Biology Reference
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fluorescent tags. The first detailed description of
in vitro
imaging of cells using
a macromolecule carrying multiple fluorescein fluorophore molecules was
provided by French et al.
64
This study established that, upon labeling of
BSA with several FITC molecules, the latter were clearly subject to
quenching with a resultant decrease of fluorescence intensity and lifetime.
The increase of fluorescein emission intensity and a drastic sixfold lengthening
of fluorescence lifetime (FL) from 0.5 to 3 ns were observed over time in cells
after dye uptake. Experiments with FITC-labeled
L
-and
D
-polylysine lead to
the conclusion that proteolysis was responsible for the fluorescence increase, as
D
-polylysine (unlike
L
-polylysine), which is noncleavable by cellular proteases,
did not result in fluorescence changes.
64
In later studies, the same FITC-
labeled probe was used for tracking migrating cells in a 3D matrix.
65
These initial studies were important in that they provided inroads for fu-
ture development of
in vivo
probes that report on proteolysis. Fluorescence
intensity and FL changes resulting from cleavage of the enzymatic macro-
molecular substrate arise from conformational and chemical changes occur-
ring in the cleaved fluorescent products. Most polypeptides have secondary
and tertiary structures defined by hydrogen-bonding and hydrophobic in-
teraction of peptide bonds and amino acid side residues. If reactive groups
within a polypeptide chain (primarily N-
-amino groups of lysines) are lo-
cated spatially close to each other, the covalent linking of fluorophores to
these groups will result in low fluorescence. Natural globular proteins such
as albumins have low segmental mobility as opposed to their synthetic poly-
and oligoamino acid counterparts. For example, polylysines exist in ex-
tended coil conformation at a pH above 8.5. The conformational flexibility
of linear or branched synthetic poly(amino acids) can potentially result in
higher numbers of interacting dyes because of (1) higher overall number
of accessible reactive groups and (2) their mobility that facilitates the forma-
tion of transient or stable dye dimers (or stacks). The probability of fluores-
cent dye quenching in the above case is high because of the ease of collisional
interaction and, more importantly, the formation of nonfluorescent dye
dimers (H- or J-dimers) or higher order aggregates (also known as “self-
quenching”). The formation of nonfluorescent aggregates is more common
for fluorochromes that are electroneutral and/or are hydrophobic such as
NIR cyanine dyes (
Fig. 9.2
). Sulfation of cyanines results in better water sol-
ubility but does not prevent the formation of nonfluorescent H-aggregates.
Therefore, the design of macromolecular imaging substrates can benefit
from using sulfated cyanines and other NIR fluorochromes because of
the overall increase of solubility of macromolecules with fluorophore-linked
e
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