Changes in activity of layer 2/3 cortical neurons in isoflurane-anesthetized mice: Real-time Ultra-large-Scale High-resolution imaging platform / 中华麻醉学杂志

Objective:To observe and analyze the changes in activity of layer 2/3 cortical neurons in isoflurane-anesthetized mice by Real-time Ultra-large-Scale High-resolution (RUSH) imaging platform.Methods:Clean-grade healthy male Rasgrf2-Cre/Ai148d mice, aged 8-12 weeks, weighing 18-25 g, were studied.The mice recovered ten days after the skull replacement surgery and proceeded to the next experiment.Imaging data of calcium fluorescence signals from layer 2/3 cortical neurons were acquired by RUSH imaging platform after fixing the head of mice.The time of imaging data acquisition in the awake state, during anesthesia with 1.2% isoflurane, and after the end of anesthesia was 100, 600 and 600 s, respectively.Imaging data were analyzed using Image J and MATLAB softwares.Results:The overall trend of activity of layer 2/3 cortical neurons decreased first and then stabilized with the inhalation of 1.2% isoflurane.The cortical neural activity were gradually increased when isoflurane inhalation was stopped.The recovery rate of neural activity was different in different brain regions after isoflurane inhalation was stopped.The recovery of neural activity in the primary motor cortex was delayed obviously.During the maintenance of anesthesia, the activities of most layer 2/3 cortical neurons in the retrosplenial cortex were weakened, however, some of the neurons became more active.Conclusions:The neural activity in the 2/3 layer of cortex in isoflurane anesthetized mice is inconsistent in observation region, brain region and single cell, suggesting that different neural pathways are involved in the process of anesthesia induction and recovery from anesthesia.

Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity Under Homeostatic Laminar Flow.

Objective: Endothelial cells (ECs) that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to EC quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level ECs regulate overall expression heterogeneity during this transition is poorly understood. Here, we profiled EC transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow. Approach and Results: Single-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human ECs under homeostatic laminar flow compared to nonflow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous, although small subsets of cells did not follow this pattern. Analysis of a subset of genes for relative protein expression revealed little congruence between RNA and protein heterogeneity changes under flow. In contrast, the magnitude of expression level changes in RNA and protein was more coordinated among ECs in flow versus nonflow conditions. Conclusions: ECs exposed to homeostatic laminar flow showed overall increased heterogeneity in RNA expression levels, while expression heterogeneity of selected cognate proteins did not follow RNA heterogeneity changes closely. These findings suggest that EC homeostasis is imposed post-transcriptionally in response to laminar flow.

Efficacy of modified Parks incision in the surgery for correction of strabismus in children / 中国基层医药

