ZEISS
3 Product and Functional Description | 3.4 Microscopy and Contrast Methods
3.4.3 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.
3.4.4 Transmitted Light Differential Interference Contrast Microscopy
The transmitted light DIC method allows for a high-contrast vivid display of transparent sample
details.
The light is linearly polarized by a polarizer and is separated into two beams in a birefringent
prism. These pass through two neighboring sample locations at a short distance and experience
different path differences there due to differences in refractive index and sample thickness. Both
beams are then combined in a second birefringent prism and have the same polarization after
passing the analyzer. Therefore both beams can interfere in the intermediate image and the path
differences are thus converted into intensity differences represented by a gray scale. A compen-
sator, e.g. λ-plate, may be used for a consecutive conversion of the gray scale in a color scale.
3.4.5 Transmitted Light PlasDIC Microscopy
PlasDIC can be used independently from the material of the sample holder.
The contrast method gives a relief-like image and is especially well suited for thicker objects. The
contrast is adjustable. It is possible to contrast the cavities of microtiter plates up to the edge. It is
not necessary to use cultivation holders with a glass base.
3.4.6 Transmitted Light Polarization
The transmitted light polarization method is used for samples which change the polarization of
the light. Such samples are called birefringent. Examples include crystals, minerals or polymers. If
such birefringent substances are observed between crossed polarizers, the birefringent portion of
the sample appears bright while its surroundings remain dark.
3.4.6.1 Detecting Birefringence
A birefringent substance can be recognized by turning the sample by 360° between crossed po-
larizers. The sample should show four bright and four dark appearances during the turning proce-
dure. During the turning procedure, interference colors appear that range from gray (mostly for
biological samples) through white, yellow and red until blue, depending on birefringence, thick-
ness as well as sample orientation. The interference colors may be of the first or of a higher order.
Instruction Manual ZEISS Axioscope 5, Axioscope 5/7 MAT | en-US | Rev. 13 | 430035-7344-001
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