 90% of cervical cancer cases and deaths occur in low- and middle-income countries that lack comprehensive national HPV immunization and cervical cancer screening programs. In these settings, it is difficult to implement screening programs due to a lack of infrastructure and shortage of trained personnel. Screening programs based on visual inspection with acetic acid (VIA) have been successfully implemented in some low-resource settings. However, VIA has poor specificity and up to 90% of patients receiving treatment based on a positive VIA exam are over-treated. A number of studies have suggested that high-resolution cervical imaging to visualize nuclear morphology in vivo can improve specificity by better distinguishing precancerous and benign lesions. To enable high-resolution imaging in low-resource settings, we developed a portable, low-cost, high-resolution microendoscope that uses a mobile phone to detect and display images of cervical epithelium in vivo with subcellular resolution. The device was fabricated for less than $2,000 using commercially available optical components including filters, an LED and triplet lenses assembled in a 3D-printed opto-mechanical mount. We show that the mobile high-resolution microendoscope achieves similar resolution and signal-to-background ratio as previously reported high-resolution microendoscope systems using traditional cameras and computers to detect and display images. Finally, we demonstrate the ability of the mobile high-resolution microendoscope to image normal and precancerous squamous epithelium of the cervix in vivo in a gynecological referral clinic in Barretos, Brazil.

Development of Functional Requirements for Ex Vivo Pathology Applications of In Vivo Microscopy Systems: A Proposal From the In Vivo Microscopy Committee of the College of American Pathologists.

CONTEXT.­: In vivo microscopy (IVM) allows direct, real-time visualization of tissue histology in living patients without the need for tissue removal, processing, or staining. The IVM technologies in clinical use include confocal microscopy and optical coherence tomography. These technologies also show promise for use with pathology specimens (ex vivo microscopy [EVM]). However, few systems designed for EVM are commercially available, at least in part because of the lack of defined minimal functional requirements (FRs). OBJECTIVE.­: To develop minimal FRs for likely high-volume pathology applications of EVM. DESIGN.­: The IVM Committee of the College of American Pathologists identified potential EVM pathology applications based on the published literature. A subcommittee of IVM and EVM early adopters and experts then defined FRs for the most likely EVM applications. RESULTS.­: Potential EVM applications include assessment of margins, adequacy of needle biopsies and aspirates for diagnosis, and transplant tissues; selection of tissue for molecular studies or biorepository; and guidance in block selection from gross specimens. The first 3 applications were selected for development of FRs. The FRs were identified based on existing laboratory practices and guidelines and input from experts in the field and included device footprint and portability, specimen preparation, imaging time, field of view or resolution, morphologic diagnostic capability, yield, accuracy, ease of use, safety, and cost. CONCLUSIONS.­: Consensus was achieved on FRs that would accommodate the selected EVM applications. Publication and dissemination of those FRs will provide guidance to engineers, researchers, and vendors on how to optimally adapt IVM technologies for EVM for widespread adoption by pathologists.

Biodistribution and toxicity assessment of intratumorally injected arginine-glycine-aspartic acid peptide conjugated to CdSe/ZnS quantum dots in mice bearing pancreatic neoplasm.

Quantum dots (QDs) conjugated with arginine-glycine-aspartic acid (RGD) peptides (which are integrin antagonists) are novel nanomaterials with the unique optical property of high molar extinction coefficient, and they have potential utility as photosensitizers in photodynamic therapy (PDT). Our group previously demonstrated significant benefits of using PDT with QD-RGD on pancreatic tumor cells. This study aimed to evaluate the biodistribution and toxicity of QD-RGD in mice prior to in vivo application. Mice with pancreatic neoplasms were intratumorally injected with varying doses of QD-RGD, and the biodistribution 0-24 h post injection was compared to that in control mice (intravenously injected with unconjugated QD). Various tissue samples were collected for toxicity analyses, which included inductively coupled plasma mass spectrometry (ICP-MS) to assess Cd2+ concentrations and hematoxylin-eosin staining for histopathological examination. Fluorescent imaging revealed relatively sufficient radiant efficiency in mice under specific conditions. The ICP-MS and HE data showed no significant signs of necrosis due to Cd2+ release by QDs. The mice survived well and had no apparent weakness or weight loss during the 4 weeks post injection. These findings provide novel insights into the biodistribution of QD-RGD and encourage profound in vivo studies regardless of safety concerns. These findings alleviate safety concerns and provide novel insights into the biodistribution of QD-RGD, offering a solid foundation for comprehensive in vivo studies.

