Part 1 Principles
Confocal Microscopy tutorial
Part 1 Principles of Confocal microscopy
2. Filter-set and its choice, limitation
A detailed internal structure of the filter-set cube containing above-mentioned filter 5, 8, 9, shown here as A, B, C and its light path is given below:
Arrow 1 represents the excitation light source of mixed wavelength.
Arrow 2 represents excitation light which passes through A and reaches the surface of Beam Splitter B, BSP is a special filter which reflects certain wavelength away but permit other wavelength to pass. A multiple-bands reflecting filter or neutral percentage splitter can be used for this role. Wavelength falling into the reflecting band or shorter than the cut-off value will be stopped and reflected to the specimen. The fluorescence light emitted from specimen is illustrated as Arrow 3. The emission is longer than excitation wavelength and cut-off value of the BSP, so the returned emission can go through the BSP towards emission filter C.
Unfortunately, these two tasks placed on the single filter are
contradictory. Reflecting certain wavelength to specimen for excitation means
blocking it from transmission.
Emission filter C. Emission filter is either a band pass filter or long pass filter, the cut-off value is longer than excitation wavelength so the residue of excitation light 2 is further prevented from reaching image plane. Strict band pass results more specific but weaker signal, while long pass leads stronger signal but more background.
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