High spatial resolution measurements of organ blood flow in small laboratory animals

Abstract

With the use of a newly developed Imaging Cryomicrotome to determine the spatial location of fluorescent microspheres in organs, we validate and report our processing algorithms for measuring regional blood flow in small laboratory animals. Microspheres (15-microm diameter) of four different fluorescent colors and one radioactive label were simultaneously injected into the left ventricle of a pig. The heart and kidneys were dissected, and the numbers of fluorescent and radioactive microspheres were determined in 10 randomly selected pieces. All microsphere counts fell well within the 95% expected confidence limits as determined from the radioactive counts. Fluorescent microspheres (15-microm diameter) of four different colors were also injected into the tail vein of a rat and the left ventricle of a rabbit. After correction for Poisson noise, correlation coefficients between the colors were 0.99 +/- 0.02 (means +/- SD) for the rabbit heart and 0.99 +/- 0.02 for the rat lung. Mathematical dissection algorithms, statistics to analyze the spatial data, and methods to visualize blood flow distributions in small animal organs are presented.

High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

“…In a second set of experiments, yellow, red, and scarlet microspheres (Molecular Probes) were applied in 40-min intervals, and microsphere deposition was imaged using a commercially available imaging cryomicrotome (Barlow Scientific; Olympia, WA) (5,14). In brief, the microtome sectioned through the frozen heart at a selected slice thickness of 30 -40 m. Fluorescent images of the newly exposed myocardial surface were digitally obtained with appropriate excitation and emission filters to isolate each of up to four different fluorescent colors.…”

“…The deposition of fluorescent microspheres in tissue slices can also be imaged, and a recently developed imaging cryomicrotome (14) opens up the opportunity to image and determine the position of all individual microspheres within a given organ or tissue piece. In fact, this image analysis compared favorably to radioactive microspheres (5).…”

On the Emission Intensity of Fluorescent Microspheres in Cardiac Tissue Images

“…In an attempt to overcome the limitations of the -destruction & extraction‖ method, fluorescence imaging of fluorescent microspheres in biomedical samples was employed for qualitative and quantitative characterization of microsphere deposition and blood flow [5][6][7]. In particular, fluorescence images of frozen thin (30 m) rabbit heart slices were shown to quantify fluorescent microspheres deposited in the cardiac tissue [7].…”

“…In particular, fluorescence images of frozen thin (30 m) rabbit heart slices were shown to quantify fluorescent microspheres deposited in the cardiac tissue [7].…”

An Optimized Method for Delivering Flow Tracer Particles to Intravital Fluid Environments in the Developing Zebrafish

“…Small fluorescent microspheres (0.02-0.04 micron diameter) have been used to perform zebrafish angiographies, 34 while much larger ones have been used to make two-dimensional strain measurements in the chick heart 35 and to measure regional blood flow in adult rainbow trout, 36 pigs, rabbits, and rats. 37 More recently, microspheres injected into the embryonic zebrafish bloodstream which had lodged into the heart wall were used to map endocardial motions using three-dimensional digital defocusing particle image velocimetry (3D-DPIV). 31 The increased use of microspheres in a variety of flow-mapping applications highlights the importance of developing a supporting infrastructure of detailed and efficient injection methodologies.…”