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3 Product and Functional Description | 3.3 Microscopy and Contrast Techniques
3 .3 Microscopy and Contrast Techniques
The availability of microscopy and contrast techniques depends on the microscope type and the
configuration.
3 .3.1 Transmitted Light Brightfield Microscopy
Transmitted light (TL) brightfield microscopy is the most common of all optical microscopy meth-
ods, since it can be used to quickly and easily examine high-contrast or stained samples (e.g.
blood smears).
In order to obtain an image as close as possible to the object, not only the so-called direct beam
bundles but also the indirect ones, i.e. the beam bundles diffracted and scattered at the prepara-
tion details, are of essential importance. According to ABBE, the larger the indirect beam compo-
nents are, the more true to the object the microscopic image is.
3 .3.2 Transmitted Light Phase Contrast Microscopy
The phase contrast method is ideal for examining thin uncolored samples, e.g. individual cells of
cell cultures. Generally, the human eye cannot detect phase differences (variations in refractive in-
dex or thickness) within the different cell components.
The phase contrast method uses the optical modulators "annular phase diaphragm" and "phase
ring" to convert the small phase differences in intensity differences which are visible to the human
eye. The interference of different beams in the intermediate image is important for the generation
of such images.
With the aid of the optically defined ring channel "annular phase diaphragm and phase ring", the
bright direct light portions are attenuated and provided with a constant phase shift. The indirect
light portions, however, which are diffracted by different cell particles, bypass this optical channel
and their phase is affected by the difference in the sample's refractive index and thickness.
In the intermediate image plane, the partial beams are thus differently affected and achieve inter-
ference and strengthen or weaken each other (constructive and destructive interference) – de-
pending on their phase. As a result, these interferences create image contents with intensity dif-
ferences visible to the human eye.
3 .3.3 Reflected Light Fluorescence Microscopy
The reflected light fluorescence method is used to show fluorescent substances in typical fluores-
cent colors in high contrast. The light originating from a high-performance light source in a re-
flected light fluorescence microscope passes through a heat protection filter onto an excitation fil-
ter (bandpass). The filtered short-wave excitation radiation is reflected by a Dichroic Beam splitter
and is focused on the sample through the objective. The sample absorbs the short-wave radiation
before emitting longer-wave fluorescence radiation (Stokes' Law). This radiation is then captured
from the image side by the objective and passes through the Dichroic Beam splitter. Last, the
beams pass through a emission filter (longpass/bandpass) and only the long-wave radiation emit-
ted by the sample passes.
The spectra of the excitation and the emission filter must match very closely. They must be in-
serted in a Reflector Module FL EC P&C reflector module together with the according Dichroic
Beam splitter.
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Instruction Manual ZEISS Axiovert 5 digital | en-US | Rev. 1 | 431030-7021-101
ZEISS
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