Quicke Peter, Reynolds Stephanie, Neil Mark, Knöpfel Thomas, Schultz Simon R, Foust Amanda J
Department of Bioengineering, Imperial College London, SW7 2AZ, UK.
Centre for Neurotechnology, Imperial College London, SW7 2AZ, UK.
Biomed Opt Express. 2018 Jul 12;9(8):3678-3693. doi: 10.1364/BOE.9.003678. eCollection 2018 Aug 1.
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.
多焦点双光子显微镜（MTPM）通过并行荧光激发提高了成像速度，相较于单焦点扫描。然而，成像荧光对串扰的敏感性严重降低了散射组织中的对比度。在此，我们提出一种源定位的MTPM方案，该方案针对散射哺乳动物脑组织中的高速功能荧光成像进行了优化。一个光栅化的子束线阵列激发用互补金属氧化物半导体（CMOS）相机成像的荧光。我们通过对光栅化图像进行时间上的过采样、利用结构化照明生成分组图像以及应用理查森- Lucy反卷积来重新分配散射光子，从而减轻散射引起的串扰。然后通过对反卷积后的图像组进行最大强度投影来检索单幅图像。该方法提高了散射脑组织中深度达112μm处的图像对比度，并在神经元钙成像期间降低了像素间的功能串扰。在我们的实验条件下，源定位在密集标记的组织中不影响信噪比（SNR）。在稀疏标记的组织中，低帧率下SNR会降低，而在高于50Hz的帧率下则无影响。我们的非扫描源定位MTPM系统能够以高SNR、100Hz的频率捕获散射脑内的荧光瞬变，将MTPM的应用范围扩展到更快、更小的功能信号。
