Flewellen James L, Zaid Irwin M, Berry Richard M
Immune Receptor Activation Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.
Department of Physics, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, United Kingdom.
Rev Sci Instrum. 2019 Feb;90(2):023705. doi: 10.1063/1.5066556.
We present a transmission-mode digital holographic microscope that can switch easily between three different imaging modes: inline, dark field off-axis, and bright field off-axis. Our instrument can be used: to track through time in three dimensions microscopic dielectric objects, such as motile micro-organisms; localize brightly scattering nanoparticles, which cannot be seen under conventional bright field illumination; and recover topographic information and measure the refractive index and dry mass of samples via quantitative phase recovery. Holograms are captured on a digital camera capable of high-speed video recording of up to 2000 frames per second. The inline mode of operation can be easily configurable to a large range of magnifications. We demonstrate the efficacy of the inline mode in tracking motile bacteria in three dimensions in a 160 μm × 160 μm × 100 μm volume at 45× magnification. Through the use of a novel physical mask in a conjugate Fourier plane in the imaging path, we use our microscope for high magnification, dark field off-axis holography, demonstrated by localizing 100 nm gold nanoparticles at 225× magnification up to at least 16 μm from the imaging plane. Finally, the bright field off-axis mode facilitates quantitative phase microscopy, which we employ to measure the refractive index of a standard resolution test target and to measure the dry mass of human erythrocytes.
我们展示了一种透射模式数字全息显微镜，它可以在三种不同的成像模式之间轻松切换：同轴、暗场离轴和亮场离轴。我们的仪器可用于：在三维空间中随时间追踪微观介电物体，如活动的微生物；定位在传统亮场照明下不可见的强散射纳米颗粒；以及通过定量相位恢复来恢复样品的地形信息并测量其折射率和干质量。全息图由一台能够进行高达每秒2000帧高速视频记录的数码相机捕获。同轴操作模式可以轻松配置为大范围的放大倍数。我们展示了同轴模式在45倍放大率下在160μm×160μm×100μm体积内三维追踪活动细菌的有效性。通过在成像路径的共轭傅里叶平面中使用新型物理掩模，我们将显微镜用于高放大倍数的暗场离轴全息术，通过在离成像平面至少16μm处定位100nm金纳米颗粒，放大倍数为225倍来证明。最后，亮场离轴模式便于进行定量相显微镜检查，我们用它来测量标准分辨率测试目标的折射率和人类红细胞的干质量。
