Department of Physics and Astronomy "Ettore Majorana", University of Catania, Catania, Italy.
Nanoscopy, CHT Erzelli, Istituto Italiano di Tecnologia, Genoa, Italy.
Microsc Res Tech. 2022 Sep;85(9):3207-3216. doi: 10.1002/jemt.24178. Epub 2022 Jun 10.
Confocal fluorescence microscopy is a well-established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal-to-noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to "virtually" perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super-resolution, and for this reason we call this method SPLIT-pinhole (SPLIT-PIN). We show with simulated data that the SPLIT-PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT-PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT-PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium. RESEARCH HIGHLIGHTS: We describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor-based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT-pinhole (SPLIT-PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope.
共聚焦荧光显微镜是一种成熟的成像技术，能够对生物样本进行薄光学切片。共聚焦显微镜中的光学切片主要由小孔的尺寸决定，小孔是放置在点探测器前面的一个小光圈。原则上，使用封闭的小孔进行成像可以提供最高程度的光学切片。实际上，在更小的小孔尺寸下，信号噪声比 (SNR) 会急剧下降，这使得使用明显小于 1 个艾里单位 (AU) 的小孔尺寸变得极具挑战性。在这里，我们介绍了一种简单的方法，可以在不显著降低 SNR 的情况下，以更小的小孔尺寸“虚拟”进行共焦成像。该方法基于以不同小孔孔径尺寸采集多个共焦图像的顺序采集，并基于相量分析进行图像处理。该方法的实现概念上类似于通过寿命调谐分离光子（SPLIT），这是一种利用相量分析实现超分辨率的技术，因此我们将这种方法称为 SPLIT-小孔（SPLIT-PIN）。我们用模拟数据表明，SPLIT-PIN 图像可以提供改进的光学切片（即，实际上更小的小孔尺寸），但相对于使用封闭小孔获得的图像具有更好的 SNR。例如，可以将在 2 和 1 AU 处采集的两个图像组合起来，以获得虚拟小孔尺寸为 0.2 AU 的 SPLIT-PIN 图像，但 SNR 更好。作为对生物成像应用的示例，我们表明 SPLIT-PIN 改善了体外肠上皮模型中顶膜的共焦成像。研究亮点：我们描述了一种增强任何共焦显微镜的光学切片能力的方法。该方法基于以不同小孔孔径尺寸顺序采集多个共焦图像。使用基于相量的通过寿命调谐（SPLIT）算法分析所得到的图像序列。SPLIT-小孔（SPLIT-PIN）方法生成具有改进的光学切片但保留 SNR 的图像。这是第一次将相量分析和 SPLIT 算法用于利用可调小孔尺寸中编码的空间信息，并改善共焦显微镜的光学切片。