Spatial, Temporal, and Functional Assessment of LC3-Dependent Autophagy in Shigella flexneri Dissemination.

Shigella flexneri disseminates within the colonic mucosa by displaying actin-based motility in the cytosol of epithelial cells. Motile bacteria form membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the bacteria escape, thereby achieving cell-to-cell spread. During dissemination, S. flexneri is targeted by LC3-dependent autophagy, a host cell defense mechanism against intracellular pathogens. The S. flexneri type III secretion system effector protein IcsB was initially proposed to counteract the recruitment of the LC3-dependent autophagy machinery to cytosolic bacteria. However, a recent study proposed that LC3 was recruited to bacteria in DMVs formed during cell-to-cell spread. To resolve the controversy and clarify the role of autophagy in S. flexneri infection, we tracked dissemination using live confocal microscopy and determined the spatial and temporal recruitment of LC3 to bacteria. This approach demonstrated that (i) LC3 was exclusively recruited to wild-type or icsB bacteria located in DMVs and (ii) the icsB mutant was defective in cell-to-cell spread due to failure to escape LC3-positive as well as LC3-negative DMVs. Failure of S. flexneri to escape DMVs correlated with late LC3 recruitment, suggesting that LC3 recruitment is the consequence and not the cause of DMV escape failure. Inhibition of autophagy had no positive impact on the spreading of wild-type or icsB mutant bacteria. Our results unambiguously demonstrate that IcsB is required for DMV escape during cell-to-cell spread, regardless of LC3 recruitment, and do not support the previously proposed notion that autophagy counters S. flexneri dissemination.

Induced Dynamic Intracranial Pressure and Cerebrovascular Reactivity Assessment of Cerebrovascular Autoregulation After Traumatic Brain Injury with High Intracranial Pressure in Rats.

OBJECTIVE: In previous work we showed that high intracranial pressure (ICP) in the rat brain induces a transition from capillary (CAP) to pathological microvascular shunt (MVS) flow, resulting in brain hypoxia, edema, and blood-brain barrier (BBB) damage. This transition was correlated with a loss of cerebral blood flow (CBF) autoregulation undetected by static autoregulatory curves but identified by induced dynamic ICP (iPRx) and cerebrovascular (iCVRx) reactivity. We hypothesized that loss of CBF autoregulation as correlated with MVS flow would be identified by iPRx and iCVRx in traumatic brain injury (TBI) with elevated ICP. METHODS: TBI was induced by lateral fluid percussion (LFP) using a gas-driven device in rats. Using in vivo two-photon laser scanning microscopy, cortical microcirculation, tissue oxygenation (NADH autofluoresence), and BBB permeability (fluorescein dye extravasation) were measured before and for 4 h after TBI. Laser Doppler cortical flux, rectal and brain temperature, ICP and mean arterial pressure (MAP), blood gases, and electrolytes were monitored. Every 30 min, a transient 10 mmHg rise in MAP was induced by i.v. bolus of dopamine. iPRx = ΔICP/ΔMAP and iCVRx = ΔCBF/ΔMAP. RESULTS: We demonstrated that iPRx and iCVRx correctly identified more severe loss of CBF autoregulation correlated with a transition of blood flow to MVS after TBI with high ICP compared to TBI without an increase in ICP. CONCLUSIONS: In TBI with high ICP, high-velocity MVS flow is responsible for the loss of CBF autoregulation identified by iPRx and iCVRx.

In vivo Confocal Laser Microscopy for monitoring of actinic keratosis treatment: a comparison with histopathologic assessment after treatment with topical 5% 5-fluorouracil.

