3D digital image microscope system-assisted vasovasostomy and vasoepididymostomy in rats.

Optimal vision and ergonomics are essential factors contributing to the achievement of good results during microsurgery. The three-dimensional (3D) digital image microscope system with a better 3D depth of field can release strain on the surgeon's neck and back, which can improve outcomes in microsurgery. We report a randomized prospective study of vasoepididymostomy and vasovasostomy using a 3D digital image microscope system (3D-DIM) in rats. A total of 16 adult male rats were randomly divided into two groups of 8 each: the standard operating microscope (SOM) group and the 3D-DIM group. The outcomes measured included the operative time, real-time postoperative mechanical patency, and anastomosis leakage. Furthermore, a user-friendly microscope score was designed to evaluate the ergonomic design and equipment characteristics of the microscope. There were no differences in operative time between the two groups. The real-time postoperative mechanical patency rates were 100.0% for both groups. The percentage of vasoepididymostomy anastomosis leakage was 16.7% in the SOM group and 25.0% in the 3D-DIM group; however, no vasovasostomy anastomosis leakage was found in either group. In terms of the ergonomic design, the 3D-DIM group obtained better scores based on the surgeon's feelings; in terms of the equipment characteristics, the 3D-DIM group had lower scores for clarity and higher scores for flexibility and adaptivity. Based on our randomized prospective study in a rat model, we believe that the 3D-DIM can improve surgeon comfort without compromising outcomes in male infertility reconstructive microsurgery, so the 3D-DIM might be widely used in the future.

High-Speed Video Cryomicroscopy for Measurement of Intracellular Ice Formation Kinetics.

Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissues. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the setup and operation of a high-speed video cryomicroscope system are described, including protocols for imaging of intracellular ice crystallization events and stochastic analysis of the ice formation kinetics in a cell population. Recommendations are provided for temperature profile design, sample preparation, and configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate best practices that have been drawn from two decades of experience with high-speed video cryomicroscopy in our laboratory.

Motion magnification analysis of microscopy videos of biological cells.

It is well recognized that isolated cardiac muscle cells beat in a periodic manner. Recently, evidence indicates that other, non-muscle cells, also perform periodic motions that are either imperceptible under conventional lab microscope lens or practically not easily amenable for analysis of oscillation amplitude, frequency, phase of movement and its direction. Here, we create a real-time video analysis tool to visually magnify and explore sub-micron rhythmic movements performed by biological cells and the induced movements in their surroundings. Using this tool, we suggest that fibroblast cells perform small fluctuating movements with a dominant frequency that is dependent on their surrounding substrate and its stiffness.

Study of Caveolae-Dependent Mechanoprotection in Human Muscle Cells Using Micropatterning and Live-Cell Microscopy.

Caveolae are plasma membrane organelles that are, among many other features, involved in mechanosensing and mechanoprotection. Different tools have been developed to study caveolae-dependent mechanoprotection and had to be adapted to the tissue or cells studied, as these structures are found in almost every type of cells. This chapter focuses on a protocol combining the use of live-cell imaging, micropatterning, hypo-osmotic shock as a mechanical stress, and dyes such as calcein-AM and propidium iodide. We used this protocol for the in vitro study of the effect of mechanical stress on membrane integrity in human muscle cells from patients bearing caveolin-3 mutations.

Automated evaluation of probe-based confocal laser endomicroscopy in the lung.

RATIONALE: Probe-based confocal endomicroscopy provides real time videos of autoflourescent elastin structures within the alveoli. With it, multiple changes in the elastin structure due to different diffuse parenchymal lung diseases have previously been described. However, these evaluations have mainly relied on qualitative evaluation by the examiner and manually selected parts post-examination. OBJECTIVES: To develop a fully automatic method for quantifying structural properties of the imaged alveoli elastin and to perform a preliminary assessment of their diagnostic potential. METHODS: 46 patients underwent probe-based confocal endomicroscopy, of which 38 were divided into 4 groups categorizing different diffuse parenchymal lung diseases. 8 patients were imaged in representative healthy lung areas and used as control group. Alveolar elastin structures were automatically segmented with a trained machine learning algorithm and subsequently evaluated with two methods developed for quantifying the local thickness and structural connectivity. MEASUREMENTS AND MAIN RESULTS: The automatic segmentation algorithm performed generally well and all 4 patient groups showed statistically significant differences with median elastin thickness, standard deviation of thickness and connectivity compared to the control group. CONCLUSION: Alveoli elastin structures can be quantified based on their structural connectivity and thickness statistics with a fully-automated algorithm and initial results highlight its potential for distinguishing parenchymal lung diseases from normal alveoli.

