Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The resolution of two-photon image scanning microscopy with Bessel beam illumination is a factor 2.13 better than in standard two-photon fluorescence. Axial resolution and optical sectioning in two-photon or three-photon fluorescence are also improved by using the image scanning modality.

中文翻译：

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具有多光子激发或贝塞尔光束照明的图像扫描显微镜

图像扫描显微镜是一种共聚焦显微镜技术，其中共聚焦针孔被检测器阵列取代，并且图像通过像素重新分配最直接地重建。在荧光模式下，检测器阵列会收集大部分荧光，因此，与具有小针孔的共聚焦显微镜相比，信噪比得到了极大的改善，而与常规荧光显微镜相比，其分辨率得到了提高。在这里，我们考虑两种情况，其中照明和检测点扩展功能不相同：使用贝塞尔光束照明和多光子显微镜。先前已经表明，对于具有大阵列的图像扫描显微镜中的贝塞尔光束照明，成像性能下降。另一方面，还已知通过贝塞尔光束照射可以提高共聚焦显微镜的分辨率。在这里，我们用贝塞尔光束照明和一小阵列对图像扫描显微镜进行分析，结果表明与传统的荧光显微镜相比，横向分辨率（点扩展函数的宽度）提高了1.78倍。我们还检查了图像扫描显微镜在两光子或三光子荧光中的行为，以及在使用贝塞尔光束照明时对于两光子激发的行为。图像扫描显微镜的光学切片效果与多光子显微镜的结合可减少样品表面的背景，从而增加穿透深度。对于两个Airy单位的探测器阵列大小，双光子图像扫描显微镜的分辨率是系数1。85更好，并且点扩散函数的峰比非共聚焦双光子荧光峰高2.84倍。三光子图像扫描显微镜的分辨率提高了2.10倍，点扩散函数的峰比非共聚焦三光子荧光峰高了3.77倍。使用贝塞尔光束照明的双光子图像扫描显微镜的分辨率比标准双光子荧光的分辨率高2.13倍。通过使用图像扫描方式，还可以改善两光子或三光子荧光的轴向分辨率和光学截面。比非共聚焦三光子荧光高77倍。使用贝塞尔光束照明的双光子图像扫描显微镜的分辨率比标准双光子荧光的分辨率高2.13倍。通过使用图像扫描方式，还可以改善两光子或三光子荧光的轴向分辨率和光学截面。比非共聚焦三光子荧光高77倍。使用贝塞尔光束照明的双光子图像扫描显微镜的分辨率比标准双光子荧光的分辨率高2.13倍。通过使用图像扫描方式，还可以改善两光子或三光子荧光的轴向分辨率和光学截面。