Multi-photon microscopy without scanning for faster than video-rate fluorescence imaging of live cells

在过去的二十年中，新的显微镜技术有了爆炸性的发展，这些技术利用激光源的高峰强度来激发用作活细胞中标记物的荧光染料。这些基于非线性光学的方法为生物学家提供了比传统成像技术更大的优势。这些包括由于更长的激发波长而对更深的组织进行成像，避免破坏性的短波长，以及光漂白的总体减少。然而，人们普遍认为，这些非线性显微镜方法必须使用激光聚焦到一个微小的斑点，然后扫描周围的标本。这将信息的捕获速率限制在大约1帧/秒。这是研究活细胞的方法的一个主要限制，因为细胞内生物化学的快速和重要变化经常被忽略。已经证明了一些提高非线性显微镜成像速度的方法，但只有一种是商业上可用的（当该技术用于生物研究实验室时，这是必不可少的）。该技术涉及将单个高强度激光束分裂成多达64个较低强度的“小光束”，然后围绕样品扫描，但不幸的是，这可能导致“拼凑被子”效应，将不必要的伪影引入图像中，并可能使解释和分析变得困难。为了提供非线性显微镜的优点，但捕获速度快，我们建议利用传感器技术中的创新，并使用聚焦不太好的激光束，这将照亮整个图像场。这种“宽场”方法为生物学家所知，但采用线性（单光子）而不是非线性（双光子）方法，因此是对终端用户熟悉的现有仪器的简单适应。我们的技术与传统荧光显微镜的关键区别在于光源，我们将从发光二极管改为高峰强度激光器（我们实验室已经有了）。我们还将对显微镜进行小的修改，并添加一个敏感的科学相机探测器。我们的计算表明，荧光的非线性激发是可能的捕获速度高达100帧/秒。我们将首先用非生物样本测试这项新技术，然后将该方法应用于两种不同的细胞类型，以研究快速和缓慢的钙信号传导事件。如果我们成功了，这项技术几乎肯定会改变细胞生物学家获取标本图像的方式，这反过来可能会对药理学和新药开发产生长期影响。

During the last twenty years, there has been an explosion in new microscopy techniques which exploit the high peak intensities from laser sources for excitation of fluorescent dyes used as markers in live cells. These methods, which are based on nonlinear optics, offer several advantages for the biologist over more traditional imaging techniques. These include imaging of deeper tissue thanks to longer excitation wavelengths, avoidance of damaging short-wavelengths, and an overall reduction in photo-bleaching. However, it has been generally accepted that these nonlinear microscopy methods must use a laser focused to a tiny spot which is then scanned around the specimen. This limits the capture rate of information to around 1 frame/second. This is a major limitation to the method for studying live cells, since rapid and important changes in the intra-cellular biochemistry are often missed.A few methods for increasing the imaging speed of nonlinear microscopy have been demonstrated, but only one is commercially available (which is essential when the technology is to be used in a biology research laboratory). This technique involves splitting a single high-intensity laser beam into up to 64 lower intensity 'beamlets' which are then scanned around the specimen, but this unfortunately can result in a 'patchwork quilt' effect which introduces unwanted artifacts into the images and can render interpretation and analysis difficult.To provide the advantages of nonlinear microscopy but at fast capture speeds, we propose to capitalize on innovations in sensor technology and use a less well-focused laser beam, which will illuminate the full image field. This 'wide-field' method is known to biologists, but in a linear (single-photon) rather than nonlinear (two-photon) approach, and therefore is a simple adaptation to existing instrumentation that is familiar to the end-user. The key difference in our technology over a conventional fluorescence microscope will be the light source, which we will change from a light-emitting diode to a high peak intensity laser (which we already have in our laboratory). We will also use small modifications to the microscope and add a sensitive scientific camera detector. Our calculations show that nonlinear excitation of fluorescence is possible at capture speeds of up to 100 frames/second. We will test this new technology with non-biological specimens initially, and then apply the method to two different cell types to study both fast and slow calcium signalling events. If we are successful, this technology is almost certain to change how cell biologists obtain images of their specimens which, in turn, will likely have a long-term impact on pharmacology and the development of new medicines.