For example, a high resolution lens such as a 60x having a numerical aperture of 1.4 may have a working distance of 170 micrometers, whereas a 20x (having a typical numerical aperture of 0.75) might offer a relatively large 660 micrometer working distance, with the ability to access more restricted areas of a specimen without physical interference. In some cases resolution may have to be compromised in order to accommodate a specimen, and to avoid damage to it or the objective lens. Certain minimum physical requirements for the specimen apply it must fit on the microscope stage, and the area of interest must be able to be placed within the working distance range of the objective lens. The primary advantage and application of the confocal microscope is in improved imaging ability of thicker specimens, although the success can be limited by the specific properties of the specimen. Although many protocols include an antibleaching agent to prevent fading of the fluorescent species, such additives may not be required with many of the more modern confocal instruments, such as the Nikon A1 HD25/A1R HD25 and C2+ systems. It is usually advisable to use the lowest laser power that is practical for imaging in order to protect the fluorophore. At a typical scan speed of one point per 1.6 microseconds, the actual illumination at any given point is generally less than in a conventional wide field epifluorescence microscope. The result is that samples may require increased staining times or stain concentrations for confocal analysis, and if evaluated in the conventional microscope, may appear to be over stained.Īlthough the illumination in the typical laser scanning confocal system appears to be extremely bright, the average illumination at any given point on the specimen is relatively moderate, due to the fact that many points are scanned per second.

Due to the optical sectioning achieved by the confocal approach, the confocal microscope undersamples the fluorescence in a thick specimen as compared to the conventional epifluorescence microscope. Most of the methods that have proven successful in preparing specimens for the conventional wide field optical microscope are specifically aimed at reducing the amount of out-of-focus fluorescence, since this produces flare in the image that greatly reduces the resolution of the features of interest. Figure 1 presents the merged three-channel image (combined red, green, and blue channels). By evaluating the image resulting from merging the three, the most effective color-to-channel assignment for illustrating the various protein domains was chosen. After three corresponding images were collected in the red, green, and blue channels of the confocal system, the images could be rearranged by copying them to different channels. The specimen was immunofluorescently labeled with antibodies to three different proteins. This is discussed in more detail below, but one elegant example of the image display possibilities is presented in Figure 1, a triple-labeled Drosophila embryo at the cellular blastoderm stage.

Regardless of the specimen preparation protocol employed, a primary benefit of the manner in which confocal microscopy is carried out is the flexibility in image display and analysis that results from the simultaneous collection of multiple images, in digital form, into a computer.

Snapgene confocal microscopy