Improved detection of fluorescently labeled microspheres and vessel architecture with an imaging cryomicrotome

Due to spectral overlap, the number of fluorescent labels for imaging cryomicrotome detection was limited to 4. The aim of this study was to increase the separation of fluorescent labels. In the new imaging cryomicrotome, the sample is cut in slices of 40 μm. Six images are taken for each cutting plane. Correction for spectral overlap is based on linear combinations of fluorescent images. Locations of microspheres are determined by using the system point spread function. Five differently colored microspheres were injected in vivo distributed over two major coronaries, the left anterior descending and left circumflex artery. Under absence of collateral flow, microspheres outside of target perfusion territories were not found and the procedure did not generate false positive detection when spectral overlap was relevant. In silico-generated microspheres were used to test the effect of background image, transparency correction, and color separation. The percentage of microspheres undetected was 2.3 ± 0.8% in the presence and 1.5 ± 0.4% in the absence of background structures with a density of 900 microspheres per color per cm3. The image analysis method presented here, allows for an increased number of experimental conditions that can be investigated in studies of regional myocardial perfusion.

[1]  J. Hales,et al.  Direct observations of the behaviour of microspheres in microvasculature. , 1977, Bibliotheca anatomica.

[2]  M. Noble,et al.  Total and Regional Coronary Blood Flow Measured by Radioactive Microspheres in Conscious and Anesthetized Dogs , 1969, Circulation research.

[3]  D. McDevitt,et al.  Simultaneous measurement of cardiac output and its distribution with microspheres in the rat. , 1976, Cardiovascular research.

[4]  Geert J. Streekstra,et al.  Diameter measurement from images of fluorescent cylinders embedded in tissue , 2008, Medical & Biological Engineering & Computing.

[5]  Edward R. Dougherty,et al.  An introduction to morphological image processing , 1992 .

[6]  J. R. Ewen,et al.  Regional blood flow measurements from fluorescent microsphere images using an Imaging CryoMicrotome , 2000 .

[7]  J I Hoffman,et al.  Some sources of error in measuring regional blood flow with radioactive microspheres. , 1971, Journal of applied physiology.

[8]  F W Prinzen,et al.  Fluorescent microspheres to measure organ perfusion: validation of a simplified sample processing technique. , 1995, The American journal of physiology.

[9]  W. Hauck,et al.  Quantitating error in blood flow measurements with radioactive microspheres. , 1989, The American journal of physiology.

[10]  R. Kloner,et al.  Fluorescent microspheres: a new tool for visualization of ischemic myocardium in rats. , 1986, The American journal of physiology.

[11]  J. R. Ewen,et al.  High spatial resolution measurements of organ blood flow in small laboratory animals. , 2000, American journal of physiology. Heart and circulatory physiology.

[12]  Han Wen,et al.  High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres. , 2004, American journal of physiology. Heart and circulatory physiology.

[13]  R W Glenny,et al.  Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. , 1993, Journal of applied physiology.

[14]  W. D. de Groat,et al.  Voltage-dependent potentiation of neuronal L-type calcium channels due to state-dependent phosphorylation. , 1995, The American journal of physiology.

[15]  G. Vlahakes,et al.  Acute and chronic microsphere loss from canine left ventricular myocardium. , 1982, The American journal of physiology.

[16]  A M Rudolph,et al.  The Circulation of the Fetus in Utero: Methods For Studying Distribution of Blood Flow, Cardiac Output And Organ Blood Flow , 1967, Circulation research.

[17]  Rene ter Wee,et al.  Organization and collateralization of a subendocardial plexus in end-stage human heart failure. , 2010, American journal of physiology. Heart and circulatory physiology.

[18]  Geert J. Streekstra,et al.  Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalisation of myocardial perfusion areas , 2005, Medical and Biological Engineering and Computing.