High speed functional imaging with source localized multifocal two-photon microscopy.
High speed functional imaging with source localized multifocal two-photon microscopy.
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DOI：
10.1364/boe.9.003678
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发表时间：
2018-08-01
影响因子：
3.4
通讯作者：
Foust AJ
中科院分区：
文献类型：
--
作者：
Quicke P;Reynolds S;Neil M;Knöpfel T;Schultz SR;Foust AJ
多焦点双光子显微镜（MTPM）通过并行荧光激发来提高单焦点扫描的成像速度。然而，成像的荧光对串扰的敏感性严重降低了散射组织中的对比度。在这里，我们提出了一个源定位MTPM计划优化的高速功能性荧光成像散射哺乳动物脑组织。光栅线阵列的小光束激发荧光成像的互补金属氧化物半导体（CMOS）相机。我们通过对光栅图像进行时间过采样，使用结构化照明生成分组图像，并应用Richardson-Lucy反卷积重新分配散射光子来减轻散射引起的串扰。然后通过去卷积图像组以最大强度投影检索单个图像。该方法增加了散射脑组织中高达112 μm深度处的图像对比度，并减少了神经元钙成像过程中像素之间的功能串扰。在我们的实验条件下，源定位不影响密集标记组织的信噪比（SNR）。在稀疏标记的组织中，SNR在低帧速率下降低，在高于50 Hz的帧速率下没有影响。我们的非去扫描源定位MTPM系统可以实现高SNR，100 Hz的散射脑中荧光瞬态捕获，将MTPM的范围扩大到更快，更小的功能信号。
Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.