Objective:To investigate the clinical efficacy of modified parks incision in the surgery for correction of strabismus in children.Methods:Sixty children patients with strabismus who received treatment in Shanxi Hospital of Integrated Traditional and Western Medicine, China between January 2015 and January 2020 were included in this study. They were randomly assigned to receive surgery with either a trapezoidal flap incision (control group, n = 30) or modified Parks incision (modified Parks group, n = 30). Time to tear film break-up, amount of tears secreted, and the angles of deviation in strabismus were compared before and after surgery. The efficacy was evaluated by corneal fluorescein staining score. Postoperative complications were compared between the two groups. Results:Before surgery, there were no significant differences in the time to tear film break-up and the amount of tears secreted between the two groups (both P > 0.05). After surgery, the time to tear film break-up and the amount of tears secreted were (9.16 ± 1.74) seconds, (7.51 ± 1.36) mm/5 minutes in the modified Parks group and they were (7.57 ± 1.45) seconds and (6.05 ± 1.14) mm/5 minutes, respectively in the control group. After surgery, the time to tear film break-up and the amount of tears secreted in each group were shortened or decreased compared with before treatment, and these two indices in the modified Parks group were significantly shorter or less than those in the control group ( t = 3.845, 4.506, both P < 0.05). Before surgery, there were no significant differences in corneal fluorescein staining score and the angles of deviation in strabismus between the two groups (both P > 0.05). After surgery, corneal fluorescein staining score and the angles of deviation in strabismus in the modified Parks group were (5.14 ± 1.51) points and (10.68 ± 1.75) PD, respectively and they were (6.25 ± 1.73) points and (15.95 ± 2.14) PD, respectively in the control group. After surgery, corneal fluorescein staining score or the angle of deviation in strabismus was increased or decreased in each group. The corneal fluorescein staining score and the angle of deviation in strabismus in the modified Parks group were significantly lower than those in the control group ( t = 2.648, 10442, both P < 0.05). Total effective rate in the modified Parks group was significantly higher than that in the control group [96.7% (29/30) vs. 80.0% (24/30), χ2= 4.043, P < 0.05]. The incidence of postoperative complications and discomfort rate in the modified Parks group were 0.0% (0/30) and 53.3% (16/30), respectively, which were significantly lower than those in the control group 13.3% (4/30) and 80.0% (24/30), χ2 = 4.286, 4.800, both P < 0.05]. Conclusion:Modified Parks incision for corrective strabismus surgery can better correct the angle of strabismus, protect the stability of tear film function, reduce postoperative complications and decrease postoperative discomfort rate compared with trapezoidal flap incision.

[Diagnostic value of fungal fluorescence staining on corneal scrapings for fungal keratitis].

Objective: To analyze the sensitivity and specificity of fungal fluorescent staining in the diagnosis of fungal keratitis, and to compare it with conventional fungal culture, in vivo confocal microscopy (IVCM) and Giemsa staining. To explore its value of clinical application. Methods: Prospective case-control study. A total of 105 consecutive patients (105 eyes) diagnosed with infectious keratitis at Beijing Tongren Hospital from August 2017 to April 2018 were included. Patients with infectious keratitis were divided into fungal keratitis (FK) group and non-fungal keratitis (NFK) group by slit lamp microscopy, corneal in vivo confocal microscopy (IVCM) examination, and the results of Giemsa staining, fluorescent staining and pathogenic culture of corneal scraping from ulcer. The sensitivity and specificity of the above-mentioned examination methods for the diagnosis of fungal keratitis were analyzed. The receiver operating characteristic curve (ROC curve) and Area Under Curve (AUC) values were calculated to determine the diagnostic value of fungal fluorescent staining for fungal keratitis. Results: Among the 105 patients with infectious keratitis, 66 were fungal keratitis, 39 were non-fungal keratitis (29 cases of bacterial keratitis and 10 cases of acanthamoeba keratitis). Isolation from fungal keratitis were mainly Fusarium spp. (43.5%), followed by Alternaria spp. (21.7%) and Aspergillus spp. (19.6%). After fluorescent staining of the ulcer smear, the background of tissue demonstrated homogeneous black or weak blue fluorescence. The cell wall of fungi showed bright blue-violet to blue fluorescence, and the morphology, structure and hyphal density were easily recognized. The sensitivity of different methods for the diagnosis of corneal fungal infection were smear fluorescence staining (97.0%), IVCM (87.9%) , Giemsa staining (86.7%), and fungal culture (69.7%); the specificity of fungal culture was the highest (100%), followed by IVCM and Giemsa staining (94.9%), and fluorescent staining (87.2%). The ascending order of AUC values was: fungal culture (0.848)

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation.

BACKGROUND: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. METHODS: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. RESULTS: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states. CONCLUSIONS: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.

Fast Adipogenesis Tracking System (FATS)-a robust, high-throughput, automation-ready adipogenesis quantification technique.

