Biophotonics_Concepts_to_Applications

sample returns through thefiber bundle, passes through the scanning and dichroic
mirror system, and travels to the photodetector through the confocal pinhole
aperture.

8.4 Fluorescence Microscopy

Fluorescence microscopy has become an important tool in biophotonics for clari-
fying many questions in the life sciences and in medicine. As Sect.6.7describes
and as Fig.8.12shows, certain atoms and molecules have the ability to absorb light
at a particular wavelength, for example,λ 1 orλ 2. This absorption occurs on the
order of 10−^15 s (femtoseconds) and elevates the molecule to a higher energy level
as indicated by the solid upward arrows. Immediately following the absorption the
molecules drop to a more stable intermediate energy level through a non-radiative
transition shown by the dashed downward arrows. This occurs on the order of
10 −^14 – 10 −^11 s. Subsequently, as indicated by the solid downward arrows, the
molecule drops back to a lower energy level after a time interval in the order of
10 −^9 – 10 −^7 s, thereby emitting light of a longer wavelength (lower energy), for
example,λ 3 orλ 4. This spectroscopic property is calledfluorescenceand is widely
used as an imaging mode in biological laser-scanning confocal microscopy. The
advantage of this technique is the ability to target specific in vitro and in vivo
structural components and dynamic chemical and biological processes [ 19 – 22 ].
Further details on its application in areas such asfluorescence spectroscopy,
fluorescence lifetime imaging, andfluorescence correlation spectroscopy are given
in Chap. 9.
Recall from Sect.6.7that thefluorescing light can originate either from internal
fluorophores or selective externalfluorophores. These externalfluorophores basi-
cally are probes that are known as dyes, labels, markers, stains, or tags. Thus,

Confocal

pinhole

apertures

Laser

Beam expander

and collimator

Camera or

photodetector

Scan focusing lens

Tissue sample

xy scanning

mechanism

Optical fiber bundle

Beam

splitter

Fig. 8.11 Setup for an opticalfiber-based confocal microscope system

8.3 Confocal Microscopy 249