BACKGROUND: Histological examination is the gold standard for actinic keratosis diagnosis; however, it is not always a feasible approach. Reflectance confocal microscopy (RCM) is a non-invasive technique that may be an alternative for monitoring actinic keratoses treatment response. Topical 5-fluorouracil is indicated for actinic keratosis multiple lesions and for field cancerization treatment. OBJECTIVES: To assess the RCM accuracy, sensibility and specificity for actinic keratosis, considering as a gold standard the histopathological examination; as well as to evaluate the efficacy of 5% 5-fluorouracil treatment. METHODS: This is a prospective study in actinic keratosis patients between August 2014 and November 2015. RCM analyses were performed in one randomly selected actinic keratosis lesion of the upper limbs by two independent observers before and after 5% 5-fluorouracil treatment. At the end of treatment and with clinical bleaching of treated lesions, histological examination was performed by two pathologists. RESULTS: A total of 50 lesions were enroled, and 40 lesions presented complete clinical bleaching after treatment and were included in the final analysis. Accuracy, sensibility and specificity means among observers were 83.8%, 84.6% and 83.3%, respectively. After 5-fluorouracil treatment, actinic keratosis was diagnosed in 45.0% (observer 1) and 32.5% (observer 2) of subjects according to RCM and in 32.5% of subjects according to histological examination. Considering RCM observers diagnosis, the concordance was substantial (k 0.637, P < 0.001). 5-fluorouracil led to a reduction in 55.0%-67.5% of actinic keratoses according to RCM analysis. CONCLUSION: This study allows to validate RCM as a non-invasive method capable of monitoring actinic keratosis therapeutic response to 5-fluorouracil, presenting efficacy comparable to histological examination. Additionally, the results suggest that 5-fluorouracil may be a satisfactory option for therapeutic control of this condition.

Volumetric laser endomicroscopy in the management of Barrett's esophagus.

PURPOSE OF REVIEW: The incidence of esophageal adenocarcinoma is on the rise despite widespread appreciation that the precursor lesion is Barrett's esophagus. Studies have shown that some patients known to have Barrett's esophagus develop cancer despite their enrollment in conventional endoscopic surveillance programs. This highlights the need for advanced endoscopic imaging to help identify early neoplasia and prevent its progression to esophageal cancer. Recently, a wide-field, second-generation optical coherence tomography endoscopic platform called volumetric laser endomicroscopy (VLE) was cleared by the Food and Drug Administration and made commercially available for advanced imaging in Barrett's esophagus. RECENT FINDINGS: The current review discusses current literature on VLE imaging in Barrett's esophagus. Based on ex-vivo studies, criteria have been established for identifying Barrett's esophagus-associated neoplasia. In addition, recent studies, case series, and case reports have demonstrated that VLE is well tolerated, efficacious, and can target neoplasia. SUMMARY: VLE is a new advanced imaging platform for Barrett's esophagus with considerable promise to target Barrett's esophagus-associated neoplasia. The following are needed to establish VLE's clinical role: studies showing incremental yield of dysplasia detection using VLE, studies to determine VLE's in-vivo diagnostic accuracy for identifying and classifying Barrett's esophagus-associated neoplasia, and studies on the cost-efficacy of VLE.

Precision real-time evaluation of bowel perfusion: accuracy of confocal endomicroscopy assessment of stoma in a controlled hemorrhagic shock model.

BACKGROUND AND AIMS: Confocal laser endomicroscopy (CLE) can provide real-time evaluation of bowel perfusion. We aimed to evaluate CLE perfusion imaging in a hemorrhagic shock model. MATERIALS AND METHODS: Five pigs were equipped to ensure hemodynamic monitoring. Three ileostomies per animal (total n = 15) were randomly created (T0). Blood was withdrawn targeting a mean arterial pressure of 40 mmHg (shock phase, T1), for 90 min. Infusion of Ringer's lactate was started and continued for 90 min (T2). At the different time points: (a) stomas' mucosa was scanned with CLE; (b) capillary lactates were measured on blood obtained by puncturing stomas' mucosa; and (c) full-thickness stomas' biopsies were sampled for histology, mitochondrial respiratory rate (V 0 = basal and V ADP = respiratory rate in excess of adenosine diphosphate), and levels of superoxide anion evaluation. Functional capillary density (FCD) was measured using ad hoc software. RESULTS: Confocal scanning provided consistent and specific imaging of bowel hypoperfusion at T1: vascular hyperpermeability (blurred and enlarged capillaries) and edema (enhanced visualization of the brush border due to increased intercellular spaces and fluorescein leakage). At the end of T2, there was an improved capillary flow. FCD-A index expressed statistically significant correlation with (1) stoma capillary lactates (p = 0.023); (2) systemic capillary lactates (p = 0.031); (3) inflammation pathology score (p = 0.048); (4) central venous pressure (p = 0.0043); and (5) pulmonary artery pressure (p = 0.01). Stoma capillary lactates (mmol/L) were significantly increased at T1 (8.81 ± 4.23; p < 0.0001) and at T2 (4.77 ± 3.13; p < 0.01) when compared to T0 inclusion values (1.86 ± 0.56). V 0 and V ADP (pmol O2/min/mg tissue) were both significantly decreased at T1 versus T0 (p < 0.007 and p < 0.01, respectively) and recovered by the end of reanimation (T2 vs. T0, p = n.s.). Mean O 2·- production (µmol/min/mg/dry tissue) increased at T1 and significantly decreased at T2. CONCLUSIONS: Confocal laser endomicroscopy (CLE) imaging could identify morphological signs congruent with biochemical markers of bowel perfusion and could be useful for assessment of stomas.