Adipogenesis is essential in in vitro experimentation to assess differentiation capability of stem cells, and therefore, its accurate measurement is important. Quantitative analysis of adipogenic levels, however, is challenging and often susceptible to errors due to non-specific reading or manual estimation by observers. To this end, we developed a novel adipocyte quantification algorithm, named Fast Adipogenesis Tracking System (FATS), based on computer vision libraries. The FATS algorithm is versatile and capable of accurately detecting and quantifying percentage of cells undergoing adipogenic and browning differentiation even under difficult conditions such as the presence of large cell clumps or high cell densities. The algorithm was tested on various cell lines including 3T3-L1 cells, adipose-derived mesenchymal stem cells (ASCs), and induced pluripotent stem cell (iPSC)-derived cells. The FATS algorithm is particularly useful for adipogenic measurement of embryoid bodies derived from pluripotent stem cells and was capable of accurately distinguishing adipogenic cells from false-positive stains. We then demonstrate the effectiveness of the FATS algorithm for screening of nuclear receptor ligands that affect adipogenesis in the high-throughput manner. Together, the FATS offer a universal and automated image-based method to quantify adipocyte differentiation of different cell lines in both standard and high-throughput workflows.

Preliminary clinical application of fluorescence microscopic imaging and computer-aided diagnosis system in the diagnosis of superficial cutaneous fungal infections / 中华皮肤科杂志

Objective@#To evaluate the accuracy of automated fluorescence microscopic imaging and computer-aided diagnosis system (AFMICADS) in the auxiliary diagnosis of superficial cutaneous fungal infections.@*Methods@#Totally, 106 outpatients and inpatients with suspected superficial fungal infections were enrolled from clinical departments of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and September 2018. A total of 126 specimens were collected, including 83 skin scales and 43 nail parings. Each specimen was divided into 3 groups to be examined by conventional fungal microscopy, culture with modified Sabouraud dextrose agar and fluorescence microscopy (artificial fluorescence microscopy and AFMICADS-based fluorescence microscopy) respectively. A positive result was defined as that conventional fungal microscopy and/or fungal culture was positive. Consistency rate, sensitivity and specificity of the 3 microscopic methods were calculated. Statistical analysis was carried out with SPSS 10.0 software by using McNemar test and Kappa test for analyzing difference in the positive rate, as well as consistency, between the 3 microscopic methods and the positive standard, and by using efficiency test for comparing the consistency rate among the 3 microscopic methods.@*Results@#Of 126 specimens, 124 (98.4%) were positive for artificial fluorescence microscopy, and 123 (97.6%) for AFMICADS-based fluorescence microscopy. Both positive rates of the above 2 microscopic methods were significantly higher than the positive rate of the positive standard (77.8%, both P < 0.001) . The sensitivity, specificity and consistency rate of AFMICADS-based fluorescence microscopy were 100%, 10.7% and 80.2% respectively, and those of artificial fluorescence microscopy were 100%, 7.1% and 79.4% respectively. Additionally, no significant difference in the consistency was observed between the AFMICADS-based and artificial fluorescence microscopy (P > 0.05) .@*Conclusion@#The accuracy of AFMICADS-based fluorescence microscopy in the diagnosis of superficial cutaneous fungal infections is similar to that of artificial fluorescence microscopy.

Preliminary clinical application of fluorescence microscopic imaging and computer-aided diagnosis system in the diagnosis of superficial cutaneous fungal infections / 中华皮肤科杂志