Motility Analysis of Trypanosomatids.

Motility analysis of microswimmers has long been limited to a few model cell types and broadly restricted by technical challenges of high-resolution in vivo microscopy. Recently, interdisciplinary interest in detailed analysis of the motile behavior of various species has gained momentum. Here we describe a basic protocol for motility analysis of an important, highly diverse group of eukaryotic flagellate microswimmers, using high spatiotemporal resolution videomicroscopy. Further, we provide a special, time-dependent tomographic approach for the proof of rotational locomotion of periodically oscillating microswimmers, using the same data. Taken together, the methods describe part of an integrative approach to generate decisive information on three-dimensional in vivo motility from standard two-dimensional videomicroscopy data.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

Multispectrum Indocyanine Green Videography for Visualizing Brain Vascular Pathology.

OBJECTIVE: Currently, neurosurgical vascular surgery frequently uses indocyanine green (ICG)-videography (VG) to evaluate the blood flow in brain vessels. Although ICG-VG delineates intravascular ICG fluorescence as a high-intensity signal in gray-scale with dark background, it is hard to identify anatomical structures, including vasculature or surgical devices simultaneously. This report developed combination of a near-infrared (NIR) camera with particular sensitivity and an optical filter to observe the blood-flow conditions and anatomical structures. METHODS: To overcome the specific issues of ICG-VG, we applied a high-sensitivity camera with a 980-nm NIR component to delineate anatomical and fluorescence images, detecting signals between 830 and 1000 nm simultaneously during operation. We used a diluted ICG phantom to evaluate fluorescence signal changes by changing wavelength of the emission light. For clinical use, we used a high-sensitivity NIR camera with a high-pass filter on a surgical microscope. The new NIR system detected signals between 770 and 1000 nm, and the lighting system illuminated objects mainly at 980-nm wavelength. Both images with the blood flow and anatomical structures were projected to the smart glasses in real time. RESULTS: In the phantom experiment, we found that the emission light with wide band widths (575-800 nm) evoked various intensities of ICG fluorescence. This new NIR system allowed us to observe ICG fluorescence and anatomical structures without image fusion or time-delay. The both information of anatomy and fluorescence was projected on wearable smart glasses. Furthermore, the new NIR system detected ICG-fluorescence signals for a longer duration than the original camera, which allowed us to achieve careful and detailed observation of more vasculature and fine vessels. CONCLUSIONS: This study proposes a new NIR system and emphasizes simultaneous observation of anatomy and fluorescence signals during operation. It paves the way for further possibilities in the development of optical systems. To understand the natural phenomena and combination of different scientific and clinical fields, it might be important to understand and combine not only fluorescence, but also natural science, optics, and background pathology. This simple system would be available for neuroendoscope and robotic surgery.

Low-Cost Stereoscopic Recordings of Neurologic Surgery Operative Microscopy for Anatomic Laboratory Training.

OBJECTIVE: Stereoscopic video recordings of operative microscopy during neuroanatomic dissections are an important component of surgical training and research in well-financed medical schools and teaching hospitals. However, the high cost of the latest operative microscopes with integrated video recording equipment can be a limiting factor in their worldwide use. The aim of the present work is to provide a simple low-cost 3-dimensional (3D) stereoscopic operative microscope recording system that can be used even in economically and resource-limited locations. This is achieved by using readily available smartphones, smartphone accessories, and computer software. METHODS: Stereoscopic recording is accomplished by attaching and aligning matched or similar smartphones to the eyepieces of an operative microscope using readily available smartphone mounting connectors. Video recordings from the smartphones are then transferred to a personal computer and processed with a video-editing software to generate stereoscopic movies that are viewed on a smartphone using virtual-reality glasses. RESULTS: The setup time to mount and align the smartphone cameras typically requires 15-30 minutes. Video image quality and 3D depth presentation is more than sufficient for surgical training and research purposes. The implementation cost ranges from $1,315-$7,066, or much less if smartphones and a computer are already available. CONCLUSIONS: The 3D video system demonstrated herein can be implemented on any type of operative microscope, including older units for which commercial stereo recording systems are not available. The system and method presented herein can be readily and affordably implemented in low-budget environments for clinical training and research.

