Center for Optical Diagnostics and Therapy, Department of Radiation Oncology, Erasmus Medical Center, Room Ee-1675, PO Box 2040, 3000 CA, Rotterdam, The Netherlands.
Lasers Med Sci. 2011 Nov;26(6):789-801. doi: 10.1007/s10103-011-0888-z. Epub 2011 Jan 29.
A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed.
生物医学光学中的一个主要挑战是对体内荧光图像进行准确的定量。荧光成像是用于确定光动力疗法中使用的光敏剂药代动力学的常用方法。然而，这种类型的成像通常没有考虑到组织体积和所研究组织的光学性质的差异和变化。为了解决这个问题，开发并应用了一种比率定量方法来监测在鼠皮褶观察室中的光敏剂间四（羟基苯基）氯（mTHPC）的药代动力学。该方法采用双波长激发和双波长检测相结合的方式。激发和检测波长选择在近红外区域。一个激发波长选择在 mTHPC 的 Q 带，而第二个激发波长接近其吸收最小值。使用两个荧光发射带；一个在 mTHPC 的次级荧光最大值处，中心在 720nm，另一个在组织自体荧光的区域。第一个激发波长用于激发 mTHPC 和自体荧光，第二个用于仅激发自体荧光，以便可以进行减法。随后，将自体荧光校正的 mTHPC 图像除以自体荧光信号，以校正组织光学性质的变化。该校正算法原则上导致校正后的荧光与光敏剂浓度之间呈线性关系。讨论了所提出的方法的局限性以及与以前发表的和经过验证的技术的比较。
