Super-resolution laser scanning microscopy through spatiotemporal modulation.

Super-resolution laser scanning microscopy through spatiotemporal modulation.
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DOI:
10.1021/nl902087d
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发表时间:
2009-11
期刊:
影响因子:
10.8
通讯作者:
Lichtman JW
Lichtman JW
中科院分区:
材料科学1区
文献类型:
--
作者:
Lu J;Min W;Conchello JA;Xie XS;Lichtman JW

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超分辨率光学显微镜在许多领域引起了研究人员的极大兴趣,特别是在生物学中,其中物理结构和分子过程的尺度低于光的分辨率的衍射极限。作为一种新兴的技术,结构照明显微镜可以通过与宽场结构照明图案的空间频率混合将不可分辨的空间频率移动到显微镜的通带中,从而使分辨率加倍。然而,这样的宽场方案通常只能成像光学薄的样品,并且与多光子过程(诸如双光子荧光)不兼容,所述多光子过程需要用聚焦激光束进行点扫描。在这里,我们提出了两个新的激光扫描显微镜的超分辨率计划,通过推广的空间非均匀成像系统的概念。一种方案,扫描图案化照明(SPIN)显微镜,采用调制的激发结合时间累积成像的非去扫描阵列检测器。另一种方案,扫描图案化检测(SPADE)显微镜,利用检测调制连同空间累积成像,在这种情况下,由一个nondesscan单元素检测器。当与多光子激发相结合时,这两种方案都可以对厚样品进行三维光学切片成像,并大大提高分辨率。
Super-resolution optical microscopy has attracted great interest among researchers in many fields, especially in biology where the scale of physical structures and molecular processes fall below the diffraction limit of resolution for light. As one of the emerging techniques, structured illumination microscopy can double the resolution by shifting unresolvable spatial frequencies into the pass-band of the microscope through spatial frequency mixing with a wide-field structured illumination pattern. However, such a wide-field scheme typically can only image optically thin samples and is incompatible with multiphoton processes such as two-photon fluorescence, which require point scanning with a focused laser beam. Here, we propose two new super-resolution schemes for laser scanning microscopy by generalizing the concept of a spatially nonuniform imaging system. One scheme, scanning patterned illumination (SPIN) microscopy, employs modulation of the excitation combined with temporally cumulative imaging by a nondescanned array detector. The other scheme, scanning patterned detection (SPADE) microscopy, utilizes detection modulation together with spatially cumulative imaging, in this case by a nondescanned single-element detector. When combined with multiphoton excitation, both schemes can image thick samples with three-dimensional optical sectioning and much improved resolution.
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