Objective To evaluate the accuracy of automated fluorescence microscopic imaging and computer-aided diagnosis system (AFMICADS) in the auxiliary diagnosis of superficial cutaneous fungal infections.Methods Totally,106 outpatients and inpatients with suspected superficial fungal infections were enrolled from clinical departments of Union Hospital,Tongji Medical College,Huazhong University of Science and Technology between July 2018 and September 2018.A total of 126 specimens were collected,including 83 skin scales and 43 nail parings.Each specimen was divided into 3 groups to be examined by conventional fungal microscopy,culture with modified Sabouraud dextrose agar and fluorescence microscopy (artificial fluorescence microscopy and AFMICADS-based fluorescence microscopy) respectively.A positive result was defined as that conventional fungal microscopy and/or fungal culture was positive.Consistency rate,sensitivity and specificity of the 3 microscopic methods were calculated.Statistical analysis was carried out with SPSS 10.0 software by using McNemar test and Kappa test for analyzing difference in the positive rate,as well as consistency,between the 3 microscopic methods and the positive standard,and by using efficiency test for comparing the consistency rate among the 3 microscopic methods.Results Of 126 specimens,124 (98.4％) were positive for artificial fluorescence microscopy,and 123 (97.6％) for AFMICADS-based fluorescence microscopy.Both positive rates of the above 2 microscopic methods were significantly higher than the positive rate of the positive standard (77.8％,both P ＜ 0.001).The sensitivity,specificity and consistency rate of AFMICADS-based fluorescence microscopy were 100％,10.7％ and 80.2％ respectively,and those of artificial fluorescence microscopy were 100％,7.1％ and 79.4％ respectively.Additionally,no significant difference in the consistency was observed between the AFMICADS-based and artificial fluorescence microscopy (P ＞0.05).Conclusion The accuracy of AFMICADS-based fluorescence microscopy in the diagnosis of superficial cutaneous fungal infections is similar to that of artificial fluorescence microscopy.

Biomateriais com aplicabilidade na ortopedia: avanços e desafios na área da infectologia / Biomaterials with applicability in orthopedics: advances and challenges in the infectology area

O controle na formação do biofilme em implantes e próteses ortopédicas continua sendo um dos grandes desafios acerca da infeção relacionada aos dispositivos na área da saúde. O objetivo desta pesquisa foi investigar biomateriais com aplicabilidade na ortopedia, visando os avanços e enfrentamentos dos desafios na área da infectologia. Uma revisão integrativa foi realizada a respeito da formação de biofilme em biomateriais de próteses de quadril com a finalidade de contribuir com as medidas de prevenção e controle aos agravos infecciosos. Além disso, a formação in vitro do biofilme em função dos biomateriais (titânio e titânio revestido com biovidro F18), microrganismos (Staphylococcus epidermidis e Candida albicans) e tempos de incubação (2, 4 e 8 horas) foi avaliada por microscopia de fluorescência. A revisão integrativa foi realizada no portal PubMed da National Library of Medicine, bem como nas bases Cochrane, Embase, Web of Science, CINAHL e LILACS com a inclusão de estudos primários sobre a temática, publicados online até novembro de 2017, em português, inglês e espanhol. Na fase experimental / laboratorial, biofilmes de S. epidermidis (ATCC 12228) e C. albicans (ATCC 90028) foram formados em corpos de prova de titânio e titânio revestido com biovidro F18 após 2, 4 e 8 horas de incubação a 37?C sob agitação orbital. As áreas das imagens dos corpos de prova, em porcentagem, recobertas com biofilme (células vivas) foram avaliadas por microscopia de fluorescência. Os dados coletados foram submetidos à análise estatística empregando-se os testes de normalidade Shapiro Wilk, U de Mann-Whitney e t de Student por meio do software IBM SPSS Statistics (versão 25) e nível de significância ?=5%. Na revisão integrativa, os resultados demonstraram que dos 16 estudos primários, 81,25% eram pesquisas experimentais in vitro e que novos biomateriais foram desenvolvidos para prevenir a formação de biofilme. Com relação à fase experimental / laboratorial, houve menor formação de biofilme por S. epidermidis e C. albicans (p<0,001) no titânio revestido com biovidro F18 do que no titânio, após 8 horas de incubação. Entretanto, houve maior formação de biofilme por S. epidermidis e C. albicans após 8 horas do que em 2 horas de incubação, tanto no titânio quanto no titânio revestido com biovidro F18 (p<0,05). Em suma, a revista da literatura mencionou o desenvolvimento de biomateriais novos para prevenir a formação de biofilme. Na fase laboratorial / experimental, o titânio revestido com biovidro F18 apresentou atividade antibiofilme em comparação com o titânio, e os tempos de incubação de 2 para 8 horas aumentaram a formação de biofilme em ambos os biomateriais. Ainda, pesquisas futuras acerca do biovidro F18 fundamentadas nos aspectos físicoquímicos, bioquímicos e microbiológicos são importantes para a elucidação dos mecanismos de ação relacionados ao controle dos biofilmes

