Understanding 3-dimensional structure also requires the ability to resolve distinct details. The axial z- resolution of a conventional fluorescence microscope is however poor — on the order of 2.
This principle is demonstrated in Figure 2. Light from fluorophores above and below the focal plane does not converge tightly enough to pass through the pinhole, and so is blocked from reaching the detector. This can improve axial resolution to around nm, as well as improve image contrast. The first design of confocal microscope uses a laser beam which passes through a single pinhole in the excitation light path, with the emission light from the illuminated point passing through a separate pinhole to a detector, a photomultiplier tube PMT.
To generate an image, this light path is raster-scanned across the sample in 2 dimensions, then an image is reconstructed from the detected light. A piezo Z-stage is then used for the z-direction. This technique generates high-quality images, but at the cost of very slow speed, on the order of 1 second per image. Additionally, the poor efficiency of the photomultiplier tubes used to detect light in laser scanning confocal combined with the highly concentrated illumination beam cause considerable photobleaching and photodamage to live cells.
Another technique is therefore needed for live cell work, or for fast processes. Spinning disk confocal microscopy solves the scanning problem by using multiple pinholes. The primary means of achieving this is through arranging pinholes in a spiral pattern, etched into an opaque disk Figure 3.
When spun, the pinholes scan across entire image rows in sequence. The holes are positioned so that every part of the image is scanned as the disk is turned.
The rotation speed of the disk, therefore, determines the maximum image acquisition speed. The parallelization of pinhole scanning not only vastly improves the speed of acquisition. But also using array-based rather than point-based scanning means the system can take advantage of the latest state-of-the-art Scientific CMOS and EMCCD cameras in place of the photomultiplier tube.
The spinning disk confocal unit is typically a self-contained module that can be added to the camera port of a microscope. The light source and cameras are then routed through the disk module. A typical setup is illustrated in Figure 4. The result of this increased speed and sensitivity over laser scanning confocal is a microscopy technique much more suited to the study of live cells, and the dynamic processes which occur within them.
Parallel scanning means irradiation of the sample is lower both on average and at peak, which leads to a considerable reduction in damage and photobleaching of the sample. Image brightness, contrast and quality of optical sectioning can all be optimized through the properties of the disk. For detection, most of the light blocked will originate from out-of-focus planes, however strong illumination and a high sensitivity camera are still vital to achieve an excellent quality image.
Both pinhole diameter and separation are also determining factors of the quality of out-of-focus rejection. Pinhole diameter determines the thickness of the vertical section that can pass through a pinhole — larger pinholes accept a larger optical section.
Pinhole separation determines the quality of rejection on longer length scales; out-of-focus light originating sufficiently far from the focal plane can enter neighboring pinholes — a process known as pinhole cross-talk. The extent of cross-talk is sample-dependent, with thick samples most strongly affected. Placing pinholes further apart reduces this effect, at the cost of reducing light transmission through the disk.
The transmittance of the excitation path of a spinning disk setup can be considerably improved with a second disk containing microlenses in the place of pinholes, as illustrated in Figure 5, which focuses illumination light through the pinholes of the primary disk.
Transmittance may be improved by an order of magnitude Inoue and Inoue This system reduces the light intensity necessary to illuminate a sample, though does not improve the detection of light from the sample. The larger the pinhole the more light can pass through, but axial resolution suffers. There are three key parameters that determine the optimal pinhole size to balance these factors:.
The optimal pinhole size for a given objective, D opt , is set as the first minimum of the Airy disk from a point source:. For a 1. Overlarge pinholes reduce axial resolution. Too small pinholes diffract illumination light excessively, and block emission light unnecessarily, reducing image contrast. Typically, disk pinholes as supplied by spinning disk unit manufacturers are optimized for either 60x or x magnification, oil-immersion objectives. Camera pixel size is therefore important to optimize resolution for the optics of the system.
In the xy plane, resolution is determined by the optical system exactly as it is for a conventional fluorescence microscope, although the increased imaging contrast may mean that in practice, resolvable details are improved. Image obtained from Leica Science Lab. Hundreds of pinholes are arranged in Archimedean spirals left , which pass over the sample as the disk spins. The pinholes have diameter D and separation distance S, changing these values can optimize the resultant image received.
Figure 3: Using a secondary micro-lens disk to improve light transmittance through the primary pinhole disk. Laser illumination is focused through the pinholes by the micro-lenses, with both disks spinning in sync over the sample. Image obtained from Graf et al. Figure 4: The effects of disk rotation speed and exposure time on the resultant image.
Image obtained from Stehbens et al. This website uses cookies. By proceeding navigation on this page, you agree to the use of cookies according to the terms of our Privacy Policy. I Agree. In , Nia et al. In this article, the authors proposed 4 distinct physical traits of cancer linked to the biomechanical abnormalities observed Read more…. It is still widely used in research and Read more…. As you might know, October is the Breast Cancer Awareness Month, an annual campaign to raise awareness about the impact of breast cancer.
Research is the key tool to fight this disease and we are Read more…. Light path in a ImageXpress Micro Confocal In conclusion, spinning disk confocal microscopy offers a clear improvement on standard laser scanning confocal microscopy and conventional fluorescence microscopy, allowing for fast and efficient imaging of live samples, dynamic processes and optical sectioning of 3D samples in 2D slices.
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