1. General information
2. Phase and differential interference contrast
3. Dark-field contrast
4. Fluorescence microscopy
5. Confocal laser scanning microscopy
6. References
1. General information
In our institute we use different versions of light microscopes: From the normal stereo microscope (Fig. 1) to the confocal laser scanning microscope. Fig. 2 shows images of the more sophisticated microscopes available in our labs: (a) Zeiss Axiovert 200, (b) Zeiss Axiotech, (c) Zeiss LSM 510 Meta confocal microscope. All of these microscopes are capable to be used as phase -, differential interference (DIC) and dark-field microscopes as well as fluorescence microscopes. In the following we present a brief discussion of the various contrast modes. For details we refer to the literature [1-3]. |
|
Fig. 2
2. Phase and differential interface contrast
An incoming wave from a light source has a given wavelength, an amplitude and a phase. Most of the incoming light does not interact with the object and is not changed. This part of the light forms the zero order contribution in the diffracted light wave. When the light wave interacts with the object, the amplitude and the phase may change. Both, amplitude and phase changes carry optical information of the diffracting object. An object, keeping the phase constant and retarding the amplitude is called amplitude object (Fig. 3). Vice versa, an object dominantly altering the phase is called phase object. Whereas images of amplitude objects are easy to visualize in a normal microscope, phase objects are hardly visible.
|
The special design of phase microscopes allows to separate the dominating zero order part of the diffracted wave pattern from the small contribution by the interaction with the phase object. Fig. 4 shows the principle setup of a phase microscope: An annular aperture (1) generates a hollow cone of light that illuminates the object. An annular l/4 phase plate (2) located in the back aperture of the objective lens shifts the phase of the non-diffracted (zero order) light with respect to the diffracted light. |
The relative phase shift is caused by a difference in optical path length within the phase plate for the non-diffracted and the diffracted rays. In addition, the difference in optical path length causes a slight difference in the wave amplitude. Depending on the relative phase shift between non-diffracted and diffracted rays constructive or destructive interference occurs. Due to the amplitude difference, the microscope generates a negative (dark) or positive (bright) contrast image of the object, respectively. Differential interference contrast (DIC) is a special method of deriving contrast in an unstained specimen from differences in index of refraction of specimen components. As with phase contrast, DIC transforms the phase shift |
|
of light, induced by the specimen refractive index, into detectable amplitude differences. An advantage of interference-derived contrast is that an object will appear bright against a dark background but without the diffraction halo associated with phase contrast. However, because DIC utilizes optical path differences within the specimen (i.e.: product of refractive index and geometric path length) to generate contrast the three-dimensional appearance may not represent reality. In other words the 3-D relief of DIC imaged specimens is an optical rather than a geometric relief.
3. Dark-field contrast In dark-field microscopy, light passing the condenser lens is restricted to far off-axis rays (Fig. 5) which (as undisturbed rays) do not fall into the objective lens. If an objective is located in the condenser focal plane diffracted rays from the objective may fall into the objective lens and become visible in the ocular lens. Hence, the specimen is displayed as bright object on a dark background. |
|
4. Fluorescence microscopy
A fluorescence microscope is basically a conventional light microscope with added features and components that extend its capabilities. Whereas a conventional microscope uses visible light to illuminate the sample and produce a magnified image of the sample, the fluorescence microscope uses light with short wavelength to illuminate the sample. The light excites fluorescence species in the sample, which then emit light of a longer wavelength. For proteins, for instance, so-called fluorophores with high quantum yield and adapted emission
|
spectra are available as labeling agents. Furthermore, the microscope has a filter that only lets through radiation with the desired wavelength that matches the fluorescing material. The radiation collides with the atoms in the specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light (Fig. 6). |
The key to the optics in a fluorescence microscope is the separation of the illumination (excitation) light from the fluorescence emission emanating from the sample which is done in the Zeiss Axiovert 200 by the help of a reflector cube (Fig. 7). A fluorescence microscope also produces a magnified image of the sample,
but the image is based on the second light source -- the light emanating from the fluorescent species -- rather than from the light originally used to illuminate, and excite, the sample. The fluorescing areas can be observed in the microscope against a dark background with high contrast. Fluorescence microscopy is a rapid expanding technique, both in the medical and biological sciences. The technique has made it possible to identify cells and cellular components with a high degree of specificity. |
|
5. Confocal laser scanning microscopy
The imaging principle in a confocal laser scanning microscope (CLSM) is very different from conventional light microscopes. In conventional microscopes, the whole specimen is continuously illuminated by a condenser. Via the objective lens and the ocular an image is created in our eyes. The key idea of the working principle of the CLSM is to restrict the illumination area by using an illumination point source. Hence, at a given time merely local information from a very small sample area can be obtained. An image of the entire specimen
surface is produced by a scanning mechanism. In addition to the small aperture used for illumination, a second small aperture is used in front of the detector. The intermediate image of the specimen in the back-focal plane of the objective lens is simultaneously located in the focal plane of the collector lens (Fig. 8) which gives rise to the name "confocal". This leads automatically to a further important characteristic of the CLSM: Light rays with an origin off the optical axis and off the focal plane are focused in a different plane, and hence, are excluded from image formation by the pinhole aperture. Therefore, the CLSM is capable of focusing in the third dimension (the depth of the sample) and by scanning different focal planes one may produce a three-dimensional image of a three-dimensional specimen. For conventional light sources, the reduction of the illumination light to a tiny point source reduces of course the illumination intensity dramatically. Therefore, in CLSMs a laser, which guarantees a small beam diameter and small beam divergence with high intensity, is generally used as a light source.
6. References
- D.B. Murphy: "Fundamentals of light microscopy and electronic imaging"
Wiley, New York - S. Inoue , K.R. Spring: "Video microscopy : the fundamentals"
Plenum Press, New York - M. Pluta: "Advanced light microscopy: Principles and basic properties"
Elsevier Science, Amsterdam
last change 04.10.2007 | Sebastian Houben | Print