The control of biofilm formation on implants and orthopedic prostheses still is one of the major challenges concerning infection related to devices in the health field. The objective of this research was to investigate biomaterials with applicability in orthopedics, aiming for advances and facing challenges in the infectology area. An integrative review was performed regarding biofilm formation on hip prosthesis biomaterials in order to contribute to the preventive and infection control measures. Moreover, the in vitro biofilm formation according to biomaterials (titanium and titanium coated with F18 bioglass), microorganisms (Staphylococcus epidermidis and Candida albicans) and incubation times (2, 4 and 8 hours) was evaluated by fluorescence microscopy. The integrative review was performed on PubMed portal from National Library of Medicine as well as on Cochrane, Embase, Web of Science, CINAHL and LILACS databases with the inclusion of primary studies about the topic, published online up until November 2017, in Portuguese, English and Spanish. In the experimental / laboratory step, S. epidermidis (ATCC 12228) and C. albicans (ATCC 90028) biofilms were formed on proof bodies of titanium and titanium coated with F18 bioglass after 2, 4 and 8 hours of incubation at 37?C under orbital shaking. The image areas of proof bodies, in percentage, coated with biofilm (living cells) were evaluated by fluorescence microscopy. The data collected were submitted to statistical analysis using normality tests Shapiro Wilk, U from Mann-Whitney and t from Student through IBM SPSS Statistics (version 25) software and significance level ?=5%. In the integrative review, the results showed that among 16 primary studies, 81.25% were in vitro experimental studies and that new biomaterials were developed to prevent biofilm formation. Regarding experimental / laboratory step, there was less biofilm formation by S. epidermidis and C. albicans (p<0.001) on titanium coated with F18 bioglass than on titanium, after 8 hours of incubation. However, there was more biofilm formation by S. epidermidis and C. albicans after 8 hours than in 2 hours of incubation, both on titanium and on titanium coated with F18 bioglass (p<0.05). In sum, the literature review mentioned the development of new biomaterials to prevent biofilm formation. In laboratory / experimental step, titanium coated with F18 bioglass presented antibiofilm activity in comparison with titanium, and the incubation times of 2 to 8 hours increased biofilm formation on both materials. Besides, future studies about F18 bioglass based on physicochemical, biochemical and microbiological aspects are important for the elucidation of action mechanisms related to biofilms control

Clinical and histological features of patients with membranoproliferative glomerulonephritis classified by immunofluorescence findings / Características clínicas e histológicas de pacientes com glomerulonefrite membranoproliferativa classificados por achados de imunofluorescência

Abstract Background: New classification for membranoproliferative glomerulonephritis has been proposed in the literature. The aim of this study was to compare the clinical, biochemical, etiology and renal biopsy findings of these patients grouped by immunofluorescence as proposed by the new classification. Methods: Patients with renal biopsy-proven membranoproliferative glomerulonephritis unrelated to systemic lupus erythematosus, diagnosed between 1999 and 2014. The patients were divided according to immunofluorescence: Immunoglobulin positive group, C3 positive only and negative immunofluorescence group. Results: We evaluated 92 patients, the majority of which were in the immunoglobulin positive group. Infectious diseases, hepatitis C virus and schistosomiasis, were the most frequent etiology. A negative immunofluorescence group had more vascular involvement in renal biopsy compare with others groups. Conclusions: The only difference between the groups was higher vascular involvement in renal biopsy in negative immunofluorescence group. These new classification was satisfactory for the finding of etiology in one part of the cases.

