tryptophan, tyrosine, phenylalanine),flavin, collagen, melanin, elastin, and NADH
(a reduced form of nicotinamide adenine dinucleotide). Table6.5lists the excitation
andfluorescence wavelengths of some common intrinsicfluorophores.
Many types of biological molecules are either nonfluorescent or the intrinsic
fluorescence is not adequate for a particular desired molecular analysis. For
example, substances such as DNA and lipids (e.g., fats, triglycerides, and vitamins
A, D, E, and K) possess no intrinsicfluorescence characteristics. In these cases
extrinsicfluorophores can be attached (generally through covalent bonding) to the
molecules to providefluorescence for experimental studies. An extrinsicfluor-
ophore is known by various names such as alabel,marker,dye,ortag. Thus the
attachment process is known aslabeling, marking,dyeing,ortagging of the
molecule. Many varieties of extrinsicfluorophores are commercially available with
individual peak wavelengths located throughout the visible spectrum. Table6.6
lists the excitation and fluorescence wavelengths of some example extrinsic
fluorophores.
The discovery ofgreenfluorescent protein(GFP) started a new era in cell
biology to monitor cellular processes in living cells and organisms usingfluores-
cence microscopy and related methodologies [ 64 – 66 ]. Subsequent to the initial use
of GFP, a number of genetic variants that exhibitedfluorescence emission in dif-
ferent regions of the visible spectrum were developed from the original GFP
nucleotide sequence. These include blue, cyan, and yellowfluorescent protein,
which go by the acronyms BFP, CFP, and YFP. Longer wavelengthfluorescent
proteins emitting in the orange and red spectral regions also have been developed
[ 67 ]. Consequently, a large number offluorescent proteins are available with
emission maxima ranging from 424 to 625 nm.
6.8 Summary
Light-tissue interaction is a complex process because the constituent tissue mate-
rials are multilayered, multicomponent, and optically inhomogeneous. The basic
effects include reflection at a material interface, refraction when light enters a tissue
structure that has a different refractive index, absorption of photon energy by the
material, and multiple scattering of photons in the material. Because diverse
Table 6.6 Excitation and
fluorescence wavelengths of
some common extrinsic
fluorophores
Molecule Excitation (nm) Fluorescence (nm)
Dansyl chloride 350 520
Fluorescein 480 510
Rhodamine 600 615
Prodan 360 440
BIODIPY-FL 503 512
Texas Red 589 615190 6 Light-Tissue Interactions