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An optically sectioning microscope designed for high speed high resolution random access multi-point scanning of single cells and microcircuits.

光学切片显微镜，专为单细胞和微电路的高速高分辨率随机访问多点扫描而设计。

基本信息

批准号：
BB/E00461X/1
负责人：
Nicholas Hartell
金额：
$ 41.21万
依托单位：
University of Leicester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE00461X%2F1
关键词：
optically sectioning microscope designed speed

项目摘要

Fluorescent molecules absorb light energy at one wavelength and emit light at a longer wavelength. This property is harnessed in fluorescence microscopy; because the excitation and emission wavelengths of light can be optically separated, structures labelled with a fluorophore can be selectively visualised. However, illumination reveals not only parts of the labelled specimen that are in focus, it reveals areas above or below the focal plane, obscuring detail and reducing resolution. Confocal microscopes circumvent this problem by optically sectioning the specimen by removing unwanted light originating above and below the focal plane. In standard confocal systems, a small point of light, usually supplied by a laser, is focussed onto the specimen and scanned point by point across the entire field of view. Emitted light is directed towards a pin hole positioned in the conjugate primary focal plane of the specimen. An image with a finite optical thickness is produced because light originating above or below the plane of focus misses the pinhole and is rejected. Standard scanning methods use two oscillating mirrors to move the laser in x and y directions. These systems can operate at relatively high frequencies that can generate two dimensional images at near video rates (~30 frames per second). However, because the laser is moved contiguously across the whole field of view, the amount of time that the laser dwells in any one position is extremely short. This limits the amount of light collected and so increases the signal to noise ratio. Thus speed is gained at the expense of image quality. Whereas these speeds may be useful for measuring fluorescence changes associated with some biological signals, measurements of action potentials, indicators of neuronal activity, with voltage sensitive dyes for example requires sampling rates of at least 1 KHz. This is clearly not feasible with standard scanning techniques. In this proposal, we will develop two different configurations of optically sectioning microscope that are each capable of very fast scanning (up to 25 KHz) but from a limited number of selected points of interest. This will be achieved by using non scanning methods that allow rapid laser positioning to any point in the field of view in under 20 micro seconds. Increased measurement speeds are achieved by taking measurements from a limited number of non-contiguous regions of a single cell or from distantly separated neurones that comprise a functioning network. This approach is termed random access scanning. The first configuration will be implemented with a programmable digital micromirror device (DMD). This represents an array of miniature mirrors that can be independently controlled at high speed. These will be used to define patterns of non-contiguous laser illumination on the specimen. Optical sectioning can be achieved because only light emitted from the focal plane of the specimen will be reflected back along the same optical path to the mirror and reflected onto a CCD camera. Mirrors will be switched rapidly to cover the whole field of view to generate an optically sectioned image. A small number of selected sites of interest of variable size can then be defined from the field of view and scanned at very high speeds, theoretically approaching 10-15 KHz. In the second configuration, speed will be increased further by positioning the laser beam with an acousto optical device (AOD). These devices use radio frequency sound waves to deflect light An AOD that is capable of deflecting light in x and y planes will be used to direct laser light to the specimen. Optical sectioning will be achieved with a DMD synchronised to the AOD positioned in the conjugate primary image plane. This system should produce maximum scan rates of 25 KHz. Successful implementation will lead towards the development of a low cost, 'solid state', high speed confocal imaging system capable of operating at very high speeds.

荧光分子在一个波长吸收光能，在更长的波长发射光。这一特性在荧光显微镜中得到了利用；因为光的激发和发射波长可以光学分离，所以用荧光团标记的结构可以有选择地可视化。然而，照明不仅显示了标记样本的焦点部分，还显示了焦平面上方或下方的区域，模糊了细节并降低了分辨率。共焦显微镜通过去除源自焦平面上方和下方的不需要的光来对标本进行光学切片，从而绕过了这个问题。在标准的共焦系统中，通常由激光提供的一小点光聚焦到样品上，并在整个视场中逐点扫描。发射的光指向位于样品的共轭主焦平面中的针孔。产生有限光学厚度的图像是因为在焦平面上方或下方发出的光错过针孔而被拒绝。标准的扫描方法使用两个振动镜来在x和y方向上移动激光。这些系统可以以相对较高的频率运行，可以以接近视频速率(约30帧/秒)的速度生成二维图像。然而，由于激光在整个视场中连续移动，激光在任何一个位置停留的时间都非常短。这限制了收集的光量，从而提高了信噪比。因此，速度是以牺牲图像质量为代价的。虽然这些速度可能有助于测量与某些生物信号相关的荧光变化，但测量动作电位、神经元活动的指示器，例如使用电压敏感染料，需要至少1 kHz的采样率。使用标准扫描技术，这显然是不可行的。在这个方案中，我们将开发两种不同配置的光学切片显微镜，每种都能够非常快地扫描(高达25 kHz)，但从有限数量的选定兴趣点进行扫描。这将通过使用非扫描方法来实现，这些方法允许在20微秒内快速定位到视野中的任何点。通过从单个细胞的有限数量的非连续区域或从组成正常网络的遥远分离的神经元进行测量，可以提高测量速度。这种方法被称为随机访问扫描。第一种配置将使用可编程数字微镜设备(DMD)来实现。这代表了一系列可以高速独立控制的微型反射镜。这些将被用来定义样品上的非连续激光照明模式。可以实现光学切片，因为只有从样品的焦平面发射的光将沿着相同的光路反射回反射镜，并反射到CCD相机上。反射镜将快速切换以覆盖整个视场，以生成光学切片图像。然后，可以从视场中定义少量不同大小的选定感兴趣位置，并以非常高的速度进行扫描，理论上接近10-15 kHz。在第二种配置中，通过使用声光器件(AOD)定位激光束，将进一步提高速度。这些设备使用射频声波来偏转光线，能够在x和y平面偏转光线的AOD将被用来将激光引导到样品上。光学切片将在DMD与位于共轭主像平面中的AOD同步的情况下实现。该系统应能产生25 kHz的最大扫描速率。成功的实施将导致开发一种低成本、‘固态’、能够以非常高的速度运行的高速共焦成像系统。