Tumor regrowth and development of keratinocytic neoplasms in patients under smoothened inhibition: in vivo assessment with reflectance confocal microscopy.

BACKGROUND: The regrowth of a tumor after complete clinical response and the development of keratinocytic neoplasms while patients are still undergoing continuous vismodegib have stressed the importance of the accurate monitoring to detect recurrences earlier and ensure the best possible outcome. OBJECTIVE: The objective of this study was to determine the role of reflectance confocal microscopy (RCM) in monitoring the response of locally advanced basal cell carcinoma (laBCC) to vismodegib and to discard secondary resistance. METHODS: Seven patients presenting with nine laBCC, were prospectively included and their response to this drug was assessed by means of clinical examination, dermoscopy, and RCM. The study was conducted at the Melanoma Unit in Hospital Clinic of Barcelona, between June 2012 and March 2013. RESULTS: Histologically confirmed lesion 10 mm or larger in diameter for which surgery was contraindicated and radiation therapy was inappropriate. The median patient age was 73 years and the most common histological type was infiltrating BCC. RCM allowed the identification of residual tumor in two lesions and to confirm complete response in the other four cases. Two patients developed new lesions within the tumor bed, they were assessed by RCM showing features of actinic keratosis which were confirmed by histopathology. CONCLUSION: The use of in vivo RCM allowed the characterization of the dynamic morphologic changes in tumor response helping to better define partial response and to differentiate it from secondary resistance. Another interesting observation was the recognition of a phenomenon characterized by the development of keratinocytic neoplasms within the tumor bed.

Basics of Confocal Microscopy and the Complexity of Diagnosing Skin Tumors: New Imaging Tools in Clinical Practice, Diagnostic Workflows, Cost-Estimate, and New Trends.

The use of reflectance confocal microscopy (RCM) and other noninvasive imaging devices can potentially streamline clinical care, leading to more precise and efficient management of skin cancer. This article explores the potential role of RCM in cutaneous oncology, as an adjunct to more established techniques of detecting and monitoring for skin cancer, such as dermoscopy and total body photography. Discussed are current barriers to the adoption of RCM, diagnostic workflows and standards of care in the United States and Europe, and medicolegal issues. The potential role of RCM and other similar technological innovations in the enhancement of dermatologic care is evaluated.

Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy.

Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs. In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection on hepatic hemodynamics. This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug's effect on inflammation by quantifying its effect on blood flow.

In vivo scanning electron microscope assessment of enamel permeability in primary teeth with and without early childhood caries.

BACKGROUND: Over the years, certain primary teeth have been shown to be highly sensitive to dental caries, while others have remained caries-free. It has been hypothesized that this may be attributed to differences in the permeability of the enamel surface. AIM: The aim of the study was to evaluate the hypothesized differences in the permeability of primary tooth enamel in children with and those without Severe Early Childhood Caries (S-ECC) using scanning electron microscopy. MATERIALS AND METHODS: Sixteen children between 3 and 6 years of age were randomly selected and divided into two groups: Group 1, children without S-ECC (n = 8), and Group 2, children with S-ECC (n = 8). In each child, 4 teeth (the maxillary right and left central and lateral incisors) were subjected to evaluation. An impression was made with polyvinylsiloxane impression material, and scanning electron microscopy was used to inspect the negative replicas for droplets. RESULTS: The results indicated higher significance when individual regions (cervical, middle, and incisal thirds) in the two groups were evaluated and compared. Similarly, the overall results showed high statistical significance between S-ECC and non-S-ECC teeth. CONCLUSION: There could be a positive relationship between the permeability of tooth enamel and the development of caries, which needs further research.