Biology Reference
In-Depth Information
fluorescence properties.
101
As mentioned in
Section 3
, the optical properties
of Nile Red show high sensitivity to the polarity of the environment. This
allows fluorescent staining of hydrophobic regions, such as lipid droplets, in
living cells. In water, Nile Red displays low fluorescence and relatively long
absorption and emission maxima with
l
max
/
l
em
¼
591/657 nm. In organic
solvents, however, compound
8
exhibits
523/565 nm and a
large increase in quantum yield.
19
This dramatic change has been exploited
to prepare fluorogenic stains for cellular membranes and lipid droplets.
20
The environmental sensitivity of phenoxazine dyes can also be used to read
out protein conformation.
102
Cresyl Violet (
63
) exhibits a
l
max
/
l
¼
em
l
max
that is
100 nm longer than the analogous rhodamine 110 (
49
), with
l
max
/
0.54.
103
Like rhoda-
mines, the fluorescence of Cresyl Violet and similar dyes can be modulated
by acylation of the aniline nitrogens. This modification dramatically shifts
the
10
4
M
1
cm
1
, and
¼
e
¼
F
¼
l
591/654 nm,
8.3
em
em
to shorter wavelengths and decreases the quantum yield, mak-
ing this dye a useful scaffold for fluorogenic molecules.
27,104
l
max
/
l
8.2. Enzyme substrates
The relatively long absorption and emission spectra of resorufin are advan-
tageous for bioassays because of reduced interference from cellular
autofluorescence. Analogous to the hydroxycoumarin and fluorescein-based
enzyme substrates, phenol substitution of resorufin causes a decrease in fluo-
rescence. Thus, resorufin has been used to construct a variety of red-shifted
enzyme substrates. These include the phosphatase substrate
64
105
and the
cytochrome P450 substrate
65
.
106
Resorufin acetate
66
is a fluorogenic
dye that can report on the activity of a variety of enzymes including esterases
and proteases.
107,108
The instability of the resorufin-ester bond and
the propensity of acyl resorufins toward nucleophilic attack
98
hamper the
utility of acyl resorufins, especially in living cells. Substitution of the
acetate ester with an acetoxymethyl ether group as in compound
67
improves stability; the extra carboxyl group increases cellular retention of
the molecule.
58
Glycosylated resorufins, such as
b
-galactosidase substrate
68
,
find wide use for many applications because of
the red-shifted
properties of the released resorufin.
109
In addition, the oxidation state of the dye can be modulated to create
fluorogenic molecules. For example, resazurin (
69
) is a strongly absorbing
(
10
4
M
1
cm
1
) but nonfluorescent molecule.
110
Reduction of this compound by endogenous oxidoreductases, such as
l
¼
602 nm,
e
¼
4.2
max
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