Optical Imaging in Biology: Basics and Applications
641
Here N is the density of the molecules per unit volume at standard temperature and
pressure, and n is the refractive index. The above equation shows that the scattering
is dependent on 1/λ
4 .
When the size of the particle is in the order of magnitude of the incident radiation,
the scattering is described by Mie scattering, which closely follows geometrical
optics. The Maxwell equations are solved in spherical coordinates to calculate the
scattering cross-section, and also the scattering anisotropy factor.
Another type of scattering which is often harnessed for imaging and materials
characterization is Raman scattering, where only the electrical component of the incident radiation interacts with the molecule or the material. The electric field induces
a dipole moment in the molecule which is given by:
π = α E loc = ε 0 α el E loc
Here α is the polarizability of the molecule and E loc is the electric field and ε 0
is the permittivity of the free space. Oscillating dipoles radiate energy in the form
of radiation with the same oscillating frequency. This scattered radiation is of three
types Rayleigh, Stokes, and anti-stokes (Fig. 1) [6]. Among these, Rayleigh scattering
(elastic scattering) is the most common where no change in the energy of the incident
and scattered photon is observed. In contrast, when there is a change in the energy
of the scattered photons the process is called inelastic or Raman scattering. In case
of energy transfer from photon to the molecule, if the scattered photons have higher
energy it is called Strokes scattering. The opposite of this is anti-strokes scattering.
As most of the molecules, stay in their fundamental energy state at room temperature
the majority of the Raman scattering is of stokes type. Both absorption and scattering
based methods such as fluorescence, Raman, and infrared have emerged as important
tools in biological imaging [7–10] and will be discussed in detail in the subsequent
sections.
3 Instrumentation
A typical bio-optical imaging system consists of an excitation unit, a light transmission unit configured to illuminate a region of interest with incident light, and a
detection unit configured to sense the resulting signals. For macro- and mesoscopic
imaging, the transmission unit is composed of an optical fiber which guides the
filtered light from the excitation unit to the sample (Fig. 2). For microscopy, the
transmission unit contains a dichroic mirror which reflects the desired excitation
wavelength through an objective onto a biological sample. The spectrometer serves
as the detection unit and houses the dispersive element and forms the central part of
an imaging system. It can have one or multiple entrance slits and can house a detector
or camera (Fig. 2). Scanning in a spectrometer can provide spatial information.
641
Here N is the density of the molecules per unit volume at standard temperature and
pressure, and n is the refractive index. The above equation shows that the scattering
is dependent on 1/λ
4 .
When the size of the particle is in the order of magnitude of the incident radiation,
the scattering is described by Mie scattering, which closely follows geometrical
optics. The Maxwell equations are solved in spherical coordinates to calculate the
scattering cross-section, and also the scattering anisotropy factor.
Another type of scattering which is often harnessed for imaging and materials
characterization is Raman scattering, where only the electrical component of the incident radiation interacts with the molecule or the material. The electric field induces
a dipole moment in the molecule which is given by:
π = α E loc = ε 0 α el E loc
Here α is the polarizability of the molecule and E loc is the electric field and ε 0
is the permittivity of the free space. Oscillating dipoles radiate energy in the form
of radiation with the same oscillating frequency. This scattered radiation is of three
types Rayleigh, Stokes, and anti-stokes (Fig. 1) [6]. Among these, Rayleigh scattering
(elastic scattering) is the most common where no change in the energy of the incident
and scattered photon is observed. In contrast, when there is a change in the energy
of the scattered photons the process is called inelastic or Raman scattering. In case
of energy transfer from photon to the molecule, if the scattered photons have higher
energy it is called Strokes scattering. The opposite of this is anti-strokes scattering.
As most of the molecules, stay in their fundamental energy state at room temperature
the majority of the Raman scattering is of stokes type. Both absorption and scattering
based methods such as fluorescence, Raman, and infrared have emerged as important
tools in biological imaging [7–10] and will be discussed in detail in the subsequent
sections.
3 Instrumentation
A typical bio-optical imaging system consists of an excitation unit, a light transmission unit configured to illuminate a region of interest with incident light, and a
detection unit configured to sense the resulting signals. For macro- and mesoscopic
imaging, the transmission unit is composed of an optical fiber which guides the
filtered light from the excitation unit to the sample (Fig. 2). For microscopy, the
transmission unit contains a dichroic mirror which reflects the desired excitation
wavelength through an objective onto a biological sample. The spectrometer serves
as the detection unit and houses the dispersive element and forms the central part of
an imaging system. It can have one or multiple entrance slits and can house a detector
or camera (Fig. 2). Scanning in a spectrometer can provide spatial information.