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个 与共焦成像相比的显著优势： 光漂白（以及因此的光毒性）和增加的深度 在样品中的渗透。 我们开发了一个多光子系统 它的特点是紧凑的全固态激光器。 该系统还具有 一个环境室，以方便活标本的研究。 两 利用激光器提供共焦成像之间的比较， （单光子激发）以及两个和三个光子激发图像。 第一个激光器是一个标准的氩离子激光器，提供5毫瓦 在488 nm时进入扫描仪的平均功率。 第二个激光器是 红外、短脉冲（120飞秒）、固态激光器， Microlase，Ltd.制造的，其在1047 nm处产生480毫瓦的平均功率。 激光扫描系统基于BioRad MRC 600共聚焦系统 并连接到Nikon Diaphot Quantum倒置显微镜。 两 检测选项可用。 发射信号可以被引导 并被内部光电倍增管检测到。 去扫描 发射提供了将“针孔”引入到 当认为有必要调整分辨率时，可以使用成像路径。 否则，发射信号可以直接通过 “凯勒”孔到安装在显微镜下方的PNff。 通过检测 直接排放，我们已经获得了大约17倍以上 信号检测比去扫描。

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.