Principles of Fluorescence Spectroscopy

656 FLUORESCENCE SENSING
Figure 19.63. Fluorescence and light images of fibroblast cells which
transiently express a pH-sensitive GFP. Top: Image at 435–485 nm.
Middle: Image at 490–685 nm. Bottom: Overlay of the fluorescence
images onto a light image. Reprinted with permission from [235].
Copyright © 2000, American Chemical Society.
was fluorescein, which is not practical for wavelengthratiometric
measurements. Wavelength-ratiometric measurements
were made possible by inclusion of sulforhodamine,
which is not sensitive to pH. Ratios of the intensi-
Figure 19.64. Emission spectra of a Pebble sensor, a polyacrylamide
bead containing fluorescein as a pH-sensitive dye, and sulforhodamine
as a pH-insensitive reference dye. Reprinted with permission
from [238]. Copyright © 1999, American Chemical Society.
ties at 530 and 590 nm can be used to determine the pH
without interference from biomolecules.
The intracellular use of Pebble sensors is shown in Figure
19.65. In this case the Pebbles were made by encapsulating
Calcium Green and sulforhodamine in polyacrylamide
beads. 239 Sulforhodamine served as a calcium-insensitive
reference fluorophore. The laser scanning confocal
images of human SYSY neuroblastoma cells are shown in
Figure 19.65. Images were recorded using filters that transmitted
the emission of sulforhodamine (right) or calcium
green (left). The cells were treated with m-dinitrobenzene,
which caused the release of calcium from the mitochondria,
which resulted in an increase in the emission from Calcium
Green but no change in the emission from sulforhodamine.
Sensors using polymer beads have also been made by
coating the outer surface of polystyrene particles. 241–245 This
has been accomplished by covalent attachment of probes to
the outer surface. Bead sensors have also been made by
coating polystyrene beads with lipids which bind the sensing
fluorophores. These particles are called Lipobeads.
19.13. IN-VIVO IMAGING
In-vivo imaging is an emerging futuristic application of fluorescence
technology. By in-vivo imaging we mean the creation
of three-dimensional fluorescence images of the internal
structures of humans or small animals. In-vivo imaging
can be traced to the suggestion by Chance and coworkers
that images could be obtained from the diffusive migration
of photons in scattering tissues. 246–248 Tissues are strongly
absorbing and strongly scattering at wavelengths below 600
nm (Figure 19.66). Tissue absorption and scattering is much