The Use of the Exoscope in Lateral Skull Base Surgery: Advantages and Limitations.

OBJECTIVE: We describe our experience using the extracorporeal video microscope, the "exoscope" for various applications within the field of lateral skull base surgery. STUDY DESIGN: A retrospective case series was performed investigating patient demographics, indications for surgery, procedure type, operative time, approach to the skull base, complications, adequacy of visualization, and surgeon comfortability. PATIENTS: Six cases were performed with a three dimensional surgical exoscope, obviating the use of a traditional binocular microscope. SETTING: Academic, tertiary referral center. MAIN OUTCOME MEASURES: Type of surgical approach, operative time, patient demographics, surgical complications, and surgeon comfortability. RESULTS: The following procedures were performed; four vestibular schwannoma resections via suboccipital craniotomy and two combined transmastoid and transtemporal approaches for temporal lobe encephalocele repairs. The average operative time was 227 and 577 minutes for temporal lobe encephalocele repairs and vestibular schwannoma cases, respectively. No intraoperative complications were encountered during these cases. None of the procedures required abandonment of the exoscope in favor of the microscope during the procedure. Advantages include high-resolution three-dimensional visualization, increased degrees of freedom for exoscope adjustment, and reduced surgeon fatigue in a fixed, unnatural posture. Limitations include decreased depth perception and increased operative time. CONCLUSION: The exoscope system is a safe and effective alternative or adjunct to the existing binocular operating microscope for lateral skull based procedures. The exoscope provides the surgeon with a comfortable, high-resolution visualization without compromising surgical exposure and patient safety. LEVEL OF EVIDENCE: 4.

Label-free in vivo pathology of human epithelia with a high-speed handheld dual-axis confocal microscope.

There would be clinical value in a miniature optical-sectioning microscope to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology for early disease detection and surgical guidance. To address this need, a reflectance-based handheld line-scanned dual-axis confocal microscope was developed and fully packaged for label-free imaging of human skin and oral mucosa. This device can collect images at >15 frames/s with an optical-sectioning thickness and lateral resolution of 1.7 and 1.1 µm, respectively. Incorporation of a sterile lens cap design enables pressure-sensitive adjustment of the imaging depth by the user during clinical use. In vivo human images and videos are obtained to demonstrate the capabilities of this high-speed optical-sectioning microscopy device.

In vivo quantification of rolling and adhered leukocytes in human sepsis.

BACKGROUND: The use of in vivo videomicroscopy at the bedside has demonstrated microcirculatory flow disturbances in sepsis. The ability of in vivo videomicroscopy to detect changes in the prevalence of rolling and adhered leukocytes that occur in sepsis is not well-described in humans. We sought to (1) develop methodology for accessing and quantifying sublingual leukocyte rolling and adherence with sidestream dark field (SDF) imaging; (2) compare the number of rolling and adhered leukocytes between patients with septic shock and non-infected controls; and (3) compare the number of rolling and adhered leukocytes between survivors and non-survivors of septic shock. METHODS: We included adult (age > 18 years) patients in the emergency department presenting with septic shock prospectively enrolled in the ProCESS trial. We recruited comparison non-infected patients as emergency department controls. Using a SDF videomicroscope, we obtained image sequences from the sublingual mucosa, quantifying rolling and adhered leukocytes per 1 mm × 1 mm visual field in a standardized 3-s clip. We report data as median and interquartile range and depicted as box plots. We compared groups using the Mann-Whitney U test, considering a p value < 0.05 significant. RESULTS: We included a total of 64 patients with septic shock and 32 non-infected controls. The median number of adhered leukocytes per field in the sepsis group was 1.0 (IQR 0-3.5) compared to 0 (0-0) in the non-infected group (p < 0.001). The median number of rolling leukocytes was 26 (10.3-42) in the sepsis group and 9.8 (4.8-17.3) in the non-infected group (p < 0.001) per field. Among the patients with sepsis (n = 64), there was an increased number of adhered leukocytes in non-survivors compared to survivors (3.0 (1-5.5) vs. 1.0 (0-3.0)) (p < 0.05); however, there was no difference in rolling leukocytes (35 (20-48) vs. 26 (10-41)) (p = 0.31). CONCLUSIONS: Our results demonstrated a higher number of rolling and adhered leukocytes in patients with septic shock when compared to non-infected controls, and an increased number of adhered leukocytes in non-survivors. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00793442 ; Registered on 19 November 2008 PG0GM076659 (US NIH Grant/Contract). First submitted 18 July 2007. First posted 2 August 2007.

