Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images.

This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

基于荧光的显微镜作为生物医学研究的标准工具之一，越来越多地受益于超分辨率方法，它提供了增强的空间分辨率，可以洞察新的生物过程。使用这些方法的一个典型缺点是需要新的、复杂的光学设置。当使用双光子荧光激发时，这一点变得更加重要，它提供了深层组织成像和出色的 z 切片。我们表明，使用开源图像重建软件可以很容易地利用双光子激光扫描显微镜中条纹照明图案的生成来实现光学超分辨率和对比度增强。这种方法的特殊吸引力在于，即使在商业双光子激光扫描显微镜的情况下，也不需要进行光学机械修改来实现这种模式。使用自定义编写的宏修改扫描软件以解决扫描镜，并结合电光调制器的快速强度切换，足以在 sCMOS 相机上完成双光子条纹照明图案的采集。我们通过将不同的最近发布的图像分辨率评估程序应用于重建的滤波宽场和超分辨率图像来演示和分析由此产生的分辨率改进。使用自定义编写的宏修改扫描软件以解决扫描镜，并结合电光调制器的快速强度切换，足以在 sCMOS 相机上完成双光子条纹照明图案的采集。我们通过将不同的最近发布的图像分辨率评估程序应用于重建的滤波宽场和超分辨率图像来演示和分析由此产生的分辨率改进。使用自定义编写的宏修改扫描软件以解决扫描镜，并结合电光调制器的快速强度切换，足以在 sCMOS 相机上完成双光子条纹照明图案的采集。我们通过将不同的最近发布的图像分辨率评估程序应用于重建的滤波宽场和超分辨率图像来演示和分析由此产生的分辨率改进。

本文是 Theo Murphy 会议问题“超分辨率结构照明显微镜（第 1 部分）”的一部分。