etc.) which are routinely used to tag antibodies, other ligands, or
even whole cells [19, 23]. Labeling can also be done using small
molecules having binding characteristics that define their spectral
properties, such as the DNA-specific fluorochromes. The scatter
and fluorescence light pulses are then converted to electrical pulse
waveforms (voltage versus time), which are digitally processed to
provide numbers representing pulse area, peak, and width values.
Pulse-width (time-of-flight) is a robust method to measure cell
sizes ([24]; see also Fig. 5).
1.4 Identifying
Specific Cell Types in
Flow Cytometry and
Sorting
In order to purify different cell types using flow sorting, we require
fluorescent tags to be associated with these cells. Natural autofluorescence can be used; for example, protoplasts from leaf mesophyll
and epidermal cells leaves can be readily sorted based on the presence/absence of chlorophyll [24, 25], since tobacco mesophyll
protoplasts contain approximately 50 chloroplasts. This raises an
interesting point: for a leaf mesophyll protoplast population that is
95% intact (which most would consider an excellent technical
accomplishment), the suspension actually contains protoplasts and
chloroplasts at a ratio of 95:250 (i.e., intact protoplasts represent
less than 30% of the total population of objects detectable by the
cytometer). This emphasizes the critical importance of purification
of intact protoplasts at least to this level prior to further analysis
using flow cytometry and sorting. The situation is actually worse
for arabidopsis, in that individual mesophyll cells are reported to
contain 80–120 chloroplasts [26]. Even better would be to routinely include a staining step with a viability dye, such as fluorescein
diacetate, and analyze and sort only viable protoplasts based on this
signal [24, 25].
Since antibodies directed against cell type-specific surface epitopes of protoplasts are still not widely available for higher plants,
considerable attention has focused on transgenic expression of FPs,
starting with the prototypical Green Fluorescent Protein (GFP)
isolated from Aequorea victoria [27]. FPs and their coding
sequences have been isolated from an ever-increasing number of
organisms, and sequence variants continue to emerge having useful
spectral and cellular characteristics (see for example [28–36], and
citations therein). FPbase (www.fpbase.org), an interactive Fluorescent Protein database, currently lists 713 Fluorescent Proteins and
543 FP spectra. An additional 28 non-Aequorea synthetic FPs are
available from ATUM (formerly DNA2.0, Inc.) (https://www.
atum.bio/catalog/reagents/protein-paint-box#__fluorescentoverview). Given the explosion of information concerning new
FPs, their ready availability, and their widespread deployment in
different organisms, it is critical to populate available databases
(cf. www.fpbase.org) with references that indicate successful expression in different organisms, including plants. Crowd sourcing
258
David W. Galbraith and Guiling Sun
even whole cells [19, 23]. Labeling can also be done using small
molecules having binding characteristics that define their spectral
properties, such as the DNA-specific fluorochromes. The scatter
and fluorescence light pulses are then converted to electrical pulse
waveforms (voltage versus time), which are digitally processed to
provide numbers representing pulse area, peak, and width values.
Pulse-width (time-of-flight) is a robust method to measure cell
sizes ([24]; see also Fig. 5).
1.4 Identifying
Specific Cell Types in
Flow Cytometry and
Sorting
In order to purify different cell types using flow sorting, we require
fluorescent tags to be associated with these cells. Natural autofluorescence can be used; for example, protoplasts from leaf mesophyll
and epidermal cells leaves can be readily sorted based on the presence/absence of chlorophyll [24, 25], since tobacco mesophyll
protoplasts contain approximately 50 chloroplasts. This raises an
interesting point: for a leaf mesophyll protoplast population that is
95% intact (which most would consider an excellent technical
accomplishment), the suspension actually contains protoplasts and
chloroplasts at a ratio of 95:250 (i.e., intact protoplasts represent
less than 30% of the total population of objects detectable by the
cytometer). This emphasizes the critical importance of purification
of intact protoplasts at least to this level prior to further analysis
using flow cytometry and sorting. The situation is actually worse
for arabidopsis, in that individual mesophyll cells are reported to
contain 80–120 chloroplasts [26]. Even better would be to routinely include a staining step with a viability dye, such as fluorescein
diacetate, and analyze and sort only viable protoplasts based on this
signal [24, 25].
Since antibodies directed against cell type-specific surface epitopes of protoplasts are still not widely available for higher plants,
considerable attention has focused on transgenic expression of FPs,
starting with the prototypical Green Fluorescent Protein (GFP)
isolated from Aequorea victoria [27]. FPs and their coding
sequences have been isolated from an ever-increasing number of
organisms, and sequence variants continue to emerge having useful
spectral and cellular characteristics (see for example [28–36], and
citations therein). FPbase (www.fpbase.org), an interactive Fluorescent Protein database, currently lists 713 Fluorescent Proteins and
543 FP spectra. An additional 28 non-Aequorea synthetic FPs are
available from ATUM (formerly DNA2.0, Inc.) (https://www.
atum.bio/catalog/reagents/protein-paint-box#__fluorescentoverview). Given the explosion of information concerning new
FPs, their ready availability, and their widespread deployment in
different organisms, it is critical to populate available databases
(cf. www.fpbase.org) with references that indicate successful expression in different organisms, including plants. Crowd sourcing
258
David W. Galbraith and Guiling Sun