Biophotonics_Concepts_to_Applications

fluorescence microscopy is a technique for viewing specimens that aredyed(also
referred to asstained),labeled,marked,ortaggedwith one or morefluorophores.
Both internal and externalfluorescent probes can be utilized in a variety of ways.
Internalfluorophoresinclude aromatic amino acids, collagen, elastin,flavins, and
NADH. Many extrinsic fluorophores (for example, dansyl chloride and the
fluorescent dyesfluorescein, rhodamine, prodan, and BIODIPY) are available for
fluorescent labeling of macromolecules such as proteins, amino acids, peptides, and
amines to assess molecular characteristics and dynamics. For example, dansyl
chloride can be excited by 350-nm light (where proteins do not absorb light) and
typically emit near 520 nm. The dyesfluorescein and rhodamine have absorption
maxima near 480 and 600 nm and emission peaks ranging from 510 to 615 nm,
respectively. On a smaller scale, thesefluorophores can be localized within a cell to
examine specific cell structures such as the cytoskeleton, endoplasmic reticulum,
Golgi apparatus, and mitochondria.
Other applications of fluorophores in fluorescent spectroscopy include the
following:

• Antibody/antigen fiber sensors for an enzyme-linked immunosorbent assay
  (ELISA) (see Sect. 7.3.2)


• Monitors of dynamic molecular processes


• Monitors of cellular integrity, endocytosis and exocytosis (the movement of
  material into and out of a cell, respectively), membranefluidity, and enzymatic
  activity


• Genetic mapping
  It should be noted that manyfluorophores are susceptible tophotobleaching
  effects. This is the result of a photochemical alteration of a dye orfluorophore
  molecule that causes it to no longerfluoresce. This photobleaching effect can occur
  anytime from after a few cycles to after many thousands offluorescent cycles.
  The basic setup, components, and light paths of an upright fluorescence
  microscope are illustrated in Fig.8.13. The four main elements are the following:


• An excitation light source, for example, a xenon arc lamp, a mercury vapor
  lamp, or a selected laser operating in a specific spectral band

S 2

S 1

S 0

Excitation

photons

λ 1 λ 2 λ 3 λ 4

Fluorescence

photons

Non-radiative transitions (10-14 -10-11 s)

Ground state

energy level

Energy levels for excited state 2

Energy levels for

excited state 1

10 -15 s^10 -9 -10-7 s

Fig. 8.12 Example of a
molecularfluorescence
processes

250 8 Microscopy