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specimen with a Ca 2 þ -sensitive fluorophore and a fluorophore that is sensitive for
another characteristic of the cell, which may also be Ca 2 þ in a di
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erent compart-
ment of the cell with di
erent concentration range, or a
second fluorophore that may be sensitive to the plasma membrane voltage in an
excitable cell (also called a potentiometric dye). Measurements of Ca 2 þ and mem-
brane voltage (resting membrane potentials and action potentials) may then be
conducted either simultaneously by exciting both fluorophores at the same time and
capture spectrally di
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erent dynamics or a di
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erent fluorescence emission signals, or sequentially by exciting
each fluorophore separately, that is, one after the other, under otherwise similar
experimental conditions. The latter approach would assume that the experimental
conditions remain the same. Several factors may necessitate this, such as an inabili-
ty to di
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erent emission signals, or an inability to excite more
than one fluorophore at any given time, for example, if the excitation spectra do not
overlap and only one excitation wavelength may be delivered at one time.
The advantage of using multiple fluorophores either simultaneously or sequen-
tially is to increase the information content of the imaging, especially how di
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erentiate between di
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erent
processes relate to each other spatially and temporally. However, several issues
may limit the applicability of such measurements. Introducing a fluorophore to the
specimen may also change the dynamics of the cellular parameter of interest,
especially in live specimens that rely on stable and constant intra- and extracellular
environments. For instance, most Ca 2 þ indicators are also Ca 2 þ chelators that
bu
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er free Ca 2 þ , and most fluorophores or the medium they are delivered in may
change biochemical and biophysical properties of the intracellular environment.
This may be accentuated by simultaneous loading with several dyes. Di
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erent dyes
may also quench, sequester, or in other ways inhibit each other. Finally, excitation
in itself may cause changes or damage to the specimen, and although this to some
degree is unavoidable, the degree of change or damage may be di
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erent or even
accentuated during sequential recordings.
The single-photon excitation and emission spectra of multiple Ca 2 þ - and
voltage-sensitive fluorescent dyes are well known. Clearly, some dyes have over-
lapping excitation or emission peaks, or present with broad excitation or emission
spectra such that even if the peaks are separated from one another, the tails of the
spectra still overlap considerably. Overlapping excitation spectra means that
di
erent dyes may be excited simultaneously, but overlapping emission spectra
may result in severely reduced signal specificity, and therefore, certain combina-
tions of fluorescent dyes may be less applicable, such as the potentiometric Di-4-
ANEPPS and Di-8-ANEPPS dyes, and the Ca 2 þ -sensitive Fluo-3 dye, all exten-
sively used by numerous laboratories for single-fluorophore purposes. All of these
dyes have single-photon excitation peaks at
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480-500 nm and emission peaks
at 520-610 nm, respectively, with especially Di-4-ANEPPS and Di-8-ANEPPS
having very broad emission spectra that peak at
610 nm, but that considerably
overlap with the Fluo-3 emission spectrum, even though the latter has its peak
at
ers from Di-4-ANEPPS and
Di-8-ANEPPS and is more narrow ( Fig. 11 A). This problem may to some degree
525 nm and therefore numerically di
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