Laser Scanning Confocal Microscope Resolution

Laser scanning confocal microscopes.

Laser scanning confocal microscope resolution. Laser scanning confocal microscopy laser scanning confocal microscopes employ a pair of pinhole apertures to limit the specimen focal plane to a confined volume approximately a micron in size. 1988 the lsm 10 a confocal system with two fluorescence channels. This equipment is able to combine a lot of the functionality of an optical microscope scanning electron. Laser scanning confocal microscope configuration basic microscope optical system characteristics have remained fundamentally unchanged for many decades due to engineering restrictions on objective design the static properties of most specimens and the fact that resolution is governed by the wavelength of light.

Confocal microscopy can be considered a bridge between these two classical methodologies. 1991 the lsm 310 combines confocal laser scanning microscopy with state of the art computer technology. Laser scanning microscopy 1982 the first laser scanning microscope from carl zeiss. Confocal microscopy most frequently confocal laser scanning microscopy clsm or laser confocal scanning microscopy lcsm is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out of focus light in image formation.

The confocal laser scanning microscope clsm is a microscope which focuses only on a single focal plane and the unfocused plane will not be visualized. It is important therefore to scan a sample at a resolution that meets the criteria of your experiment and conforms to the anatomy of the. In the confocal laser scanning microscope the highest frequency to be sampled f is imposed by the optical system and for a particular resolution specification. In some cases specimens should be sampled at more than 2 3 times the highest information frequency to allow for the possibility that the highest frequency was misjudged.

The result is a high resolution large depth of field color image with nanometer level height resolution for accurate profile and roughness measurements. With confocal laser scanning microscopy clsm we can find out even more. Capturing multiple two dimensional images at different depths in a sample enables the. Relatively thick specimens can be imaged in successive volumes by acquiring a series of sections along the optical z axis of the microscope.

In the past the traditional laser microscope excited the whole thickness of the sample resulting in saturated blurry images and sometimes visualizing false colocalization images. The prototype of the lsm 44 series is now on display in the deutsches museum in munich. Clsm combines high resolution optical imaging with depth selectivity which allows us to do optical sectioning. Illustrated in figure 1 are a series of images that compare selected viewfields in traditional widefield and laser scanning confocal fluorescence microscopy.

Within the resolution of the clsm confocal laser scanning microscope set parameters any variation in actual sample intensities will be averaged or integrated into one intensity value in any image voxel. Fluorescent microscopy not only makes our images look good it also allows us to gain a better understanding of cells structures and tissue.