Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two‐photon fluorescence microscopy

H. Kano, H. M. Voort, M. Schrader, Geert M. P. van Kempen, S. Hell
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引用次数: 35

Abstract

High-quantum-efficiency photodetection, millisecond pixel dwell time stage scanning and image restoration by maximum-likelihood estimation are synergetically combined and shown to improve the resolution of two-photon excitation microscopy 2–4 fold in all directions. Measurements of the two-photon excitation point-spread function (PSF) of a 1.4 aperture oil immersion lens are carried out by imaging fluorescence beads with a diameter of one seventh of the excitation wavelength (830 nm) and subsequent deconvolution with the bead object function. The proposed method of resolution increase is applied to beads as well as to rhodamine labelled actin fibres in mouse fibroblast cells. As the resolution improvement is not based on the non-linear effect of two-photon excitation, the results imply a comparable resolution increase in single-photon excitation confocal microscopy. In the fibroblasts, we established a three-fold improvement in axial resolution, namely from 840 nm before, to 280 nm after restoration (full-width at half-maximum). Actin fibres with axial distances of 850 nm, otherwise difficult to discern, are fully separated. In the lateral direction, images of fluorescence beads of about 110 nm diameter are restored to the real dimensions of the beads with an accuracy of better than one pixel (41 nm).
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雪崩光电二极管检测与对象扫描和图像恢复提供2-4倍的分辨率增加在双光子荧光显微镜
高量子效率的光探测、毫秒像素停留时间阶段扫描和最大似然估计图像恢复协同结合,在各个方向上都将双光子激发显微镜的分辨率提高了2-4倍。采用直径为激发波长(830nm)的七分之一的荧光珠成像,并与珠目标函数反褶积,测量了1.4孔径油浸透镜的双光子激发点扩展函数(PSF)。提出的提高分辨率的方法适用于珠以及罗丹明标记的肌动蛋白纤维在小鼠成纤维细胞。由于分辨率的提高不是基于双光子激发的非线性效应,结果表明单光子激发共聚焦显微镜的分辨率提高相当。在成纤维细胞中,我们发现轴向分辨率提高了三倍,即从修复前的840 nm提高到修复后的280 nm(全宽半最大值)。轴向距离为850纳米的肌动蛋白纤维完全分离,否则很难识别。在横向上,将直径约为110 nm的荧光珠的图像恢复到珠的真实尺寸,精度优于1像素(41 nm)。
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