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

在过去的二十年里，利用激光光源的高峰值强度来激发用作活细胞标记的荧光染料的新显微技术出现了爆炸性的发展。这些基于非线性光学的方法为生物学家提供了比更传统的成像技术更多的优势。这些措施包括由于激发波长较长而对更深层次的组织进行成像，避免破坏短波长，以及总体上减少光漂白。然而，人们普遍认为，这些非线性显微镜方法必须使用聚焦到一个微小光点的激光，然后在样品周围扫描。这将信息的捕获速率限制在1帧/秒左右。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的变化经常被遗漏。已经证明了几种提高非线性显微镜成像速度的方法，但只有一种方法是商业上可用的(当该技术要用于生物研究实验室时，这是必不可少的)。这项技术包括将一束高强度的激光分割成高达的低强度“光束”，然后在标本周围扫描，但不幸的是，这可能会导致“拼接被子”效应，在图像中引入不想要的伪影，从而使解释和分析变得困难。为了提供非线性显微镜的优势，但在快速捕获速度下，我们建议利用传感器技术的创新，使用聚焦较少的激光光束，以照亮整个图像场。这种“宽视场”方法对生物学家来说是已知的，但采用的是线性(单光子)而不是非线性(双光子)的方法，因此是对终端用户熟悉的现有仪器的简单适应。与传统荧光显微镜相比，我们技术的关键区别将是光源，我们将把光源从发光二极管改为高峰值强度激光(我们实验室已经有了)。我们还将对显微镜进行微小的修改，并增加一个灵敏的科学相机探测器。我们的计算表明，在高达100帧/秒的捕获速度下，荧光的非线性激发是可能的。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类型的细胞，以研究快钙和慢钙信号事件。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得标本图像的方式，反过来，这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.