Chemistry Reference
In-Depth Information
11.3 dIfferent types of organIc-based chroMophores
and fluorophores for bIoIMagIng
Before we discuss various advanced molecular imaging and sensing tools for the
in vitro
and
in vivo
studies of life processes,
it is perhaps necessary for us to take a look at the basic building blocks of these tools: the numerous chromophores and fluo-
rophores that have been developed over the last few centuries.
11.3.1
natural dyes as staining for early optical Microscopy
Early microscopists had already recognised the necessity to increase the contrast of transparent tissue specimens in order to
aid the observation and visual discrimination of different tissue and cellular edifices [15-18]. Colorimetric staining of spec-
imens or specific regions of a specimen have rapidly become the mainstream approach. Natural dyes from plants and insects,
such as extracts of saffron, hematoxylins from logwood, and carminic acids from cochineal, were amongst the first batch of
staining agents for microscopy (Figure 11.5; hereafter, the absorption maxima, λ
abs
, and emission maxima, λ
em
, reported are
measured in aqueous solution at neural pH unless otherwise stated).
They were then followed by a rapid exploration of a wide range of natural and synthetic organic dyes of better abilities to
reveal more detailed cellular and subcellular features in all kinds of tissues, micro- and macro-flora, and fauna. In essence,
the history of microscopic staining is the history of the development of synthetic organic dyestuffs [19].
11.3.1.1 Azine dyes
Manuveine, invented in 1856, was the first synthetic chemical dye belonging to the azine dye family
(more precisely, the diaminophenazine family, Figure 11.6) and was first used as a staining agent in optical microscopy in
1862. Other common staining agents in the family include Neutral red and Safranine O. Both produce red fluorescence and
are useful staining agents for cell nuclei and as counterstains for Gram-negative bacteria [20-24]. The related thiazine family
of the dyes has been utilised, in combination with xanthene dyes, for the identification of blood cells and hematopoietic tis-
sues since 1882 [25-27]. Such a thiazine-eosinate neutral staining technique is able to produce characteristic purple-colour
staining due to the formation of neutral ion-pairs between the blue-colour cationic thiazine dyes and the red-colour anionic
xanthene dyes, the nuclear chromatin of cells, and the cytoplasmic granules of polymorphonuclear leukocytes that were pro-
liferated by acute bacterial infection. The mostly used thiazine dye for this purpose is Methylene Blue, which is transformed
into Azure B upon prolonged boiling of its aqueous solution for ion-pairing with the xanthene dye Eosin-Y.
O
O
OH
HO
O
O
OH
HO
HO
O
HO
OH
HO
O
HO
OH
O
OH
O
HO
O
O
HO
OH
HO
OH
β - Gentobiose crocetin of saffron (λ
abs
= 427, 452 nm; λ
em
= 543 nm, ethanol)
O
O
HO
OH
OH
O
OH
HO
HO
OH
OH
O
O
OH
HO
OH
HO
OH
Hamatoxylin from the heartwood
of the logwood tree
(λ
abs
= 445 nm, pH<7.0; 560 nm, pH > 7.0)
Carminic acid from cochineal
(λ
abs
= 490 nm; 590 nm, pH=1.7)
fIgure 11.5
Natural dyes from plants and insects applied as staining agents in the early stage of development of microscopy.
