Biophotonics_Concepts_to_Applications

• An excitation optical filter, which passes only the selected spectral band
  (≈40 nm wide) needed for absorption by thefluorophore being used


• A dichroic mirror (or beamsplitter), which deflects short-wavelength light from
  the excitation source to the specimen and passes longer-wavelengthfluorescent
  light from thefluorophore to the photodetector


• An emissionfilter, which blocks light from the excitation wavelengths and
  passes only the spectral region in which thefluorescent emission occurs
  As described in Chap. 9 ,fluorescence lifetime imaging microscopy(FLIM) is an
  advanced version of the fluorescence microscopy technique. By generating
  fluorescence images based on the differences in the exponential decay times of
  fluorescence from excited molecules, FLIM can detect fluorophore-fluorophore
  interactions and environmental effects onfluorophores.

8.5 Multiphoton Microscopy.

In conventionalfluorescence microscopy, a single photon is used to excite afluor-
ophore from a ground state level to a higher energy state. Typically this requires
using photons from the ultraviolet or blue-green spectral range. Because such
photons have high energies that could damage certain biological materials, the
concept ofmultiphoton microscopywas devised [ 23 – 25 ]. For example, consider the
case of two-photon as shown in Fig.8.14. This excitation process yields the same
results as single-photon excitation, but is generated by the simultaneous absorption
of two less energetic photons (typically in the infrared spectral range) if there is
sufficiently intense laser illumination. For multiphoton excitation the sum of the
energies of the two photons needs to be greater than the energy gap between the
ground state and the excited states of the molecule under investigation. Three-photon

Dichroic mirror

Viewer or camera

Light

source

Specimen

Excitation

filter

Emission

filter

Fig. 8.13 Basic setup and
components afluorescence
microscope

8.4 Fluorescence Microscopy 251