1098 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 48, NO. 10, OCTOBER 2001 Quantifying Spatial Localization of Optical Mapping Using Monte Carlo Simulations Lei Ding, Student Member, IEEE, Robert Splinter, and Stephen B. Knisley*, Member, IEEE Abstract—Optical mapping techniques used to study spatial dis- tributions of cardiac activity can be divided into two categories. 1) Broad-field excitation method, in which hearts stained with voltage or calcium sensitive dyes are illuminated with broad-field excita- tion light and fluorescence is collected by image or photodiode ar- rays. 2) Laser scanning method, in which illumination uses a scan- ning laser and fluorescence is collected with a photomultiplier tube. The spatial localization of the fluorescence signal for these two methods is unknown and may depend upon light absorption and scattering at both excitation and emission wavelengths. We mea- sured the absorption coefficients , scattering coefficients , and scattering anisotropy coefficients at representative excita- tion and emission wavelengths in rabbit heart tissue stained with di-4-ANEPPS or co-stained with both Rh237 and Oregon Green 488 BAPTA 1. Monte Carlo models were then used to simulate ab- sorption and scattering of excitation light and fluorescence emis- sion light for both broad-field and laser methods in three–dimen- sional tissue. Contributions of local emissions throughout the tissue to fluorescence collected from the tissue surface were determined for both methods. Our results show that spatial localization de- pends on the light absorption and scattering in tissue and on the optical mapping method that is used. A tissue region larger than the laser beam or collecting area of the array element contributes to the optical recordings. Index Terms—Cardiac tissue, integrating spheres, Kubelka- Munk, Monte Carlo, optical mapping, spatial localization. I. INTRODUCTION O PTICAL mapping is used to study the spatial distribution of transmembrane potentials or intracellular calcium in the heart. Methods for optical mapping utilize either broad-field excitation or laser scanning. For broad-field excitation, hearts stained with voltage- or calcium-sensitive dye are illuminated over a wide area with light at wavelengths appropriate for exci- tation of the dye while fluorescence is collected with an imager or photodiode array [1], [2]. For laser scanning, illumination is performed with a narrow laser beam that scans various locations Manuscript received July 18, 2000; revised June 27, 2001. This work was sup- ported in part by the National Institutes of Health (NIH) under Grant HL52003 and Grant HL67728, in part by a grant from the Whitaker Foundation, and in part by the American Heart Association under Grant 9740173N. Asterisk indi- cates correponding author. L. Ding is with the Department of Electrical Engineering, Georgia Institute of Technology, Atlanta, GA 30332 USA. R. Splinter is with the Laser and Applied Technologies Laboratory, Carolinas Medical Center, Charlotte, NC 28203 USA, and the Department of Physics, University of North Carolina at Charlotte, Charlotte, NC 28223 USA. *S. B. Knisley is with the Departments of Biomedical Engineering and Medicine, University of North Carolina at Chapel Hill, CB#7575, 152 MacNider Hall, Chapel Hill, NC 27599 USA (e-mail: knisley@bme.unc.edu). Publisher Item Identifier S 0018-9294(01)08274-X. while fluorescence is collected with a photomultiplier tube [3]. In both cases, intensity of the fluorescence signal collected by the photo-detector from the dye-stained cells changes in propor- tion to changes in calcium or transmembrane potentials of the cells. With the methods mentioned, spatial localization of an in- dividual fluorescence signal to a small region of tissue is at- tempted by either restricting the area where excitation light is applied to a desired region with a small laser beam or by re- stricting the collection of emitted fluorescence to a desired re- gion by projecting photons that exit a selectively small surface area onto a single respective photodiode or charge-coupled de- vice array element placed in an image plane. However, the signal may not just represent the tissue located under the excitation laser beam or the epicardial region imaged by the array element. The signal may also contain fluorescence from tissue outside of this location due to scattering of excitation and emission light. Also, given that light scattering may not be identical at excita- tion and emission light wavelengths, the localization may differ in these two methods. The localization may be important for in- terpretation of optical recordings used to study cardiac activity or arrhythmias. Here, we estimated spatial localization (e.g., fraction of the signal that originates in a given tissue location) for the broad-field excitation and laser scanning methods with various sizes of the laser beam or imaging array element. To quantify the spatial localization involves answering two questions: 1) given a slab of tissue and the light source, what is the amount of excitation light reaching each location in the tissue?; and 2) what is the contribution of fluorescence emitted from each location to signals collected by the photo-detector? Both of the questions require understanding of light interaction with biological tissues, which has been a focus area in med- ical applications using light, such as laser surgery and photo- dynamic therapy [4], [5]. Numerous mathematical models have been developed to estimate fluence rates in tissue, or reflection and transmission of light in tissue. Among them, Monte Carlo models may be the most accurate and flexible [6]–[8]. In this study, we chose to answer the questions by simulating the prop- agation of excitation light and emitted fluorescence in the heart tissue with the help of Monte Carlo computer models. Six op- tical properties of the tissue were needed to perform this model, which include the absorption coefficients , the scattering co- efficients , and the scattering anisotropy coefficient at both excitation and emission wavelengths. The absorption and scat- tering coefficients are defined as the probability/unit path length that a photon will encounter an absorption or scattering event [9]. The anisotropy coefficient is used to describe the direction of the scattering event [9]. A value of close to zero indicates 0018–9264/01$10.00 © 2001 IEEE