A novel multi-scale multiparametric technology for high speed fluorescence imaging of excitable tissues

许多生物学过程涉及在广泛的空间尺度上的相互作用。例子包括许多神经元相互作用产生的大脑活动，或单个肌细胞在产生心跳中的作用。细胞活动取决于多个参数的复杂相互作用，我们希望同时监测这些参数，以了解它们在系统行为中的作用。心脏病是这些考虑特别重要的一个领域。心脏细胞（在显微镜下几乎可见，因此被称为“微观”尺寸）可以产生电活动，该电活动被传递到相邻细胞，导致形成穿过整个心脏的电波阵面（“宏观”行为）。一些细胞的不规则激活，通常是由更小的离子处理蛋白质（它们具有“亚显微镜”大小，本身无法通过显微镜观察到）的故障引起的，可能会导致器官水平功能的中断。反过来，宏观活动的变化影响单个细胞和离子通道，使（亚）微观和宏观事件之间的相互作用复杂化。这种相互作用中的任何不匹配都可以表现为心律紊乱，这是发达国家失能和死亡的主要原因。鉴于相互作用的复杂性，对潜在机制的研究仍然很困难。目前，用于测量可兴奋组织（如心脏）中活动模式的技术受到组织施加的约束的限制。细胞活动非常快（需要比人眼检测到的帧速率快50倍的帧速率），可用的光信号相当微弱（人眼无法检测到，需要超灵敏的检测器）。此外，使用荧光探针测量细胞活性，荧光探针将相关信息解码为大背景信号之上的小“涟漪”。这需要使用将光强度划分为数千个灰度级的检测器系统（以便可以分辨绝对强度的非常小的差异）。这在很大程度上是使用具有低空间分辨率（低至16x16像素）的探测器来实现的。虽然低分辨率探测器可以用于许多实验问题，但需要显著更高的分辨率来研究同一样品中微观（单个细胞）和宏观（整个心脏）活动之间的关系。该建议将焦点从单独改进检测器转移到改变生物样品被照射的方式以及荧光被收集的方式。通过逐个像素地精确改变激发光定位、定时、强度和波长，可以显著提高系统的性能。新成像技术的核心是数字反射镜设备（DMD），通常用于电影放映系统（DMD包含一百万个微小的反射镜，其投影角度每秒可被操纵数千次）。DMD允许精确控制到达生物样品的光强度以及检测器上的图像位置。通过在成像小视场（细胞）和大视场（大组织区域）之间交替，可以高速获得多尺度图像。该技术与一种新型的振荡照明源相结合，该光源允许从同一样品中的不同荧光探针进行顺序捕获，从而允许多参数测量。总的来说，拟议的成像技术将使人们有可能研究几个细胞的活动如何有助于全球行为，反之亦然，同时观察几个可测量的变量。新的成像系统可以应用于生物医学研究内外的研究，针对迄今为止实验无法解决的各种问题。

Many biological processes involve interactions over a wide range of space scales. Examples include the generation of brain activity by interactions of many neurons, or the role of individual myocytes in generating the heart beat. Cell activity depends on complex interactions of multiple parameters, which we would like to monitor simultaneously, to understand their role in system's behaviour. Heart disease is an area where these considerations are particularly critical. A heart cell (just about visible in a microscope and, hence, said to be of 'microscopic' dimensions) can generate electrical activity, which is passed on to neighbouring cells, resulting in the formation of an electrical wave front that traverses the whole heart ('macroscopic' behaviour). Irregular activation of a few cells, often caused by malfunctions in the even smaller ion-handling proteins (they are of 'sub-microscopic' size and can not themselves be seen by microscopy), may cause disruptions of organ level function. In turn, changes in the macroscopic activity affect individual cells and ion channels, complicating the interaction between (sub-)microscopic and macroscopic events. Any mismatch in this interaction can manifest itself as a heart rhythm disturbance, which is the major cause of incapacitation and death in the developed world. Given the complexity of interactions, the study of underlying mechanisms has remained difficult. At the present time, technologies for measuring patterns of activity in excitable tissues (like the heart) are limited by tissue-imposed constraints. Cell activity is very fast (requiring frame rates that are fifty times faster than what the human eye can detect), and the usable optical signal is rather faint (undetectable by the human eye, and requiring super-sensitive detectors). Further, cell activity is measured using fluorescent probes, which decode relevant information as a small 'ripple' on top of a large background signal. This necessitates the use of detector systems that divide light intensity into thousands of grey levels (so that very small differences in absolute intensity can be resolved). This is largely achieved using detectors with a low spatial resolution (as low as 16x16 pixels). Although low resolution detectors can be used for many experimental problems, a significantly higher resolution would be needed to study the relationship between microscopic (individual cell) and macroscopic (whole heart) activity in one and the same sample. This proposal shifts the focus from improving the detector alone, to changing the ways in which the biological sample is illuminated, and in which fluorescent light is collected. By precisely varying excitation light localization, timing, intensity and wavelength, on a pixel by pixel basis, it is possible to dramatically increase the performance of system. The core of the new imaging technology is a digital mirror device (DMD), commonly used in movie projection systems (DMDs contain a million tiny mirrors whose projection angle can be manipulated a thousands of times per second). The DMD allows precise control of the light intensity that reaches the biological sample, as well as the location of image on the detector. By alternating between imaging small (cells) and large fields of view (large tissue areas), a multi-scale image can be obtained at high speed. This technology is coupled to a novel oscillating illumination source that allows sequential capture from different fluorescence probes in the same sample, allowing multi-parameter measurements. Taken together, the proposed imaging technology will make it possible to investigate how the activity of a few cells contributes to global behaviour, and vice versa, observing several measurable variables simultaneously. The new imaging system can be applied to studies, both within and outside bio-medical research, targeting a wide range of problems that, until now, were experimentally inaccessible.