MULTIPHOTON MICROSCOPY TOOL TO STUDY PHOTODAMAGE

在堪萨斯州生物物理学会会议上的海报演示 密苏里州城市(1998年2月22日至26日)。生物群落。J.74，A189(1998)。 多光子显微镜(MPM)提供了三维分辨率 亚细胞结构的光学成像和生物监测 用于研究光化学损伤机制的功能。它是 MPM中的损伤可能是由双光子吸收引起的 事件、来自更高阶多光子事件或来自单光子 事件。剂量(Int)定义为峰值的n次方 样本处的辐照度乘以 细胞内的焦点体积。(辐照度i，单位为光子/S((m2，和 N是单次激发事件所需的光子数，或 激励顺序。)为了检测观察到的最小光损伤，我们 已经选择了一种对扰动敏感的增殖试验 细胞新陈代谢和防御机制以及死亡 细胞。使用这个分析，我们正在产生一种内在的动作 监测DNA合成开始的光损伤光谱 不添加染料对培养的HeLa细胞的抑制作用。我们所拥有的罪孽 还没有确定损伤在所有波长的激发顺序， 这个作用谱表示为n=2，定义了双光子 事件。测量到的损伤阈值是超过数量级的 典型的成像剂量。我们可以对这些数据进行评估以确定 使用一般剂量-反应方程的激发阶数n，其中 S为归一化生存，剂量D定义为Int。 初步分析表明，观察到的损坏是由 不同波长的不同吸收物质--700 nm 损伤似乎来自单光子事件，在740 nm处看起来 来自双光子事件。由NIH在DRBIO提供支持(RR04224) 和国家科学基金(BIR，8800278)。

Poster Presentation at the Biophysical Society Meeting at Kansas City, MO (Feb 22-26, 1998). Biophys. J. 74, A189 (1998). Multiphoton microscopy (MPM) provides three-dimensional resolution for optical imaging of subcellular structures and monitoring of biological functions with which to study photochemical damage mechanisms. It is possible that damage during MPM arises from two-photon absorption events, from higher order multiphoton events, or from one-photon events. Doses (Int) are defined as the nth power of the peak irradiance at the sample multiplied by the total dwell time of the focal volume in a cell. (The irradiance, I, is in photons/s((m2, and n is the number of photons required for a single excitation event, or the excitation order.) To detect the minimal observed photodamage, we have selected a proliferation assay that is sensitive to perturbations of cellular metabolism and defense mechanisms as well as to dead cells. Using this assay, we are generating an intrinsic action spectrum of photodamage by monitoring the onset of DNA synthesis inhibition in cultured HeLa cells without added dyes. Sin ce we have not yet determined the excitation order of damage at all wavelengths, this action spectrum is expressed with n = 2, defining two-photon events. The measured damage thresholds are orders of magnitude above typical imaging doses. We can evaluate this data to determine the excitation order, n, using the general dose-response equation , where S is the normalized survival and the dose, D, is defined as Int. Preliminary analysis suggests that the observed damage is arising from different absorbing species at different wavelengths -- at 700 nm damage appears to be from one-photon events and at 740 nm it appears to be from two-photon events. Supported at DRBIO by the NIH (RR04224) and NSF (BIR 8800278).