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

无需扫描的多光子显微镜对活细胞的荧光成像速度比视频速率更快

• 批准号: BB/M018903/1
• 负责人: Gail McConnell
• 金额: $ 13.9万
• 依托单位: University of Strathclyde
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM018903%2F1
• 关键词: Multi; photon; microscopy; without; scanning

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.

在过去的二十年中，新的显微镜技术出现了爆炸性的增长，这些技术利用激光源的高峰值强度来激发用作活细胞标记的荧光染料。与更传统的成像技术相比，这些基于非线性光学的方法为生物学家提供了多种优势。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微小点的激光，然后在样本周围进行扫描。这将信息捕获率限制在 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而重要的变化经常被错过。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成多达 64 个较低强度的“小束”，然后在样本周围进行扫描，但不幸的是，这可能会导致“拼凑被子”效应，从而在图像中引入不需要的伪影，并使解释和分析变得困难。为了提供非线性显微镜的优势，但以快速捕获速度，我们建议利用传感器技术的创新并使用 聚焦不太好的激光束，将照亮整个图像场。这种“宽视场”方法为生物学家所熟知，但采用的是线性（单光子）而不是非线性（双光子）方法，因此是对最终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主要区别在于光源，我们将光源从发光二极管改为高峰值强度激光器（我们实验室中已经有了）。我们还将对显微镜进行一些小的修改，并添加一个灵敏的科学相机探测器。我们的计算表明，荧光的非线性激发在高达 100 帧/秒的捕获速度下是可能的。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的细胞类型，以研究快速和慢速的钙信号传导事件。如果我们成功，这项技术几乎肯定会改变细胞生物学家获取样本图像的方式，这反过来又可能对药理学和新药的开发产生长期影响。

项目成果

3D mapping of intensity field about the focus of a micrometer-scale parabolic mirror.

• DOI: 10.1364/oe.23.002375
• 发表时间: 2014-11
• 期刊: Optics express
• 影响因子: 3.8
• 作者: Alison McDonald;G. McConnell;D. Cox;E. Riis;P. Griffin

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

• DOI: 10.1371/journal.pone.0147115
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Amor R;McDonald A;Trägårdh J;Robb G;Wilson L;Abdul Rahman NZ;Dempster J;Amos WB;Bushell TJ;McConnell G
• 通讯作者: McConnell G