Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.
Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.
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DOI：
10.1371/journal.pone.0043942
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hartell NA
中科院分区：
文献类型：
--
作者：
Martial FP;Hartell NA
共焦显微镜通常用于生物标本的高分辨率荧光成像。大多数标准的共焦系统扫描穿过样品的激光，并收集通过单个针孔的发射光，以产生样品的光学部分。逐点的顺序扫描限制了图像采集的速度，即使是最快的商业仪器也难以解析快速细胞事件的时间动力学，如钙信号。已经引入了各种方法来提高共焦成像的速度。例如，尼普科夫磁盘显微镜使用旋转磁盘上的针孔或狭缝阵列来实现并行扫描，这显著提高了采集速度。在这里，我们报道了一种显微镜模块的开发，它利用数字微镜器件作为空间光调制器，以单台相机以受相机帧速率限制的速度以高空间和轴向分辨率提供可编程的共焦光学切片。数字微镜充当固态尼普科夫盘，但增加了改变针孔大小和间隔的能力，并在逐个镜面的基础上控制光强度。在发射路径中使用凹凸镜而不是透镜，克服了DMD设备固有的散光，提高了光收集效率，并确保图像收集是消色差的，从而使图像在不同波长上完美对准。与非激光光源相结合，这可以实现低成本、高速、多波长的图像采集，而不需要复杂的波长相关图像对准。微镜还可以用于可编程照明，从而允许荧光蛋白在空间上定义的光激活。我们演示了使用该系统进行高速钙成像，使用的是单波长钙指示器和遗传编码的比率钙传感器。
Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.
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影响因子：
1.9
作者：
Jiang, Shihong;Walker, John
通讯作者：
Walker, John
DOI：
10.1046/j.1365-2818.2001.00945.x
发表时间：
2001-11-01
期刊：
JOURNAL OF MICROSCOPY-OXFORD
影响因子：
--
作者：
Heintzmann, R;Hanley, QS;Jovin, TM
通讯作者：
Jovin, TM
DOI：
10.1073/pnas.0509378103
发表时间：
2006-03-21
影响因子：
11.1
作者：
Tallini, YN;Ohkura, M;Kotlikoff, MI
通讯作者：
Kotlikoff, MI
影响因子：
3.5
作者：
Bansal, Vivek;Patel, Saumil;Saggau, Peter
通讯作者：
Saggau, Peter
影响因子：
3.6
作者：
Liang, MH;Stehr, RL;Krause, AW
通讯作者：
Krause, AW