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

许多生物过程涉及在广泛的空间尺度上的相互作用。例子包括通过许多神经元的相互作用产生大脑活动，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。在心脏病领域，这些考虑尤为重要。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，并将其传递给邻近的细胞，从而形成穿越整个心脏的电波前（“宏观”行为）。少数细胞的不规则激活，通常是由更小的离子处理蛋白（它们是“亚微观”大小，不能在显微镜下看到）的故障引起的，可能导致器官水平功能的破坏。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能表现为心律紊乱，这是发达国家丧失行为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施加的限制。细胞活动非常快（需要比人眼能检测到的帧速率快50倍），而可用的光信号相当微弱（人眼无法检测到，需要超灵敏的探测器）。此外，使用荧光探针测量细胞活动，荧光探针将相关信息解码为大背景信号上的小“涟漪”。这就需要使用将光强度分成数千个灰度级的探测器系统（这样就可以分辨出绝对强度的微小差异）。这在很大程度上是通过低空间分辨率（低至16 × 16像素）的检测器实现的。虽然低分辨率检测器可以用于许多实验问题，但要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，需要显着更高的分辨率。这一建议将重点从单独改进检测器转移到改变生物样品的照明方式和荧光灯的收集方式。通过精确地改变激发光的位置、时间、强度和波长，在逐像素的基础上，可以显着提高系统的性能。新成像技术的核心是一个数字镜像装置（DMD），通常用于电影放映系统（DMD包含一百万个微小的镜子，其投影角度每秒可以操纵数千次）。DMD可以精确控制到达生物样品的光强度，以及探测器上图像的位置。通过交替成像小（细胞）和大视场（大组织区域），可以高速获得多尺度图像。该技术与一种新型振荡照明源相结合，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，提出的成像技术将使研究几个细胞的活动如何影响整体行为成为可能，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，在实验上无法达到的广泛问题。

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.