Randomized controlled trial comparing embryo culture in two incubator systems: G185 K-System versus EmbryoScope.

OBJECTIVE: To study whether the closed culture system, as compared with a benchtop incubator with similar culture conditions, has a positive impact on intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Randomized controlled trial. SETTING: University hospital. PATIENT(S): A total of 386 patients undergoing ICSI cycles with at least six mature oocytes were randomized. INTERVENTION(S): Of these patients, 195 were assigned to the group with culture in a time-lapse imaging (TLI) system (EmbryoScope) and 191 to the group with culture in the G185 K-System (G185). MAIN OUTCOME MEASURE(S): Rate of implantation (primary endpoint) and embryo morphology grade. RESULT(S): No significant differences were found in the implantation rates. The proportion of high-grade embryos on day 2 was significantly higher in the TLI group compared with the G185 group (40.4% vs. 35.2%). The impact of the incubator on embryo morphology remained significant in multivariate analysis, which took into account the woman's age, the rank of attempt, and the smoking status (TLI vs. G185: odds ratio = 1.27; 95% confidence interval, [1.04-1.55]). No difference was found in the mean number of frozen embryos, even though the total proportion of frozen embryos was significantly higher in the TLI group than in the G185 group (29.5% vs. 24.8%). CONCLUSION(S): No difference in implantation rate was found between the two incubators for fresh cycles. It remains to be determined whether the observed differences in embryo morphology and the total number of embryos cryopreserved would translate into higher cumulative outcomes with subsequent frozen embryo transfers. CLINICAL TRIAL REGISTRATION NO: NCT02722252.

DIY: "Do Imaging Yourself" - Conventional microscopes as powerful tools for in vivo investigation.

Intravital imaging has been increasingly employed in cell biology studies and it is becoming one of the most powerful tools for in vivo investigation. Although some protocols can be extremely complex, most intravital imaging procedures can be performed using basic surgery and animal maintenance techniques. More importantly, regular confocal microscopes - the same that are used for imaging immunofluorescence slides - can also acquire high quality intravital images and movies after minor adaptations. Here we propose minimal adaptations in stock microscopes that allow major improvements in different fields of scientific investigation.

A Test-and-Not-Treat Strategy for Onchocerciasis in Loa loa-Endemic Areas.

BACKGROUND: Implementation of an ivermectin-based community treatment strategy for the elimination of onchocerciasis or lymphatic filariasis has been delayed in Central Africa because of the occurrence of serious adverse events, including death, in persons with high levels of circulating Loa loa microfilariae. The LoaScope, a field-friendly diagnostic tool to quantify L. loa microfilariae in peripheral blood, enables rapid, point-of-care identification of persons at risk for serious adverse events. METHODS: A test-and-not-treat strategy was used in the approach to ivermectin treatment in the Okola health district in Cameroon, where the distribution of ivermectin was halted in 1999 after the occurrence of fatal events related to L. loa infection. The LoaScope was used to identify persons with an L. loa microfilarial density greater than 20,000 microfilariae per milliliter of blood, who were considered to be at risk for serious adverse events, and exclude them from ivermectin distribution. Active surveillance for posttreatment adverse events was performed daily for 6 days. RESULTS: From August through October 2015, a total of 16,259 of 22,842 persons 5 years of age or older (71.2% of the target population) were tested for L. loa microfilaremia. Among the participants who underwent testing, a total of 15,522 (95.5%) received ivermectin, 340 (2.1%) were excluded from ivermectin distribution because of an L. loa microfilarial density above the risk threshold, and 397 (2.4%) were excluded because of pregnancy or illness. No serious adverse events were observed. Nonserious adverse events were recorded in 934 participants, most of whom (67.5%) had no detectable L. loa microfilariae. CONCLUSIONS: The LoaScope-based test-and-not-treat strategy enabled the reimplementation of community-wide ivermectin distribution in a heretofore "off limits" health district in Cameroon and is a potentially practical approach to larger-scale ivermectin treatment for lymphatic filariasis and onchocerciasis in areas where L. loa infection is endemic. (Funded by the Bill and Melinda Gates Foundation and others.).

