Chemistry Reference
In-Depth Information
R
1
N
+
H
N
H
2
N
Mauveine A: R
1
, R
2
=H
Mauveine B: R
1
= Me; R
2
=H
Mauveine B
2
: R
1
= H; R
2
=Me
Mauveine C: R
1
, R
2
=Me
R
2
Mauveine dyes
λ
abs
(nm)
λ
em
(nm)
Azine & thiazine dyes
Remarks
Mauveine
∼ 548
Neutral red
454 (pH 8.1)
529 (pH 5.8)
640
50% aqueous
EtOH
Safranine O
530
587
50% aqueous
EtOH
Methylene blue
656 (H2O)
661 (MeOH)
Azure B
639
MeOH
fIgure 11.6
The azine and thiazine dye families.
HO
O
O
COOH
Fluorescein
λ
abs
(nm)
λ
em
(nm)
Hydroxyxanthene dyes
Remarks
Fluorescein
494
521
/
Eosin-Y
516
538
/
Erythrosin B
525
555
pH >7.0
Rose bengal B
548
567
pH >7.0
Calcium-green
TM
-1
506
531
When bound with Ca
2+
fIgure 11.7
The hydroxyxanthene dyes.
11.3.1.2 Xanthene dyes
The first anionic hydroxyxanthene dye, fluorescein (Figure 11.7), was synthesised in 1871.
Soon afterward, a series of structurally related organic dyes appeared and were found to be extremely important in micros-
copy staining, biolabelling, and bioimaging applications because of their outstanding luminescent properties and biocompat-
ibility. As mentioned in the previous section, the brominated hydroxyxanthene, Eosin-Y, is used, coupled with Methylene
Blue, in the thiazine-eosinate staining. The iodinated hydroxyxanthene Erythrosin, and the mixed chlorinated iodinated
hydroxyxanthene rose Bengal are also important chromophores and luminophores in microscopy staining, bioimaging, and
chemosensing. Besides these hydroxyxanthenes, another eminent subfamily of xanthene is the aminoxanthene dyes—the
rhodamines. These dyes will be discussed in the later sections concerning fluorescent bioimaging.
11.3.1.3 Triarylmethines
Another class of organic dyes that were developed and applied as microscopy staining agents
around the same period of time was the triarylmethines (Figure 11.8) [28]. representatives of this dye family include Fuchsine,
Aniline Blue, Methyl Violet, and Malachite Green. Basic Fuchsine is routinely being used as a nuclear and mucin stain in his-
tology [29, 30]. It is also used as the visualising reagent for enzyme labelling in immunostaining. Aniline Blue is useful in
staining of collagen [31-33]. In combination with Eosin-Y, Aniline Blue can also be used to assess sperm viability [34]. On the
other hand, Crystal Violet (i.e., Methyl Violet 10B) is the primary staining agent in the Gram staining of bacteria [35, 36],
Malachite Green is used as a staining agent for bacterial spores [37, 38], and as a counterstain in the Gimenez staining of bacte-
rial infection in tissues [39, 40]. It is widely used, paired with glutaraldehyde, as a fixative-stain for phospholipids as well [41].
