Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Summary

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.

Abstract

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.

Introduction

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 investigations^1,2,3,4,5. 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) cameras^9,10. 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.

Protocol

All animal experiments were approved and conducted according to the Institutional Animal Care and Use Committee guidelines of Indiana University, and adhered to the NRC guide for the care and use of animals. 1. Surgical Preparation for Intravital Microscopy NOTE: This is not a survival surgery. Once section 1 "Surgical preparation for intravital microscopy” is begun, work cannot be paused until the completion of section 2 "Intravital mic…

Representative Results

STAFF analysis generates a complete census of microvascular velocities across entire microscope fields over periods of time extending from seconds to minutes. Representative results are presented in Figure 1, Figure 2, Figure 3, and Figure 4. Figure 1 shows an example of a time series of the microvascular network in the liver of a mouse, the generation of the skeletonized i…

Discussion

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 …

Materials

#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