Molecular Devices ImageXpress Confocal HT.ai, Руководство пользователя

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Chapter 1: ImageXpress Confocal HT.ai System
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Fluorescence Imaging
Fluorescence is a property of certain classes of molecules (fluorophores) in which photons of a
specific wavelength are absorbed (excitation), and, as a result, photons are emitted at a longer
wavelength (emission) a very short time later. The utility of fluorescence imaging in biological
applications stems from the ability to conjugate fluorescent molecules with biologically
significant probe molecules, so that visualization of the combined fluorophore in the specimen
highlights the specific distribution of the molecules in question.
By attaching different probes to a set of dye molecules with non-overlapping excitation and
emission spectra, you can stain a specimen with multiple fluorophores and either
simultaneously or sequentially image different structures or substances within the same
specimen. The excitation and emission peaks for each dye or fluorescent protein in a given
environment are physical characteristics of that molecule, and their specific properties
determine the initial selection of the optical components to use, such as the emission and
excitation filters and the dichroic mirror.
Confocal Imaging
In standard widefield microscopy, fluorescent objects above or below the focal plane are
seen as out-of-focus objects and increase the background fluorescence. Confocal microscopy
uses point-source illumination and a pinhole aperture ahead of the detector to reject
out-of-plane signals.
The main advantage of confocal imaging is the rejection of photons from out of focus portions
of the sample and the resulting improved resolution and reduced background. By changing
the size of the pinhole in front of the detector you can achieve a suitable balance between the
degree of confocality and the sensitivity and speed of the instrument. A smaller pinhole gives
better axial resolution, while a larger pinhole increases the collection efficiency resulting in
higher sensitivity.
Excitation and Emission Filters
In the ImageXpress Confocal HT.ai system, the excitation filters are located inside the light
source. The emission filters and the dichroic mirrors are located within two separate,
controllable wheels inside the confocal module.
To selectively excite one fluorophore more intensely than another or to minimize excitation
channel crosstalk, it is necessary to provide illumination that contains only photons with a
wavelength range that matches the excitation spectrum of the target fluorophore. A bandpass
filter in the illumination optical path (called the excitation filter, since it filters the excitation light)
restricts the illumination spectrum to a narrow range of wavelengths.
Similarly, when imaging the illuminated sample, it is desirable to collect only the emission
photons from the target fluorophore, rejecting as much as possible any reflected or scattered
excitation light, any light from other dyes, and autofluorescence from the sample and
substrate. This is done by placing a filter in the collection light path, called the emission filter.
Emission filters can either be a bandpass filter (for maximum specificity) or a longpass filter (to
maximize the amount of emission light collected).