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

荧光分子吸收一种波长的光能，发射更长波长的光。这种特性在荧光显微镜中被利用;因为光的激发和发射波长可以被光学分离，所以用荧光团标记的结构可以被选择性地可视化。然而，照明不仅揭示了标记的样本的部分焦点，它揭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。共焦显微镜规避这个问题，通过光学切片的标本，消除不必要的光起源以上和以下的焦平面。在标准的共焦系统中，通常由激光提供的一个小光点聚焦到样本上，并在整个视场中逐点扫描。发射的光被引导朝向位于样本的共轭主焦平面中的针孔。产生具有有限光学厚度的图像是因为源自焦平面上方或下方的光错过针孔并被拒绝。标准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高的频率下工作，可以以接近视频速率（每秒约30帧）生成二维图像。然而，由于激光器在整个视场中连续移动，因此激光器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，因此增加了信噪比。因此，速度是以牺牲图像质量为代价的。尽管这些速度可用于测量与某些生物信号相关的荧光变化，但使用电压敏感染料测量动作电位（神经元活动的指标）例如需要至少1 KHz的采样率。这显然是不可行的标准扫描技术。在这个建议中，我们将开发两种不同配置的光学切片显微镜，每种都能够非常快速地扫描（高达25 KHz），但从有限数量的选定的兴趣点。这将通过使用非扫描方法来实现，该方法允许在20微秒内将激光快速定位到视场中的任何点。通过从单个细胞的有限数量的非连续区域或从构成功能网络的远距离分离的神经元进行测量来实现增加的测量速度。这种方法被称为随机接入扫描。第一种配置将采用可编程数字EEPROM器件（DMD）实现。这代表了一个可以高速独立控制的微型反射镜阵列。这些将用于定义样本上的非连续激光照明模式。可以实现光学切片，因为只有从样本的焦平面发出的光将被反射回沿着相同的光路反射到镜子上，并反射到CCD相机上。反射镜将迅速切换，以覆盖整个视场，从而生成光学切片图像。然后，可以从视场中定义少量可变尺寸的所选感兴趣部位，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，能够在x和y平面偏转光的AOD将用于将激光引导到样品。光学切片将使用DMD实现，DMD与位于共轭主图像平面中的AOD同步。该系统应产生25 KHz的最大扫描速率。成功的实施将导致发展的低成本，“固态”，高速共焦成像系统能够在非常高的速度下运行。

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.