Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 377
Figure 10.28. Transillumination fluorescence microscope image (left) and white light microscope image (right) of GFP crystals. The arrows indicate
the direction of the direction of the incident polarization. The bars are 30 µm. The upper schematic shows the orientation of GFP (green) and the red
shapes the orientation of the chromophore transition moment of the crystals. Revised from [64].
GPI, the anisotropy appears to be independent of the total
intensity. For FR-TM the anisotropy is lower where the
intensities are higher. FR-TM was not expected to form
clusters. The decrease in anisotropy with increasing intensity
(density) is consistent with the top panel in Figure 10.26
for randomly distributed proteins. FR-GPI was expected to
exist in small clusters. The constant anisotropy independent
of intensity (density) is consistent with the cluster model in
the lower panel of Figure 10.26. These results show how the
principles of anisotropy and RET are being extended to cellular
imaging.
10.9. TRANSITION MOMENTS
The concept of a transition moment is somewhat abstract.
When fluorophores are observed in random solutions the
nonzero anisotropy values prove that the transition
moments exist, but the data do not indicate the direction of
the moment in the molecular structure. However, the direc-
tion of the transition moment can be determined if the fluorophores
are oriented. A dramatic example 63–64 is shown
for GFP in Figure 10.28. The left panels show microscopic
images of the emission from GFP crystals. Depending on
the orientation of the incident polarization the crystals are
either bright or dark. This result shows that the chromophore
in GFP is aligned along the long axis of the crystals.
The orientation of GFP and its chromophore in the
crystals is shown in the upper schematic. The β-barrel of
GFP is oriented at an angle to the long axis, but the transition
moments are aligned along the long axis.
Also shown in Figure 10.28 (right) are color images of
the crystals when transilluminated with polarized white
light. The crystals aligned with the incident polarization are
yellow. The crystals are clear or very pale blue when rotated
90E from the incident polarization. This phenomenon is
called dichroism. The yellow color is the result of the bluelight
absorption of GFP that occurs primarily when the transition
moments are aligned with the incident polarization.