CONSULT: MULTI PHOTON MICROSCOPY: DEEP TISSUE IMAGING IN STERILE ENVIRONMENT

在堪萨斯生物物理学会会议上的海报介绍 城市，密苏里州（1998年2月22日至26日）。 Biophys. J. 74，A189（1998）。 多光子显微镜（MPM）提供三维分辨率， 亚细胞结构的光学成像和生物监测 研究光化学损伤机制的功能。 是 MPM过程中的损伤可能来自双光子吸收 事件，从高阶多光子事件，或从单光子事件 事件 剂量（Int）定义为峰的n次幂 样品处的辐照度乘以总停留时间， 细胞中的焦点体积。 (The辐照度I的单位为光子/秒（（m2，和 n是单个激发事件所需的光子数，或 激发顺序）。为了检测最小的光损伤，我们 我选择了一种对干扰敏感的增殖测定法 细胞新陈代谢和防御机制的影响 细胞 使用这种分析，我们产生了一种内在的行为， 通过监测DNA合成的开始来获得光损伤的光谱 在没有添加染料的培养的HeLa细胞中的抑制。 因为我们有 还没有确定在所有波长处的损伤的激发顺序， 该作用光谱用n = 2表示，定义双光子 事件 测得的损伤阈值比 典型的成像剂量。 我们可以评估这些数据来确定 激发阶数，n，使用一般剂量响应方程，其中 S是标准化生存期，剂量D定义为Int. 初步分析表明，所观察到的损害是由于 不同波长下的不同吸收物质--在700 nm处 损伤似乎来自单光子事件，在740 nm处， 来自于双光子事件 由NIH在DRBIO支持（RR 04224） 和NSF（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).