Confocal microscopy BT 501

Sanjukta Patra

Wide field Focal plane Point illumination Pin hole

Confocal microscopy
The principle of confocal imaging was patented in 1957 by Marvin Minsky Imaging technique to increase resolution and contrast by using point illumination Uses a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane Enables the reconstruction of three-dimensional structures from the obtained images

Focal planes

Blue rays - lenses inside the microscope focus light from the focal point of one lens to another point Red rays - represent light from another point in the sample, which is not at the focal point of the lens, but gets imaged by the lenses of the microscope The image of the red point is not at the same location as the image of the blue point Blue point- the point directly at the focus of the lens Screen with a pinhole at the other side of the lens system, at the image of the blue point, then all of the light from the original blue point will pass through this pinhole Most of the light from the red point is still out of focus at this screen, and gets blocked by the pinhole

Fluorescence microscope- entire specimen is flooded evenly with light All parts of the specimen in the optical path are excited at the same time - resulting fluorescence is detected including a large unfocused background A confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal Only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution increases

Much of the light from sample fluorescence is blocked at the pinhole, so increased resolution is at the cost of decreased signal intensity so long exposures are often required As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular rectangular pattern of parallel scanning lines in the specimen. The achievable thickness of the focal plane is defined mostly by 1) wavelength of the used light divided by the numerical aperture of the objective lens 2) optical properties of the specimen The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples

Confocal Adding a pinhole/screen combination The focal point of the objective lens of the microscope forms an image where the pinhole is, these two points are known as "conjugate points" (the sample plane and the pinhole/screen are conjugate planes) The pinhole is conjugate to the focal point of the lens, thus it is a confocal pinhole

RAY DIAGRAM

How does it work?

Light source: laser is used to provide the excitation light (to get very high intensities) The laser light (blue) reflects off a dichroic mirror Dichroic mirror performs the critical function of both reflecting the excitation laser light to the scanning mirrors and the transmission of the returning emitted fluorescence light to the the confocal aperture. The laser hits two mirrors which are mounted on motors to scan the laser across the sample Fluorophore in the sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that are used to scan the excitation light (blue) from the laser The emitted light passes through the dichroic and is focused onto the pinhole The light that passes through the pinhole is measured by a detector Never a complete image of the sample is formed-- at any given instant, only one point of the sample is observed The detector is attached to a computer which builds up the image, one pixel at a time. In practice, this can be done perhaps 3 times a second, for a 512x512 pixel image The limitation is in the scanning mirrors

Advantage of confocal microscope??? By having a confocal pinhole, the microscope is efficient at rejecting out of focus fluorescent light The image comes from a thin section of sample (small depth of field) By scanning many thin sections through your sample, you can build up a very clean three-dimensional image of the sample A similar effect happens with points of light in the focal plane, but not at the focal point -- emitted light from these areas is blocked by the pinhole screen A confocal microscope has slightly better resolution horizontally, as well as vertically

Confocal microscopes types:

Confocal laser scanning microscopes Confocal laser scanning microscopes can have a programmable sampling density Spinning-disk (Nipkow disk) confocal microscopes Programmable Array Microscopes (PAM) Spinning disk and PAM use a fixed sampling density defined by the camera resolution Imaging frame rates are typically very slow for laser scanning systems (less than 3 frames/second) Commercial spinning-disk confocal microscopes achieve frame rates of over 50 per second a desirable feature for dynamic observations as live cell imaging

Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically complex objects For opaque specimens, this is useful for surface profiling, while for non-opaque specimens, interior structures can be imaged For interior imaging, the quality of the image is greatly enhanced over simple microscopy because image information from multiple depths in the specimen is not superimposed The CLSM achieves a controlled and highly limited depth of focus

Uses
CLSM is widely-used in numerous biological science disciplines, from cell biology and genetics to microbiology and developmental biology Clinically, CLSM is used in the evaluation of various eye diseases, and is particularly useful for imaging, qualitative analysis, and quantification of endothelial cells of the cornea It is used for localizing and identifying the presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis In the pharmaceutical industry, it is used to follow the manufacturing process of thin film pharmaceutical forms, to control the quality and uniformity of the drug distribution