New version page

CORNELL BIOPL 4440 - Lecture Notes

This preview shows page 1-2-3-4-5 out of 15 pages.

View Full Document
View Full Document

End of preview. Want to read all 15 pages?

Upload your study docs or become a GradeBuddy member to access this document.

View Full Document
Unformatted text preview:

Probably in the long run, it is worth getting the most stable, flexible, and, consequently, expensive microscope stand.The specimen is placed on the stage. The stage may move the specimen in the XY direction (and this may be motorized), the stage may glide freely on a stationary base, or the stagemay be circular and rotate around its axis. The bright field microscope is equipped with one or more light sources that can illuminate the specimen (Davidson, 1990). The light source is usually a quartz halogen bulb, although mercury vapor and xenon lamps may also be used in special cases. The light source is usually placed between a parabolic mirror and a collector lens. The light source is placed at the center of curvature of the parabolic mirror so that the rays that go backwards are focused back on the bulb. The collector lens is used to project an image of the filament onto the front focal plane of the condenser (Köhler illumination) or onto the specimen itself (critical or confocal illumination). The diameter of the field illuminated by the light source is controlled by the field diaphragm, and the number and angle of the illuminating rays is controlled by the aperture diaphragm. 1202. The Optical Paths of the Light Microscope I will briefly discuss two types of microscope illumination; Köhler and critical. In practice, Köhler illumination is used in most microscopes, and a specialized form of critical illumination is used in confocal microscopes. Köhler illumination provides a uniformly illuminated, bright field of view, which is important when one uses an uneven light source, like a coiled tungsten filament. Prior to 1893, microscopists used sunlight or oil lamps to illuminate their specimens, and very slow film to photograph them. The exposures needed to expose the filmwere as long as five hours. Thus August Köhler was motivated to find a way to obtain the brightest image possible so that he could continue his work investigating the taxonomic position of the mollusk, Syphonaria, by taking good photomicrographs of the taxonomically-important gills. Köhler devised a method in which an image of the source is formed by the collector lens at the front focal plane of the condenser and an image of the field diaphragm is formed in the plane of the specimen. The condenser collimates the light beam, which originates from any point on the source. Each point on the source forms a collimated beam of light that illuminates the entire field of view. The points on the center of the source form a collimated beam that is parallel to the optical axis. The points further and further away from the optical axis make collimated beams that strike the object at greater and greater angles. Thus, the specimen is illuminated by both parallel and oblique illumination. In critical illumination, an image of the light source is focused in the plane of the specimen. The illumination is intense, but it is uneven unless a ribbon filament is used. Critical illumination does not require a substage condenser. In critical illumination, each point in the object acts as a point source of light. Therefore, nearby points will form two overlapping images of Airy discs, the intensity of which will be the sum of the two intensities. When the microscope is set up for Köhler illumination, the following optical conditions result: The collector lens (Ls) focuses an image of the light source onto the condenser aperture 121diaphragm (Dc; Fig 5-14). The condenser lens (Lc) focuses an image of the field diaphragm (Ds) in the plane of the specimen (Fig 5-15). And the objective lens, the ocular, and the refractive elements of the eye together form an image of the specimen on the retina (Fig 5-15). Note that Figure 5-14 illustrates the imaging of the light that originates from any given point of the light source, while Figure 5-15 illustrates the imaging of light that comes from the whole filament and is converged at the field diaphragm by the collector lens. These conditions give rise to two sets of optical paths and conjugate image planes: The illuminating rays, which are equivalent to the 0th-order diffracted light (Fig 5-14), and the image-forming rays, which are equivalent to the sum of the 1st and higher order diffracted light (Fig 5-15).When the microscope is adjusted for Köhler illumination, we get the following advantages:1. The field is homogeneously bright even if the source is inhomogeneous.2. The working NA of the condenser and the size of the illuminated field can be controlled independently. Thus, glare and the size of the field can be reduced without affecting resolution.1223. The specimen is illuminated by a converging set of plane wave fronts, each arising from separate points of the light source imaged at the front focal plane of the condenser. This gives rise to good lateral resolution and fine optical sectioning, which yields good axial resolution.4. The front focal plane of the condenser is conjugate with the back focal plane of the objective lens, a condition needed for optimal contrast enhancement.I will demonstrate the pathway of the illuminating rays. The light source is placed a distance equal to twice the focal length of the parabolic mirror so that the ray that travels "backwards" is focused back onto the filament (Fig. 5-7). As shown in Figures 5-7 and 5-14, the collector lens produces a magnified, inverted, real image (S2) of the light source (S1) onto the condenser aperture diaphragm. That is, any given point of the filament is focused to a point at theaperture diaphragm.The condenser is designed so that the aperture diaphragm is placed at the front focal plane (f') of the condenser lens. Therefore, light emanating from a point in the plane of the aperture diaphragm emerges from the condenser as parallel rays (or as a plane wave). All together, the points in the front focal plane of the condenser give rise to a converging set of plane waves. The angle of each member of the set of plane waves is related to the distance of the point to the center of the aperature.The plane waves emerging from the condenser traverse the specimen and enter the objective lens. The objective lens converts the plane waves to spherical waves (Figure 5-3, 5-4) and the spherical waves converge on the back focal plane of the objective lens (S3). Thus each point at the back focal plane of the objective lens is conjugate with a corresponding point in the plane of the condenser aperture diaphragm and a point on the


View Full Document
Loading Unlocking...
Login

Join to view Lecture Notes and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Lecture Notes and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?