Molecular Devices ImageXpress Pico, Installation Manual

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ImageXpress Pico Personal Imaging System Installation Guide
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Fluorescence Imaging
Fluorescence is a property of certain classes of molecules (fluorochromes, fluorescent
proteins, or dyes) 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 active probe molecules, so that application
of the combined dye/probe molecule (fluorophore) to the specimen highlights the specific
substances or regions to which the probe is targeted.
By attaching different probes to a set of dye molecules with non-overlapping excitation and
emission spectra, one can stain a specimen with multiple fluorophores, and either
simultaneously or sequentially image different structures or substances within the same
specimen. The absorption 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 be used, such as the emission
and excitation filters, and the dichroic mirror.
Excitation and Emission Filters
In the ImageXpress Pico System, the excitation and emission filters are located in a filter
cube.
To selectively excite one fluorophore more intensely than another, or to minimize excitation
channel crosstalk, it is necessary to provide illumination containing only photons with a
wavelength range matched to the absorbance (excitation) spectrum of the target dye. A
bandpass filter in the illumination optical path (called the excitation filter, since it filters the
excitation light) is used to restrict 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).