DCS-120 共焦 FLIM 系统

说明

DCS-120 共焦 FLIM 系统采用ps 二极管激光器激发、振镜快速扫描、共焦检测和bh 的多维 TCSPC 技术进行 FLIM。它以前所未有的时间分辨率、前所未有的重现性、高空间分辨率、高灵敏度和接近理想的光子效率记录荧光寿命图像。荧光寿命可检测到 25 ps；衰变数据可解析为 4096 个时间通道，宽度可达 405 fs。像素格式可增至 2048 x 2048。

DCS-120 系统可用于尼康、蔡司和奥林巴斯的倒置显微镜。它还可用于将现有的传统显微镜转换为带有 TCSPC 检测功能的全功能共聚焦或多光子激光扫描显微镜。由于其光束扫描速度快、灵敏度高，DCS-120 系统可用于活细胞成像。

DCS-120 的功能包括在两个完全平行的波长通道中同时记录 FLIM 或稳态荧光图像、激光波长复用、多波长 FLIM、时间序列 FLIM、通过时间马赛克 FLIM 进行超快速时间序列记录、空间马赛克 FLIM、Z-Stack FLIM、磷光寿命成像（PLIM）、荧光寿命瞬时扫描（FLITS）和 FCS 记录。应用重点是代谢成像，即利用荧光团与其分子环境相互作用所产生的寿命变化。典型应用包括离子浓度测量、FRET 实验、代谢成像、快速生理效应成像和植物生理学。

数据分析由 bh 最新的SPCImage NG FLIM分析软件执行。SPCImage NG 是时域分析和相位分析的结合。通过在 GPU（图形处理器）上运行计算，几秒钟内就能得到结果。其他功能还包括通过相位图或二维时域直方图进行图像分割，以及系统 IRF 自动建模。因此，无需通过记录实际 IRF 进行重复校准。

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DCS-120 共焦和多光子 FLIM 系统用户手册

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bh FLIM 系统的应用领域是分子成像。典型的应用包括离子浓度、pH 值或局部粘度成像，通过 FRET 进行蛋白质相互作用实验，以及通过 NADH 和 FAD 的荧光衰减结合氧气测量进行代谢成像。在这些应用中，bh FLIM 系统具有高灵敏度、高时间分辨率、高定时稳定性，并能将多指数衰减曲线分解为各个组成部分。其他优势还包括能够记录毫秒级的快速生理效应 FLIM，以及同时记录多个激发和发射波长。

原则

Confocal Scanning

In a conventional fluorescence microscope fluorescence is excited and detected in a double cone throughout the entire depth of the sample, see figure below. The sharp image seen in the focal plane is therefore surrounded by out-of-focus haze from above and below the focal plane. For structural imaging of single cells this may still be acceptable. For FLIM, however, even a small amount of out-of focus blur is unacceptable because it adds unwanted decay components to the fluorescence decay in the focal plane. For FLIM of thick tissue the situation becomes entirely hopeless. The out-of-focus blur becomes entirely overwhelming, and results in an almost complete loss in image contrast and a total mixup of decay components.

The solution to the out-of-focus problem is confocal detection. The principle is illustrated in the figure below, left. A laser beam is focused into the sample by the microscope objective lens. Although this beam excites fluorescence through the entire depth of the sample the fluorescence is brightest in the focus of the laser beam. The light from the focus of the laser beam is collected back through the microscope lens, separated from the laser beam by a dichroic mirror, and focused into a pinhole in the upper focal plane of the microscope lens. Only light from the focal plane can pass the pinhole – light from other planes is not focused into the pinhole and, consequently, cannot pass it with any appreciable efficiency.

Scanning
The principle of confocal detection solves the problem of out-of-focus light but, taken by itself, does not provide an image of the sample. To obtain an image of the an object in the focal plane confocal detection has to be combined with scanning. Scanning can be achieved by different techniques, such as moving the sample, moving the objective lens, or deflecting the laser beam and the detection beam by fast moving mirrors. The third option is the only one which is applicable to imaging of biological samples. It is fast, it does not exert mechanical forces to the sample, and it can be used for any object that can be placed under the microscope. Confocal detection in combination with beam scanning is illustrated in the figure below, left and right. The laser beam is deflected by a pair of fast-moving galvanometer mirrors. The scan lens projects the beam down towards the microscope lens. As the mirrors are moving, the beam angle in the plane of the microscope lens changes, and so does the position of the laser focus in the sample plane. The fluorescence goes back through the same beam path. Being reflected at the galvanometer mirrors, it forms a stationary beam of light coincident with the laser beam, see figure below, left. The fluorescence is separated from the laser by a dichroic mirror, and projected into a pinhole in a plane conjugate with the focal plane in the sample. Please see figure below, right.

As explained above, confocal detection in combination with scanning solves the problem of out-of-focus light in a microscope. However, scanning does more than that. It also avoids that light scattered in the sample causes lateral crosstalk between the pixels. Surprisingly, this is rarely mentioned in the microscopy literature. Consider an imaging system that simultaneously illuminates the entire sample and detects an image from the focal plane by a camera. In all pixels of this image the camera will detect scattered light from all other pixels in the illuminated area, see below, left. A scanning system – scanning the sample by a focused laser beam and detecting light only from the excited spot – records only light from the current pixel (right). Even if a part of this light is scattered in the sample the imaging system will assign the scattered photons to the correct pixel.

The supression of laterally scattered light means that already scanning alone – without the help of confocal detection – massively improves the image quality in optically thick samples. An example is shown in the figure below. From left to right, it shows images of a pig skin sample recorded by an ordinary camera, by scanning and detection without a pinhole, and by confocal detection though a pinhole. The camera image (left) shows nothing from inside the sample, the scan image (middle) shows the internal structure, and the confocal image (right) shows the internal structure without out-of-focus haze.

Combination with TCSPC FLIM
The bh FLIM systems use a multi-dimensional TCSPC technique. That means, single photons of the light passing the pinhole are detected, the times of the photons within the excitation pulse period and the position of the laser beam in the moment of the photon detection are determined, and a photon distribution over these parameters is build up. The result is an array of pixels, each containing photon numbers in a large number of consecutive time channels. Please see figure below.

The TCSPC-FLIM recording process works at any scan rate, achieves a near-ideal photon efficiency, delivers a beautifully resolved decay function in every pixel, and reaches an extremely high time resolution. The signal-to-noise ratio depends only on the photon rate available from the sample and the total acquisition time. By adding additional dimensions to the photon distribution, multi-wavelength FLIM images, multi-excitation-wavelength images, combined fluorescence / phosphorescence image, and images of fast physiological effects in the sample can be accumulated. As all bh FLIM systems, the DCS-120 TCSPC system has two parallel TCSPC channels detecting in different wavelength intervals or under different angle of polarisation.
Please see The bh TCSPC Handbook and Handbook of the DCS-120 Confocal and Multiphoton FLIM Systems for details.

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