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

许多生物过程涉及大范围空间尺度上的相互作用。例如，通过许多神经元的相互作用来产生大脑活动，或者单个肌细胞在产生心跳中的作用。细胞活动依赖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。心脏病是其中一个特别重要的考虑因素。心脏细胞(在显微镜下几乎可以看到，因此被称为“微观”尺寸)可以产生电活动，并将其传递给邻近的细胞，导致形成横跨整个心脏的电波阵面(“宏观”行为)。少数细胞的不规则激活通常是由于更小的离子处理蛋白(它们是亚微观大小，本身不能用显微镜看到)中的故障引起的，可能会导致器官水平的功能中断。反过来，宏观活动的变化会影响单个细胞和离子通道，使(次)微观事件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能表现为心律紊乱，这是发达国家丧失能力和死亡的主要原因。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，测量可兴奋组织(如心脏)活动模式的技术受到组织强加限制的限制。细胞活动非常快(需要比人眼能检测到的帧速度快50倍的帧速率)，而可用的光学信号相当微弱(人眼无法检测到，需要超灵敏的探测器)。此外，细胞活动是用荧光探针测量的，荧光探针将相关信息解码为大背景信号上的一个小“涟漪”。这就需要使用探测器系统，将光强度分成数千个灰度级(这样就可以解决绝对强度的极小差异)。这在很大程度上是使用低空间分辨率(低至16x16像素)的探测器实现的。虽然低分辨率探测器可以用于许多实验问题，但需要更高的分辨率来研究同一样本中微观(单个细胞)和宏观(整个心脏)活动之间的关系。这项提议将重点从单独改进探测器转移到改变生物样本的照明方式和荧光收集方式。通过在逐个像素的基础上精确地改变激发光的局部化、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镜子设备(DMD)，通常用于电影放映系统(DMD包含100万个微小的镜子，其投影角度可以每秒操作数千次)。DMD允许精确控制到达生物样本的光强度，以及探测器上图像的位置。通过交替成像小视场(细胞)和大视场(大组织区域)，可以高速获得多尺度图像。这项技术与一种新型的振荡照明源相耦合，该光源允许从同一样品中的不同荧光探针顺序捕获，从而允许多参数测量。综上所述，拟议的成像技术将使研究少数细胞的活动如何影响全球行为成为可能，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，针对到目前为止在实验上无法解决的广泛问题。

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.