DEVELOPMENT OF TWO PHOTON IMAGING SYSTEM FOR STUDY OF LIVING SPECIMENS

我们正在探索多光子成像的潜在应用 用于生物学或临床研究。多光子成像提供了两种 与共聚焦成像相比的显著优势：减少了总成本 光漂白(因此光毒性)和增加的深度 试件内的穿透力。我们已经开发出一种多光子系统 它的特点是紧凑型全固态激光器。该系统还具有 为活体标本研究提供便利的环境室。二 激光被用来比较共焦成像 (单光子激发)和双光子和三光子激发图像。 第一个激光器是标准的氩离子激光器，输出功率为5毫瓦。 进入扫描仪的平均功率为488mN。第二个激光器是一种 由以下公司制造的短脉冲(120fsec)红外固态激光器 Microlase，Ltd.，在1047纳米处产生480毫瓦的平均功率。 激光扫描系统基于BioRad MRC600共焦系统 并与尼康Diaphot量子倒置显微镜相耦合。二 检测选项可用。发射信号可以被定向 回到扫描头并由内部PMT检测到。去扫描 该排放物提供了将“针孔”引入 当被认为需要修剪分辨率时，成像路径。 否则，发射信号可以直接通过 将“凯勒”孔安装在显微镜下的PNff上。通过检测 直接排放，我们已经获得了大约17倍的 信号检测优于去扫描。

We are exploring the potential applications of multiphoton imaging for biological or clinical research. Multiphoton imaging offers two significant advantages over confocal imaging: reduced total photobleaching (and hence phototoxicity) and increased depth of penetration within a specimen. We have developed a multiphoton system that features a compact, all-solid-state laser. The system also has an environmental chamber to facilitate studies of live specimens. Two lasers are utilized to provide comparisons between confocal imaging (one-photon excitation) and two and thr-ee-photon excitation images. The first laser is a standard argon ion laser which delivers 5 mwatts average power at 488 mn into the scanner. The second laser is an infrared, short-pulse (120fsec), solid-state laser manufactured by Microlase, Ltd. which produces a mean power of 480 mwatts at 1047 nm. The laser scanning system is based on a BioRad MRC 600 confocal system and is coupled to a Nikon Diaphot Quantum inverted microscope. Two detection options are available. The emission signal may be directed back into the scanhead and detected by the internal PMTs. Descanning the emission provides the option of introducing the "pinhole" into the imaging pathway when deemed necessary to trim the resolution. Otherwise, the emission signal can be passed directly through the "Keller" hole to a PNff mounted below the microscope. By detecting the emission directly, we have gained approximately 17 times more signal detection than descanning.