Structured Illumination Enhances Resolution and Contrast in Thick Tissue Fluorescence Imaging - eScholarship (2010)

JBO Letters Structured illumination enhances resolution and contrast in thick tissue fluorescence imaging Amaan Mazhar, a,b David J. Cuccia, c Sylvain Gioux, d,e Anthony J. Durkin, b John V. Frangioni, e and Bruce J. Tromberg a,b, * a University of California, Irvine, Department of Biomedical Engineering, Irvine, California 92612 University of California, Irvine, Beckman Laser Institute and Medical Clinic, Laser Microbeam and Medical Program, Irvine, California 92612 c Modulated Imaging, Inc., Technology Incubator Office, Irvine, California 92612 d Boston University, Department of Biomedical Engineering, Boston, Massachusetts 02215 e Beth Israel Deaconess Medical Center, Department of Medicine, Division of Hematology/Oncology, Boston, Massachusetts 02215 b Abstract. We introduce a noncontact imaging method uti- lizing multifrequency structured illumination for improv- ing lateral and axial resolution and contrast of fluorescent molecular probes in thick, multiple-scattering tissue phan- toms. The method can be implemented rapidly using a spatial light modulator and a simple image demodulation scheme similar to structured light microscopy in the dif- fraction regime. However, imaging is performed in the multiple-scattering regime utilizing spatially modulated scalar photon density waves. We demonstrate that by in- creasing the structured light spatial frequency, fluores- cence from deeper structures is suppressed and signals from more superficial objects enhanced. By measuring the spatial frequency dependence of fluorescence, back- ground can be reduced by localizing the signal to a buried fluorescent object. Overall, signal-to-background ratio 共SBR兲 and resolution improvements are dependent on spa- tial frequency and object depth/dimension with as much as sevenfold improvement in SBR and 33% improvement in resolution for ⬃1-mm objects buried 3 mm below the surface in tissue-like media with fluorescent background. © 2010 Society of Photo-Optical Instrumentation Engineers. 关DOI: 10.1117/1.3299321兴 Keywords: diffuse optics; near infrared; photon migration; spatial modulation . Paper 09396LR received Sep. 8, 2009; revised manuscript received Dec. 3, 2009; accepted for publication Dec. 4, 2009; published on- line Mar. 2, 2010. There is considerable interest in the development of in vivo near-infrared 共NIR兲 fluorescence imaging and tomography for clinical and preclinical research. 1,2 In the NIR, photon pen- etration is maximized due to reduced light scattering and rela- tively low absorption of intrinsic tissue chromophores such as water, hemoglobin, and lipid. * Address all correspondence to: Bruce J. Tromberg. Tel: 949-824-8367; Fax: 949-824-6969; E-mail: bjtrombe@uci.edu Journal of Biomedical Optics NIR excitation and subsequent emission can significantly improve imaging depth compared to visible fluorescent probes. Despite this advantage, accurate mapping of subsur- face 共i.e., mm to cm兲 fluorescence with high spatial resolution remains a critical challenge. This is due to the fact that lateral and axial localization of fluorescence is “blurred” by multiple light scattering. In order to address this limitation, imaging systems that utilize multiple detectors, source scanning, and time/frequency domain techniques have been successfully implemented based on diffuse optical tomography principles. 3–5 In this work, we demonstrate a simple alternative nonscan- ning method using multifrequency structured illumination pat- terns to improve axial and transverse resolution of buried fluorescent inhomogeneities. Our approach utilizes similar principles to technologies employing structured illumination developed for the diffraction regime. 6,7 However, in this case, lateral and axial resolution improvements are obtained from thick, turbid materials using principles of scalar photon den- sity waves propagating in the diffusion regime. 8–10 A spatial light modulator is introduced at the source, and a number of sinusoidal illumination patterns with different spa- tial frequencies, k , are projected over a tissue sample at three offset phases 共typically 0 deg, 120 deg, and 240 deg 兲. During data analysis, the images at each phase, I 1,2,3 , are demodu- lated using Eq. 共1兲 in order to analyze the AC component of the remitted light. This concept has also been applied by Neil et al. for the purposes of optical sectioning microscopy. 11 兩 M AC 共 x i 兲兩 f xi = 关共 I 1 − I 2 兲 2 + 共 I 2 − I 3 兲 2 + 共 I 3 − I 1 兲 2 兴 1/2 . In the spatial frequency domain, the reflected AC compo- nent of the spatially modulated waves can be used to charac- terize the modulation transfer function 共MTF兲 of the sample. 8,9 We now extend this concept to fluorescence imag- ing where structured light illumination specifically impacts the excitation fluence rate. A forward fluence model for spa- tially modulated light—specifically, sinusoidal waves—can be adapted from a 1-D second-order Helmholtz equation in order to calculate fluence rate 共 ␸ 兲 as a function of depth, z : 8 d 2 ␸ 0 共 z 兲 − ␮ ⬘ ef 2 f ␸ 0 共 z 兲 = − 3 ␮ tr q 0 共 z 兲 , dz 2 In these equations, q is the source, ␮ tr = 共 ␮ a + ␮ s ⬘ 兲 is the transport coefficient, ␮ a is the absorption coefficient, ␮ s ⬘ = ␮ s 共1 − g兲 is the reduced scattering coefficient, g is the mean cosine of the scattering angle 共具cos ␪ 典兲 , k is the spatial fre- quency of the illumination pattern, ␮ ef f = 关3 ␮ a ␮ tr 兴 1/2 , and ␮ ⬘ ef f = 关 ␮ 2 ef f + k 2 兴 1/2 . The solution for this equation is: ␸ 0 共 z 兲 = 3P 0 a ⬘ exp 共 − ␮ tr z 兲 + C exp 共 − ␮ ⬘ ef f z 兲 , ␮ ⬘ ef 2 f / ␮ tr 2 − 1 where a ⬘ is the reduced albedo 共 ␮ s ⬘ / ␮ tr ⬘ 兲, is the incident opti- cal power, and C is a constant determined by the choice of a boundary condition. A normalized fluence rate profile for 1083-3668/2010/15共1兲/010506/3/$25.00 © 2010 SPIE January/February 2010 Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 10/27/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx Vol. 15共1兲