Resumo Introdução: Uma nova classificação para glomerulonefrite membranoproliferativa foi proposta na literatura. O objetivo deste estudo foi comparar os achados clínicos, bioquímicos, etiológicos e da biópsia renal desses pacientes agrupados por imunofluorescência, conforme proposto pela nova classificação. Métodos: Pacientes com glomerulonefrite membranoproliferativa comprovada por biópsia renal, não relacionada ao lúpus eritematoso sistêmico, diagnosticados entre 1999 e 2014. Os pacientes foram divididos de acordo com a imunofluorescência: grupo positivo por imunoglobulina, grupo positivo por C3 apenas e grupo com imunofluorescência negativa. Resultados: avaliamos 92 pacientes, a maioria dos quais estava no grupo de imunoglobulina positiva. Doenças infecciosas, o vírus da hepatite C e a esquistossomose, foram as etiologias mais frequentes. Um grupo com imunofluorescência negativa apresentou maior comprometimento vascular na biópsia renal quando comparado com os outros grupos. Conclusões: a única diferença entre os grupos foi o maior envolvimento vascular na biópsia renal no grupo de imunofluorescência negativa. Esta nova classificação foi satisfatória para a descoberta de etiologia em uma parte dos casos.

Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. SUMMARY: Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

Deep Tissue Imaging with Multiphoton Fluorescence Microscopy.

We present a review of imaging deep-tissue structures with multiphoton microscopy. We examine the effects of light scattering and absorption due to the optical properties of biological sample and identify 1,300 nm and 1,700 nm as ideal excitation wavelengths. We summarize the availability of fluorophores for multiphoton microscopy as well as ultrafast laser sources to excite available fluorophores. Lastly, we discuss the applications of multiphoton microscopy for neuroscience.

Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes.

RATIONALE: Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE: To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS: Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Untangling autophagy measurements: all fluxed up.

Autophagy is an important physiological process in the heart, and alterations in autophagic activity can exacerbate or mitigate injury during various pathological processes. Methods to assess autophagy have changed rapidly because the field of research has expanded. As with any new field, methods and standards for data analysis and interpretation evolve as investigators acquire experience and insight. The purpose of this review is to summarize current methods to measure autophagy, selective mitochondrial autophagy (mitophagy), and autophagic flux. We will examine several published studies where confusion arose in data interpretation, to illustrate the challenges. Finally, we will discuss methods to assess autophagy in vivo and in patients.

The application of acridine orange fluorescent staining method in circulating tumor cell / 天津医药

Objective To establish the screening platform of circulating tumor cells (CTCs) using acridine orange fluo?rescent (AO-F) dyeing method, and to apply it in the screening of peripheral blood CTCs in patients with kidney cancer. Methods Twenty-seven patients with metastatic renal cell carcinoma was included in this study. Primitive tumor cells and kidney cancer cell line 769-P were cultured with different concentrations of fetal bovine serum. Smears were prepared and observed under fluorescence microscopy. The percentage of AO-F positive staining of 769-P cells under 5 random sights was calculated. The sensitivity of AO-F staining to cells was evaluated. The 5 mL morning fasting venous blood was obtained from 10 subjects with healthy check-up. The 1×106 cell suspension was prepared. The logarithmic phase of renal tumor cells was used to prepare tube containing 500, 200, 100, 50 and 10 tumor cell suspension, which were mixed with 1×106 nucleated cells to establish CTCs model of renal cancer. AO-F staining method was used to detect the expression of AO-F positive cells. The correlation between expression of AO-F positive cells and clinical parameters was analyzed. Results The prima?ry cells and cell line 769-P showed similar bright color and morphological characteristics. The percentage of AO-F positive staining in 769-P cells was 93%±3%under 5 random sights. The recovery rates (%) of four groups (500, 200, 100 and 50 tu?mor cell suspension) were 10.2±3.8, 9.2±2.3, 10.8±2.6 and 10.5±1.9, respectively. There were no significant differences in recovery rates between four groups (P>0.05). The group of 10 tumor cell suspension could find AO-F positive staining cells occasionally. Zero case was positive in controls. Nine of 27 patients were positive and the rate was 33.33%. There were no significant statistical differences in AO-F positive rates between gender, age, tumor size, pathological pattern, Furhman stage, metastasis of lung and presence of tumor (P>0.05). Conclusion It is confirmed that the method of CTCs staining with AO-F, which has high specificity and reproducibility, is feasible to detect CTCs and worthy of being studied. There is a certain reference value to predict tumor recurrence and metastasis.

