Massively parallel interferometric confocal microscopy using degenerate lasers

描述（由申请人提供）：共聚焦显微镜使用几何原理生成散射生物组织的横截面图像。传统的反射共焦显微镜使用串行像素采集，从而限制了采集速度。像素采集的并行化将大大提高成像速度，并使新的生理学研究成为可能。例如，大规模并行反射共聚焦显微镜将是非常宝贵的纤毛驱动的粘液生理学在呼吸系统中的新兴领域。事实上，在假设驱动的睫状体生物学方面，在~1 μ m尺度的高速（0.1至1 kHz帧速率）成像方法中存在关键的方法学差距。传统的共聚焦显微镜在样品照明和光检测中使用物理针孔，通过简单的几何原理生成横截面图像。虽然物理针孔的使用可以部分并行化，但使用物理针孔不可能具有完整的无扫描并行化。然而，并行干涉共焦显微镜允许整个视场成像而无需扫描。在干涉共焦显微镜中，产生虚拟干涉针孔，并且相互不相干的空间模式充当独立的和可并行的共焦成像通道。然而，缺乏具有低空间相干性（许多独立的空间模式）和每个空间模式的高亮度的源是大规模并行干涉共焦显微镜的关键障碍。传统激光器 显示出高的空间相干性，而低空间相干性的光源，例如热源和发光二极管（LED）不能提供必要的亮度。最近，我们已经表明，简并Nd：YAG激光器可以支持多达105个相互不相干的激射模式（独立的成像通道）。因此，使用一个专门设计的简并Nd：YAG激光器，我们将建立一个大规模并行干涉共焦成像系统，具有100 Hz的帧速率成像在~1 μ m的分辨率制度。我们期望操作原理允许未来缩放到>kHz帧速率范围。我们将使用倍频的532 nm光在~104（100 x 100）个平行、独立的成像通道中进行干涉反射共焦成像。选择~104个成像通道作为设计规格，因为内窥镜检查中使用的几种常用成像光纤束具有~104个成像芯。作为一个初步的演示，我们将图像纤毛生理学在非洲爪蟾（青蛙）胚胎。像呼吸上皮表面一样，爪蟾胚胎的表皮（皮肤）也有纤毛，并产生定向的流体流动。我们也会对单个纤毛的运动进行成像 使用干涉测量使能的多普勒流动成像来量化纤毛驱动的流体流动。我们的设计驱动的研究将支持呼吸纤毛生理学新诊断的未来发展。我们的共聚焦显微镜的退化激光器的发展支持未来的研究退化激光器的其他种类的医学成像（例如，光学相干断层扫描，HiLo结构照明，全息显微镜）。

DESCRIPTION (provided by applicant): Confocal microscopy uses geometric principles to generate cross-sectional images of scattering biological tissue. Traditional reflectance confocal microscopes use serial pixel acquisition, thereby limiting acquisition speeds. Parallelization of pixel acquisition would dramatically increase imaging speeds and enable the study of new kinds of physiology. For example, massively parallelized reflectance confocal microscopy would be invaluable in the emerging field of cilia-driven mucus physiology in the respiratory system. Indeed, in terms of hypothesis-driven ciliary biology, there is a critical methodological gap in high-speed (0.1 to 1 kHz frame rates) imaging methods at the ~1 um scale. Traditional confocal microscopy uses physical pinholes in sample illumination and in photo detection to generate cross-sectional images by straightforward geometric principles. While the use of physical pinholes can be partially parallelized, it is impossible to have complete, scan-free parallelizatio using physical pinholes. However, parallel interferometric confocal microscopy allows an entire field-of-view to be imaged without scanning. In interferometric confocal microscopy, virtual interferometric pinholes are generated, and mutually incoherent spatial modes act as independent and parallelizable confocal imaging channels. However, the lack of sources with low spatial coherence (many independent spatial modes) and high brightness per spatial mode is a critical barrier to massively parallel interferometric confocal microscopy. Traditional lasers exhibit high spatial coherence, while low spatial coherence sources such as thermal sources and light-emitting diodes (LEDs) do not provide the necessary brightness. Recently, we have shown that degenerate Nd: YAG lasers can support as many as ~105 mutually incoherent lasing modes (independent imaging channels). Therefore, using a specifically-designed degenerate Nd: YAG laser, we will build a massively parallel interferometric confocal imaging system with a 100 Hz frame rate for imaging in the ~1 um resolution regime. We expect the principles of operation to allow future scaling into the >kHz frame rate regime. We will use frequency-doubled 532 nm light for interferometric reflectance confocal imaging in ~104 (100 x 100) parallel, independent imaging channels. ~104 imaging channels is chosen as a design specification because several commonly-used imaging fiber bundles used in endoscopy have ~104 imaging cores. As an initial demonstration, we will image ciliary physiology in Xenopus (frog) embryos. Like respiratory epithelial surfaces, the epidermis (skin) of Xenopus embryos is ciliated and generates directional fluid flow. We will image the motion of individual cilia as well quantify cilia-driven fluid flow using interferometry-enabled Doppler flow imaging. Our design-driven research will support the future development of novel diagnostics in respiratory ciliary physiology. Our development of a degenerate laser for confocal microscopy supports future research in degenerate lasers for other kinds of medical imaging (e.g. optical coherence tomography, HiLo structured illumination, holographic microscopy).