Biology Reference
In-Depth Information
phosphate-buffered saline or equivalent buffer for imaging. For live-cell experi-
ments, samples may be imaged through the glass bottom of a culture plate suitable
for fluorescence imaging acquisition or an upright microscope with a water immer-
sion objective.
1.3.2
Necessary experimental controls: defining the QB for
monomeric moiety
After determination of detector signal broadening due to intrinsic characteristic of
the detector (
Section 1.3.5
), the first step in the analysis is to obtain an estimate
of the monomeric QB of the fluorescent label. Remember that the monomeric
QB will vary depending on the choice of fluorophore and the microscope image
collection settings (PMT voltage, laser power, filters, etc.); thus, it must be charac-
terized for each set of experiments. Different options to determine the monomeric
QB are presented in the succeeding text.
1.3.2.1
Determining the monomeric QB for receptors tagged with
fluorescent proteins
The main objective when choosing a bright fluorophore suitable for determining QB
is to avoid ones that have the tendency to oligomerize, which is a problem for several
fluorescent proteins, including early versions of GFP (
Zacharias, Violin, Newton, &
Tsien, 2002
). Several DNA constructs have been developed to express monomeric
fluorescent proteins (e.g., monomeric GFP, mGFP). These constructs are either tar-
geted to the membrane (e.g., farnesylated mGFP (
Zacharias et al., 2002
) or glyco-
sylphosphatidylinositol anchored (
Kondoh, 2002
)) or expressed in the cytoplasm.
Whether a construct is suitable as a control for the monomeric brightness can be
verified. To do this, simply collect an image time series and analyze each frame to
verify if the QB stays constant within the detector error. If the fluorophores exist in its
oligomerized form, one should observe a time-dependent decrease in the density as
reported by SpIDA due to fluorophore photobleaching. In contrast, the QB will re-
main constant if the construct is truly monomeric. Steps for monomeric QB deter-
mination for receptor tagged with fluorescent protein are described in
Fig. 1.4
.
Note that this approach to test the validity of a monomeric sample is not suitable
for fluorescently labeled antibodies since most labeled antibodies are labeled by sev-
eral fluorescent molecules.
1.3.2.2
Determining the monomeric QB for antibody labeling using
detection of a reference monomeric protein
Because the fluorescence of labeled antibodies does not react in a dichotomic manner
(on/off) and that photobleaching will induce a change in the brightness of single
probes, it is important to minimize prealteration of the sample before imaging and
also to minimize the photobleaching during imaging. Determining the QB of the
probe can be done using a protein known to be monomeric that has been reveled with
the same secondary antibody used for the protein of interest.
