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

荧光分子吸收一个波长的光能，发射波长较长的光。这种性质是利用荧光显微镜；由于光的激发和发射波长可以光学分离，用荧光团标记的结构可以选择性地可视化。然而，照明不仅会显示出被标记标本聚焦的部分，还会显示出焦平面以上或以下的区域，从而模糊细节并降低分辨率。共聚焦显微镜通过去除源自焦平面上下的不需要的光来对标本进行光学切片，从而避免了这个问题。在标准的共聚焦系统中，通常由激光提供的一个小光点被聚焦到样品上，并在整个视场中逐点扫描。发射的光被指向位于试样的共轭主焦平面上的针孔。产生有限光学厚度的图像是因为来自聚焦平面上方或下方的光错过了针孔而被拒绝。标准的扫描方法使用两个振荡镜在x和y方向上移动激光。这些系统可以在相对较高的频率下工作，可以以接近视频速率（~30帧每秒）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。这限制了光的收集量，从而增加了信噪比。因此，速度是以牺牲图像质量为代价获得的。虽然这些速度可能对测量与某些生物信号相关的荧光变化有用，但测量动作电位（神经元活动指标），例如使用电压敏感染料，需要至少1 KHz的采样率。这显然是不可行的标准扫描技术。在本提案中，我们将开发两种不同配置的光学切片显微镜，每种显微镜都能够非常快速扫描（高达25 KHz），但从有限数量的选定感兴趣点。这将通过使用非扫描方法来实现，该方法允许在20微秒内快速激光定位到视场中的任何一点。提高测量速度是通过测量单个细胞的有限数量的非连续区域或遥远的神经元组成的功能网络来实现的。这种方法称为随机存取扫描。第一种配置将使用可编程数字微镜设备（DMD）实现。这代表了一组可以在高速下独立控制的微型镜子。这些将用于定义试样上非连续激光照射的图案。光学切片可以实现，因为只有从样品的焦平面发出的光将沿着相同的光路反射到镜子和反射到CCD相机上。镜子将迅速切换以覆盖整个视野，从而生成光学切片图像。然后可以从视场中定义少量可变大小的选定感兴趣的地点，并以非常高的速度扫描，理论上接近10-15 KHz。在第二种配置中，通过用声光装置（AOD）定位激光束，速度将进一步提高。这些装置使用射频声波来偏转光，一个能够偏转x和y平面上的光的AOD将用于将激光引导到样品上。光学切片将实现与定位在共轭主像平面上的AOD同步的DMD。该系统应产生25千赫的最大扫描速率。成功的实施将导致低成本的发展，“固态”，高速共聚焦成像系统能够以非常高的速度运行。

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.