To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.
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.
The microvasculature plays a critical role in physiology, ensuring effective perfusion of tissues under changing conditions. Microvascular dysfunction is associated with myriad conditions including long-term cardiovascular morbidity and mortality, development of dementia, and disease of both liver and kidney and thus is a key factor of interest in a broad range of biomedical investigations1,2,3,4,5. While multiple techniques have been used to evaluate tissue perfusion, only intravital microscopy enables data collection at the temporal and spatial resolution necessary to characterize blood flow at the level of individual capillaries.
Microvascular flow can be visualized in fluorescence microscopy either by the movement of fluorescent microspheres or by the movement of red blood cells against the background of membrane-impermeant fluorescent markers (e.g., fluorescently-labeled dextran or albumin)6,7. Microvascular flow can be imaged in superficial cell layers using widefield microscopy, or at depth using either confocal or multiphoton microscopy. However, capillary flow rates are such that the passage of red blood cells cannot generally be captured at speeds less than 60 frames/s. Since most laser scanning confocal and multiphoton microscopes require 1–5 s to scan a full image field, this speed can generally be accomplished only by limiting the field of view, sometimes to a single scan line8. The process of limiting measurements to selected capillary segments (1) has the potential to introduce selection bias and (2) makes it impossible to capture spatial and temporal heterogeneity in the rates of capillary blood flow. In contrast, images of capillary networks can be collected at speeds exceeding 100 fps using widefield digital microscopes equipped with scientific complementary metal oxide semiconductor (sCMOS) cameras9,10. These inexpensive systems, common in typical biomedical laboratories make it possible to image microvascular flow across entire two-dimensional networks, essentially continuously. The problem then becomes one of finding an analysis approach that is capable of extracting meaningful quantitative data from the massive and complex image datasets generated by high-speed video microscopy.
To enable analysis of full-field flow data we have developed STAFF, novel image analysis software that can continuously measure microvascular flow throughout entire microscope fields of image series collected at high speed11. The approach is compatible with a variety of different experimental systems and imaging modalities and the STAFF image analysis software is implemented as a macro toolset for the FIJI implementation of ImageJ12. The underlying principle used here to visualize microvascular flow is that first, some contrast must be provided to be able to image the red blood cells within capillaries. In our studies, contrast is provided by a bulk fluorescent probe that is excluded by the red blood cells. The velocity of flow can then be quantified from the displacement of the red blood cells that appear as a negative stain within the fluorescently labeled plasma in images collected at high speed from a living animal8. We then use STAFF to make plots of distance along each capillary segment over multiple intervals of time called kymographs, then detect the slopes present in the kymographs13, and from those slopes calculate the rates of microvascular flow. The approach can be applied to images collected from any capillary bed that can be accessed for imaging. Here we describe the application of IVM and STAFF to studies of blood flow in the liver.
There are multiple critical steps in this protocol. First, minimization of motion during intravital imaging of the liver is essential for generating movies that are usable for capillary flow analysis using STAFF. Due to the proximity of the diaphragm, short periods of respiration-induced motion occur, with the secured liver returning to its initial position after each breath. Securing the surgically exposed liver against the coverslip-bottomed dish using gauze, then imaging from below using an inverted microscope serves …
The authors have nothing to disclose.
Studies presented here were supported by funding from the National Institutes of Health (NIH U01 GM111243 and NIH NIDDK P30 DK079312). Intravital microscopy studies were conducted at the Indiana Center for Biological Microscopy. We thank Dr. Malgorzata Kamocka for technical assistance with microscopy.
#5 forceps | Fine Science Tools | 11251-20 | Dumont #5 Inox Forceps |
C57BL/6 mice | Jackson Labs | male 9-12 weeks old | |
Cannula | Instech | BTPE-10 | Polyethylene Tubing .011x.024in |
CMOS camera | Hamamatsu | C11440-42U30 | 4.0LT Scientific CMOS |
Coverslip-bottomed dish | Electron Microscopy Sciences | WillCo Dish glass bottom GWST5040 | |
Dissecting scissors | Fine Science Tools | ||
Fiji ImageJ Image analysis software | https://fiji.sc/ ; https://downloads.imagej.net/fiji/Life-Line/fiji-win64-20170530.zip | ||
Fluorescein dextran | Thermo Fisher, Invitrogen | D1822 | Dextran, Fluorescein, 70,000 MW, Anionic, Lysine Fixable |
Gauze sponge | Fisher | 22-415-504 | 2×2 inch Dukal sterile gauze sponges |
Heating pad | Reptitherm | RH-4 | between mouse and stage |
Heating pad | Sunbeam | 000732-500-000U | over mouse |
Inverted epifluorescence microscope | Nikon | Nikon TiE inverted microscope | |
Isis Rodent electric shaver | Braun Aesculap | GT420 | |
Isofluorane | Abbott GmbH | PZN4831850 | |
Luer stub adapter | Fisher | 14-826-19E | Catheter adapter |
Micro scissors | Castro Viejo | ||
Microscope objective | Nikon | Plan Fluor 20x, NA 0.75 water immersion | |
Needle | Fisher | 30 Ga.x1/2" | |
Needle holder | Olsen-Hegar | ||
Objective heater | BioScience Tools | MTC-HLS-025 | Temperature controller with objective heater |
Rectal thermometer | Braintree Scientific, INC | TH-5A | Mouse Body Temperature monitoring |
STAFF macros | https://github.com/icbm-iupui/STAFF | ||
Suture string | Harvard Bioscience | 723288 | silk black suture, 6-0, spool |