instrument developments, termed Spectral Analyzers, employ
either diffraction gratings or prisms (SONY), or arrays of focusing
mirrors aligned with paired dichroic elements (Cytek), to spatially
distribute the entire fluorescence emission spectra across detector
arrays, each detector having a bandwidth 1/3 to 1/2 of that of the
detectors in a conventional cytometer. The SONY SA3800 is
equipped with a 32-channel PMT spanning 500–800 nm, a separate 2-channel PMT spanning 420–500 nm, forward and side
scatter detectors, and four spatially separated lasers (emitting at
405, 488, 561, and 638 nm). The SONY ID7000™ Spectral Cell
Analyzer is configured with separate light paths for up to seven
lasers (320, 355, 405, 488, 561, 637, and 808 nm), and up to
188 detectors spanning 360–920 nm. Finally, the Cytec Aurora
comes equipped with five lasers (355, 405, 488, 561, and 640 nm),
64 fluorescence detectors, and three scatter detectors. In spectral
analysis, each cell produces a spectral signature as an intensity vector
of length equal to the numbers of detection channels. Different
fluorochromes are distinguished by deconvolution across these
vectors, and this same process is used to identify and remove contributions from autofluorescence. At this point, the only limiting
factor appears to be the availability of sufficient numbers of spectrally distinct fluorochromes.
Fluorescent proteins producing green and yellow fluorescence
(Aequorea GFP, non-Aequorea GFPs, and sequence variants such
as YFP) are efficiently excited at 488 nm. DsRed, the canonical
RFP, and other red FPs derived from DsRed, are reasonably excited
at around 568 nm. The brightest CFP Aequorea variants are optimally excited at around 433 nm, but can also be excited at 405 nm
(preferred) and 457 nm. The original Aequorea BFP variant was of
low brightness and poor photostability, but more recent BFP variants are considerably brighter and more photostable [53, 54], but
require excitation with a UV laser, and UV-excited autofluorescence and/or toxicity can be issues. For FP sequence variants that
emit fluorescence in the orange, red, and the far-red portions of the
visible spectrum, including mOrange, tdTomato, mStrawberry,
mCherry (all derived from DsRed; [55]), TagRFP and TurboRFP
[56], and E2-Crimson, mNeptune, TurboFP650 and NirFP
[57, 58], solid state lasers producing light between 514 and
642 nm provide excellent excitation.
In considering how many different FPs might be simultaneously analyzed in flow cytometry, six appears to be the current
conceptual limit based on mammalian cell systems (W. Telford,
T. Hawley, pers. commun.; [59]). This requires excitation at
405 nm (for CFP variants), 488 nm (for GFP/YFP variants),
561 nm (mKate, Katushka, or mRFP1), and 628 nm (mNeptune,
E2-Crimson, and NirFP). In conventional flow cytometry, utilizing
discrete fluorescence detectors screened by different combinations
of optical filters, one limitation relates to the degree of overlap of
Flow Cytometry and Sorting in Arabidopsis
261
either diffraction gratings or prisms (SONY), or arrays of focusing
mirrors aligned with paired dichroic elements (Cytek), to spatially
distribute the entire fluorescence emission spectra across detector
arrays, each detector having a bandwidth 1/3 to 1/2 of that of the
detectors in a conventional cytometer. The SONY SA3800 is
equipped with a 32-channel PMT spanning 500–800 nm, a separate 2-channel PMT spanning 420–500 nm, forward and side
scatter detectors, and four spatially separated lasers (emitting at
405, 488, 561, and 638 nm). The SONY ID7000™ Spectral Cell
Analyzer is configured with separate light paths for up to seven
lasers (320, 355, 405, 488, 561, 637, and 808 nm), and up to
188 detectors spanning 360–920 nm. Finally, the Cytec Aurora
comes equipped with five lasers (355, 405, 488, 561, and 640 nm),
64 fluorescence detectors, and three scatter detectors. In spectral
analysis, each cell produces a spectral signature as an intensity vector
of length equal to the numbers of detection channels. Different
fluorochromes are distinguished by deconvolution across these
vectors, and this same process is used to identify and remove contributions from autofluorescence. At this point, the only limiting
factor appears to be the availability of sufficient numbers of spectrally distinct fluorochromes.
Fluorescent proteins producing green and yellow fluorescence
(Aequorea GFP, non-Aequorea GFPs, and sequence variants such
as YFP) are efficiently excited at 488 nm. DsRed, the canonical
RFP, and other red FPs derived from DsRed, are reasonably excited
at around 568 nm. The brightest CFP Aequorea variants are optimally excited at around 433 nm, but can also be excited at 405 nm
(preferred) and 457 nm. The original Aequorea BFP variant was of
low brightness and poor photostability, but more recent BFP variants are considerably brighter and more photostable [53, 54], but
require excitation with a UV laser, and UV-excited autofluorescence and/or toxicity can be issues. For FP sequence variants that
emit fluorescence in the orange, red, and the far-red portions of the
visible spectrum, including mOrange, tdTomato, mStrawberry,
mCherry (all derived from DsRed; [55]), TagRFP and TurboRFP
[56], and E2-Crimson, mNeptune, TurboFP650 and NirFP
[57, 58], solid state lasers producing light between 514 and
642 nm provide excellent excitation.
In considering how many different FPs might be simultaneously analyzed in flow cytometry, six appears to be the current
conceptual limit based on mammalian cell systems (W. Telford,
T. Hawley, pers. commun.; [59]). This requires excitation at
405 nm (for CFP variants), 488 nm (for GFP/YFP variants),
561 nm (mKate, Katushka, or mRFP1), and 628 nm (mNeptune,
E2-Crimson, and NirFP). In conventional flow cytometry, utilizing
discrete fluorescence detectors screened by different combinations
of optical filters, one limitation relates to the degree of overlap of
Flow Cytometry and Sorting in Arabidopsis
261