Factores predictivos de conversión en la apendicectomía videolaparoscópica / Predictive factors of conversion in the videolaparoscopic appendectomy

Introducción para el tratamiento de pacientes con apendicitis aguda, la vía videolaparoscópica se ha convertido en la técnica de elección. Desafortunadamente, en ocasiones es necesario convertir el procedimiento a cirugía convencional. Objetivo: identificar los factores predictivos de conversión en la apendicectomía videolaparoscópica. Métodos: se realizó un estudio longitudinal, prospectivo, de cohorte, en 131 pacientes operados de apendicitis aguda mediante cirugía videolaparoscópica en el Servicio de Cirugía General del Hospital Provincial Docente Clinicoquirúrgico Saturnino Lora Torres de Santiago de Cuba, desde enero del 2010 hasta diciembre del 2014. Para identificar dichos factores, el análisis de los datos se basó en la construcción de un modelo multivariado (regresión logística multivariable). Resultados: se halló predominio de los pacientes jóvenes del sexo masculino. La construcción del modelo de regresión logística estuvo sustentado por 8 variables posiblemente predictoras de conversión, de las cuales 4 resultaron altamente influyentes. Se estimó una sensibilidad de 70,6, una especificidad de 97,4 y un porcentaje global predictivo de 93,9 del modelo de regresión calculado. Conclusiones: los factores de mayor influencia para la conversión de cirugía videolaparoscópica a convencional fueron: laparotomía previa en hemiabdomen inferior, presencia de adherencias diagnosticadas por laparoscopia, ubicación retrocecal y apendicitis perforada

Introduction: the videolaparoscopic procedure has become the election technique for the treatment of patients with acute appendicitis. Unfortunately, in occasions it is necessary to convert the procedure to conventional surgery. Objective: to identify the predictive factors of conversion in the videolaparoscopic appendectomy. Methods: a longitudinal, prospective, cohort study was carried out in 131 patients operated on for acute appendicitis by means of videolaparoscopic surgery in the General Surgery Service of Saturnino Lora Torres Teaching Clinical Surgical Provincial Hospital in Santiago de Cuba, from January, 2010 to December, 2014. The analysis of the data was based on the construction of a multivaried model to identify these factors (multivariable logistical regression). Results: there was a prevalence of young male patients. The construction of the pattern of logistical regression was sustained by 8 possibly predictive variables of conversion, 4 of which were highly influential. Sensibility of 70.6, a specificity of 97.4 and a predictive global percentage of 93.9 of the calculated regression pattern were considered. Conclusions: the most influencial factors for the conversion of videolaparoscopic surgery to conventional surgery were: previous laparotomy in lower hemiabdomen, presence of adherences diagnosed by laparoscopy, retrocaecal location and perforated appendicitis

Microcirculatory Bed, Microcirculation, and Smoking-Associated Endothelial Dysfunction in Young Adults.

Computer-assisted video biomicroscopy of bulbar conjunctiva was employed to examine the sequelae of endothelial dysfunction manifested by microcirculatory bed abnormalities and microcirculation disturbances. The signs of endothelial dysfunction provoked by tobacco smoking in young adults disappeared after cessation of smoking, which resulted in pronounced widening of arterioles and capillaries as well as moderation of intravascular erythrocyte aggregation.