Chemistry Reference
In-Depth Information
O
O
N
S
N
N
O
+
Na
λ
abs
= 500, 534 nm;
λ
em
= 577 nm at pH=7.0, 500 nm at pH>7.6
-
SO
3
fIgure 11.15
Structure of Merocyanine 540.
O
N
HN
λ
abs
= 553 nm;
λ
em
= 570 nm, methanol
O
N
+
O
Cl
fIgure 11.16
Structure of Celltrack
TM
CM-DiI.
-
SO
3
-
-
SO
3
O
3
S
2
+
N
-
3
1
O
3
S
N
2
4
N
+
3
5
N
1
+
K
+
K
O
O
O
O
N
O
N
N
O
Cy3 (λ
abs
= 550 nm;
λ
em
= 570 nm)
Cy5 (λ
abs
= 649 nm;
λ
em
= 670 nm)
O
O
O
O
O
O
fIgure 11.17
The reactive cyanine stains Cy3 and Cy5.
The other two symmetrical cyanine polymethine stains that are widely used as fluoro-tagging agents for nucleic acids,
peptides, proteins, and antibodies are Cy3 and Cy5 (Figure 11.17). They are water soluble and possess the reactive succin-
imidyl ester groups that can form covalent linkages with the amino groups of their substrates. Cy3 is red fluorescing, while
Cy5 fluoresces in the far-red region.
Styryl polymethine dyes generally contain a styrene core with electron donor and acceptor attached at each end.
Interestingly, other polymethine stains that possess longer methane chains, such as a naphthyl or pyridyl ring, are also
referred to as styryl dyes. Important members of the group include DASPMI and the FM series of lipophilic membrane stains.
The 4-(4-(dimethylamino)styryl)-
N
-methylpyridinium iodide (DASPMI) is a hemicyanine whose pyridine and phenyl rings
respectively contain and carry the acceptor and donor nitrogens (Figure 11.18). It is a very useful stain for metabolically
active mitochondria in live cells [98-100]. It can also be used to assess the activity of membrane cation transporters [101].
The FM series of styryl polymethine stains are water-soluble lipophilic fluorescent staining agents commonly used in the
imaging of plasma membranes and vesiculation. They show minimal cytotoxicity and are non-fluorescent in aqueous media.
The strong green fluorescence from the FM1-43 and red fluorescence from the FM4-64 are observable only when these
staining agents are inserted into the outer leaflet of the surface membranes of live cells [102, 103]. They are commonly used
in synaptosomal studies, because they are internalised, during the recycling of synaptic vesicles, into neuron terminals that
have been actively releasing neurotransmitters [104, 105].
Coumarin dyes possess a benzo-2-pyrone fluorophore with a carbonyl moiety as an electron acceptor (Figure 11.19). It
is a large dye family with most members having very high photoluminescent quantum efficiency. Some of the family members