In vivo imaging of the mouse neurovascular unit under chronic cerebral hypoperfusion.

BACKGROUND AND PURPOSE: Proper brain function is maintained by an integrated system called the neurovascular unit (NVU) comprised cellular and acellular elements. Although the individual features of specific neurovascular components are understood, it is unknown how they respond to ischemic stress as a functional unit. Therefore, we established an in vivo imaging method and clarified the NVU response to chronic cerebral hypoperfusion. METHODS: Green mice (b-act-EGFP) with SR101 plasma labeling were used in this experiment. A closed cranial window was made over the left somatosensory cortex. To mimic chronic cerebral hypoperfusion, mice were subjected to bilateral common carotid artery stenosis operations using microcoils. In vivo real-time imaging was performed using 2-photon laser-scanning microscopy during the preoperative period, and after 1 day and 1 and 2 weeks of bilateral common carotid artery stenosis or sham operations. RESULTS: Our method allowed 3-dimensional observation of most of the components of the NVU, as well as dynamic capillary microcirculation. Under chronic cerebral hypoperfusion, we did not detect any structural changes of each cellular component in the NVU; however, impairment of microcirculation was detected over a prolonged period. In the pial small arteries and veins, rolling and adhesion of leukocyte were detected, more prominently in the latter. In the deep cortical capillaries, flow stagnation because of leukocyte plugging was frequently observed. CONCLUSIONS: We established an in vivo imaging method for real-time visualization of the NVU. It seems that under chronic cerebral hypoperfusion, leukocyte activation has a critical role in microcirculation disturbance.

Osteogenetic ability of mouse spermatogonial stem cells cultured in vitro / 中国组织工程研究

BACKGROUND:Spermatogonial stem cells are a kind of adult stem cells, which have self-renewal and differentiation potential, and can be differentiated into specific cells in vitro, suggesting that the spermatogonial stem cells may be possibly differentiated into osteoblasts. But the related research has not been reported. OBJECTIVE:To observe the biological characterization and osteogenic process of mouse spermatogonial stem cells cultured in vitro. METHODS:Spermatogonial stem cells were obtained from the testicle of mice aged 15-20 days, and were cultured on the feeder layer from bone marrow stroma cells in vitro. When cultured for 3 days, the cells were cultured in the conditioned medium (experimental group) and basic medium (control group). The cells proliferation capability and osteogenic property were examined by phase-contrast microscope, alkaline phosphatase activity and type I col agen immunofluorescence staining. RESULTS AND CONCLUSION:Spermatogonial stem cells proliferated faster in the experiment group than in the control group. cells grew rapidly in colony-like shape in the conditioned medium at 3-6 days, the three-dimensional feeling enhanced, cellmass and clusters continued to increase in size, the extracellular matrix was increased in number and the cytoplasmic bridge was not obvious. After culture for 15 days, cells in the two groups were positive for alkaline phosphatase staining that the cytoplasmic membrane was dyed black. Under the fluorescent microscope, green fluorescence was visible in the experimental group, suggesting the cells in the experimental group was positive for type I col agen, but negative in the control group, which is similar with the biological characteristics of osteoblasts. These findings indicate that spermatogonial stem cells possess the osteogenic capability under induction conditions, which are expected to provide seed cells for bone tissue engineering.

Transgenic mice for cGMP imaging.

RATIONALE: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. OBJECTIVE: To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. METHODS AND RESULTS: Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken ß-actin/ß-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. CONCLUSIONS: These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.