A simple automated method for continuous fieldwise measurement of microvascular hemodynamics.

Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using high-speed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature.

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

Image processing software for 3D light microscopy.

Advances in microscopy now enable researchers to easily acquire multi-channel three-dimensional (3D) images and 3D time series (4D). However, processing, analyzing, and displaying this data can often be difficult and time- consuming. We discuss some of the software tools and techniques that are available to accomplish these tasks.

Non-mydriatic confocal retinal imaging using a digital light projector.

A digital light projector is implemented as an integrated illumination source and scanning element in a confocal non-mydriatic retinal camera, the Digital Light Ophthalmoscope (DLO). To simulate scanning, a series of illumination lines are rapidly projected on the retina. The backscattered light is imaged onto a 2-dimensional rolling shutter CMOS sensor. By temporally and spatially overlapping the illumination lines with the rolling shutter, confocal imaging is achieved. This approach enables a low cost, flexible, and robust design with a small footprint. The 3rd generation DLO technical design is presented, using a DLP LightCrafter 4500 and USB3.0 CMOS sensor. Specific improvements over previous work include the use of yellow illumination, filtered from the broad green LED spectrum, to obtain strong blood absorption and high contrast images while reducing pupil constriction and patient discomfort.

Voxx: a PC-based, near real-time volume rendering system for biological microscopy.

Confocal and two-photon fluorescence microscopy have advanced the exploration of complex, three-dimensional biological structures at submicron resolution. We have developed a voxel-based three-dimensional (3-D) imaging program (Voxx) capable of near real-time rendering that runs on inexpensive personal computers. This low-cost interactive 3-D imaging system provides a powerful tool for analyzing complex structures in cells and tissues and encourages a more thorough exploration of complex biological image data.

Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy.

BACKGROUND: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches. METHODS: We used two-photon microscopy and advanced imaging software to render three-dimensional (3D) images of in situ nerves, blood vessels, and hair follicles labeled with various fluorescent markers. Archived frozen human skin biopsy specimens were cryosectioned up to 150 micro m in thickness and fluorescently stained with rhodamine- or fluorescein-labeled antibodies or lectins. Optical sections were collected by two-photon microscopy and the resulting data sets were analyzed in three dimensions using Voxx software. RESULTS: Reconstructed image volumes demonstrated the complex 3D morphology of nerves, blood vessels and adnexal structures in normal mucocutaneous tissue. CONCLUSION: Two-photon microscopy and Voxx rendering software allow for detailed 3D visualization of structures within human mucocutaneous biopsy specimens, as they appear in situ, and facilitate objective interpretation of variations in their morphology. These techniques may be used to investigate disorders involving cutaneous structures that are difficult to visualize by means of traditional microscopy.

Renal cysts of inv/inv mice resemble early infantile nephronophthisis.

Cystic kidney disease has been linked to mutations in the Invs gene in mice with inversion of embryonic turning (inv/inv) and the INVS (NPHP2) gene in infants with nephronophthisis type 2 (NPHP2). The inv mouse model features multiorgan defects including renal cysts, altered left-right laterality, and hepatobiliary duct malformations transmitted in an autosomal recessive manner. Affected mice usually die of renal and liver failure by postnatal day 7. Although cardiopulmonary and liver anomalies have been carefully detailed, renal cysts have yet to be fully characterized in inv/inv. By use of three-dimensional visualization by two-photon microscopy, this study provides the first comprehensive analysis of in situ cyst formation and progression in inv/inv kidneys. At embryonic day 15, there is dilatation of Bowman's capsule followed temporally by corticomedullary cysts involving collecting ducts, proximal tubules, and thick ascending limbs. Collecting ducts of newborn inv/inv mice are uniformly and diffusely cystic from medulla to cortex, with normal diameters found only at their most proximal tips. Proximal tubules form fusiform cysts that alternate with segments of normal or narrowed caliber along torturous convolutions. Because defective cilia have been linked to situs inversus and cystogenesis, we examined inv/inv cilia by scanning and transmission electron microscopy. The former detected monocilia of expected length in cystic collecting ducts and proximal tubules; the latter demonstrated the usual 9 + 2 pattern in respiratory cilia. The inv mutant mouse has renal cysts resembling infantile NPHP2 and will provide broader insight into the role cilia play in renal cystogenesis.