effects of overexpression of any protein, particularly in an inappropriate context, (2) aggregation and precipitation of FPs,
(3) mis-targeting [43], and (4) the FP itself producing reactive
oxygen species during maturation [44] and possibly acting as an
inappropriate electron donor [45]. Conditions of illumination
(intensity, spectrum, day-length) may represent significant additional variables, and, for plants, the requirement that the illumination intensities ultimately support heterotrophic photosynthesis is
an additional complication.
Targeting to subcellular organelles, such as the endoplasmic
reticulum [37] or the nucleus [39, 41, 46–49], enhances the signalto-noise ratio, since background autofluorescence in the blue-green
part of the spectrum is generally distributed diffusely throughout
the cytoplasm. This advantage in detection sensitivity does not
translate to flow cytometric measurements when using the entire
cell, but is a factor when analyzing nuclei within cell-free homogenates [48, 49].
In flow cytometry and sorting, optimizing the choice of FPs
depends on three major factors: (1) the numbers of different excitation sources and independent fluorescence detectors available on
the cytometer, (2) the spectral properties of the different FPs and of
any autofluorescent components within the tissue, and (3) whether
or not the FPs can be successfully expressed at levels sufficient for
detection and without pleiotropic side-effects.
The first laser integrated into flow cytometers was an argon gas
laser providing excitation at 488–nm, with a second HeNe gas laser
providing light at 633–640 nm. Since that time, laser development
has been rapid, and low cost solid-state lasers now reliably provide
excitation lines spanning the entire visible spectrum (355–785 nm;
[50]). The use of solid-state lasers has greatly reduced the price of
cytometers and increased their capabilities. The latest generation of
instruments (such as the Beckman-Coulter CytoFlex) are manufactured with the designed maximum of lasers and detector channels,
with the cost of the instrument to the end user not being defined by
this hardware content, but by the choice of the electronic dongle
that selects the numbers of illumination sources and detector channels that are active.
Flow cytometric optical design is now largely converging
around the following laser lines: Ultraviolet (355 nm), Violet
(405 nm), Cyan (488 nm), Green (532 nm), Yellow (561 nm),
and Red (633 or 640 nm). Spatial separation of the illumination
and detection light paths further increases the flexibility of the
instrument. In terms of detection, flow cytometers traditionally
employed combinations of optical filters (cut-on, cut-off, band
pass, and dichroic elements), to direct specific wavelength bands
of the emitted fluorescence to individual photomultiplier detectors.
Improved sensitivity particularly in the red accompanied adoption
of avalanche photodiodes as detectors [51, 52]. The latest
260
David W. Galbraith and Guiling Sun
(3) mis-targeting [43], and (4) the FP itself producing reactive
oxygen species during maturation [44] and possibly acting as an
inappropriate electron donor [45]. Conditions of illumination
(intensity, spectrum, day-length) may represent significant additional variables, and, for plants, the requirement that the illumination intensities ultimately support heterotrophic photosynthesis is
an additional complication.
Targeting to subcellular organelles, such as the endoplasmic
reticulum [37] or the nucleus [39, 41, 46–49], enhances the signalto-noise ratio, since background autofluorescence in the blue-green
part of the spectrum is generally distributed diffusely throughout
the cytoplasm. This advantage in detection sensitivity does not
translate to flow cytometric measurements when using the entire
cell, but is a factor when analyzing nuclei within cell-free homogenates [48, 49].
In flow cytometry and sorting, optimizing the choice of FPs
depends on three major factors: (1) the numbers of different excitation sources and independent fluorescence detectors available on
the cytometer, (2) the spectral properties of the different FPs and of
any autofluorescent components within the tissue, and (3) whether
or not the FPs can be successfully expressed at levels sufficient for
detection and without pleiotropic side-effects.
The first laser integrated into flow cytometers was an argon gas
laser providing excitation at 488–nm, with a second HeNe gas laser
providing light at 633–640 nm. Since that time, laser development
has been rapid, and low cost solid-state lasers now reliably provide
excitation lines spanning the entire visible spectrum (355–785 nm;
[50]). The use of solid-state lasers has greatly reduced the price of
cytometers and increased their capabilities. The latest generation of
instruments (such as the Beckman-Coulter CytoFlex) are manufactured with the designed maximum of lasers and detector channels,
with the cost of the instrument to the end user not being defined by
this hardware content, but by the choice of the electronic dongle
that selects the numbers of illumination sources and detector channels that are active.
Flow cytometric optical design is now largely converging
around the following laser lines: Ultraviolet (355 nm), Violet
(405 nm), Cyan (488 nm), Green (532 nm), Yellow (561 nm),
and Red (633 or 640 nm). Spatial separation of the illumination
and detection light paths further increases the flexibility of the
instrument. In terms of detection, flow cytometers traditionally
employed combinations of optical filters (cut-on, cut-off, band
pass, and dichroic elements), to direct specific wavelength bands
of the emitted fluorescence to individual photomultiplier detectors.
Improved sensitivity particularly in the red accompanied adoption
of avalanche photodiodes as detectors [51, 52]. The latest
260
David W. Galbraith and Guiling Sun