INSTRUMENT DESCRIPTION
Laser TIRF 3
Operating Principle
Carl Zeiss
02/2014
423683-7144-001
25
If the angle
Φ
exceeds a critical value, total reflection will occur (Fig. 2-1/
B
). As
Φ
2
is 90° at this point, the
critical angle
Φ
K
is:
2
1
sin
*
n
n
k
=
φ
If the light, for instance, makes the transition from the cover slip (n = 1.518) to water (n = 1.33), the
resulting critical angle will be 61°.
Under the conditions of total reflection, a standing evanescent wave (Fig. 2-1/
4
) is generated in the
medium of lower refractive index. The intensity of this wave decreases exponentially with the distance to
the interface. Fluorophores being farther away from the interface will be excited less intensely due to this
exponential decrease. Because of the very small volume of the electromagnetic field, background
fluorescence known from epi-fluorescence is clearly reduced and image contrast considerably enhanced.
This results in a correspondingly high Z resolution.
Therefore, the Laser TIRF imaging system is a valuable tool for examining processes of individual
molecules or objects at surfaces. It allows, for instance, the study of events or processes which take place
very closely to the cell membrane or in cell-free systems at the surface of a cover slip.
Advantages of TIRF microscopy:
−
No interference by background fluorescence
−
Higher resolution
−
Higher contrast
Fields of application:
−
Cell biology (cell physiology / neurobiology / neurophysiology)
−
Molecular biology
−
Developmental biology
−
Biochemistry
Typical applications:
−
Detection of cell adhesion structures
−
Single Molecule Detection
−
Analysis of membrane dynamics
−
Analysis of endocytosis and exocytosis processes (import and export of soluble components
in / from the cell), such as, e.g., vesicular transport mechanisms
Содержание TIRF 3
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