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Laser Scanning/Spinning Disk Confocal Microscopy
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Ozawa, P. Jacquet, M. Jacquey, Y. Kobayashi, T. Udem, R. Spinning disk confocal microscopy c illuminates the sample with a rotating pattern of 1,'s of pinholes for complete simultaneous confocal illumination. Figure 1c illustrates how the sample is illuminated, and so light detected, at multiple points simultaneously. This was originally proven by Felgett in spectroscopy and shows that using parallel detection delivers enhanced sensitivity.
A publication by Wang provides a quantitative comparison of point and disk scanning systems for imaging live-cell specimens. Figure 2. In addition to the fundamental disk containing an array of pinholes there is a second collector disk with a matching pattern of microlenses. This technology improvement, in combination with using an electron multiplying CCD detector, results in spinning disk technology being the ideal solution for fast live cell confocal imaging.
Figure 2 shows how the dual spinning disk confocal laser scanner operates. Unlike a conventional laser-scanning microscope, where a narrow laser beam sequentially scans the sample, in SDCLM an expanded beam illuminates an array of microlenses arranged on a collector disk. Each microlens has an associated pinhole laterally co-aligned on a second pinhole disk and axially positioned at the focal plane of the microlenses. The disks are fixed to a common shaft that is driven at high speed by an electric motor.
When the disks spin, and the scanner is coupled to a microscope with the pinhole disk located in its primary image plane, an array of focused laser beams scan across the specimen. Yokogawa realised the benefit of this approach and created their CSU spinning disk confocal unit. Andor's Dragonfly Multi-Modal Confocal takes this to the next level by improving the throughput, homogeneity of illumination and pinhole spacing to handle a wider variety of specimens from single molecules, to yeast to 3D model organs organoids.
The pinholes and microlenses are arranged in a pattern, which scans a field of view defined by the array aperture size and the microscope objective magnification.
Introductory Confocal Concepts | MicroscopyU
The scanning laser beams excite fluorescent labels in the specimen. Fluorescence emission will be most intense where this array is focused - the focal plane. Some fraction of this light will return along the excitation path where it will be preferentially selected by the same 'confocal' pinholes. A dichroic mirror, which reflects emission wavelengths, is located between the two disks.
An Overview of Spinning Disk Confocal Microscopy
Confocal Microscopy Methods and Protocols. Editors view affiliations Stephen W. Front Matter Pages i-xi. Pages Confocal Microscopy on the Internet. Kristin L. Hazelwood, Christopher S. Murphy, Zachary C.
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