Other Biomedical Imaging Techniques
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produces diffraction patterns that result in blurring of each demarcated item in the
field of view. When the diffraction patterns of two adjacent items overlap, the items
cannot be distinguished with any reasonable accuracy. This limitation is called the
Rayleigh criterion (also referred to as the Abbe criterion). The Rayleigh criterion
gives the boundary conditions for the smallest angle that separates two objects that
can be observed clearly for a round aperture/lens as a function of wavelength, λ, and
aperture diameter, D, as shown in Equation 18.3:
l
q min = 1 22
.
(18.3)
D
This equation states that the resolution of the optical microscopes is restricted by the
wavelength of the light. In addition, since the images created by microscopes need
to be perceived and interpreted by the human eyes, the resolution of this technology
is also limited by the limitations of the human eyes. Specifically, due to the spacing
of the rods and cones in the retina of the eye, the maximum useful magnification of
the optical microscope is only 600 times. Despite these limitations, the compound
microscope has allowed biologists to examine specimens and objects whose sizes
are within micrometer range. Such objects include cells and some of their organelles.
One of the main disadvantages of optical microscopy is the fact that a tissue
slice is needed. This requires biopsy, i.e., removal of a sample from the biological
medium, which is an invasive process.
Nowadays, virtually every professional microscope has an accessory that will
provide a mounting alternative for a camera to record the histology image for filing
and image processing. All image processing techniques discussed in the previous
chapters are used to improve the quality of the captured images. A representative
image of an optical microscope with camera attached is shown in Figure 18.2. A histology image of an aneurysm in heart muscle captured by an optical microscope is
shown in Figure 18.3.
The demand for increased detail and resolution has led to the development of
several other imaging techniques, mainly initially based on the principle of optical microscopy. Next, we discuss one of these technologies called fluorescent
microscopy.
18.3 FLUORESCENT MICROSCOPY
The phenomenon of fluorescence was discovered by the end of nineteenth century
by the British scientist George G. Stokes (1819–1903). Stokes observed that several
chromophores emit light after illumination. Specifically, he noted that the emitted
light from some biological samples after illumination has a longer wavelength than
the irradiation source. This observation led to a new generation of microscopes that
allow imaging of biological tissues based on the fluoresce emission of the objects in
the sample.
Several biological molecules and objects, such as pigments, resins, and vitamins, exhibit a phenomenon known as autofluorescence. Other tissues may not be
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produces diffraction patterns that result in blurring of each demarcated item in the
field of view. When the diffraction patterns of two adjacent items overlap, the items
cannot be distinguished with any reasonable accuracy. This limitation is called the
Rayleigh criterion (also referred to as the Abbe criterion). The Rayleigh criterion
gives the boundary conditions for the smallest angle that separates two objects that
can be observed clearly for a round aperture/lens as a function of wavelength, λ, and
aperture diameter, D, as shown in Equation 18.3:
l
q min = 1 22
.
(18.3)
D
This equation states that the resolution of the optical microscopes is restricted by the
wavelength of the light. In addition, since the images created by microscopes need
to be perceived and interpreted by the human eyes, the resolution of this technology
is also limited by the limitations of the human eyes. Specifically, due to the spacing
of the rods and cones in the retina of the eye, the maximum useful magnification of
the optical microscope is only 600 times. Despite these limitations, the compound
microscope has allowed biologists to examine specimens and objects whose sizes
are within micrometer range. Such objects include cells and some of their organelles.
One of the main disadvantages of optical microscopy is the fact that a tissue
slice is needed. This requires biopsy, i.e., removal of a sample from the biological
medium, which is an invasive process.
Nowadays, virtually every professional microscope has an accessory that will
provide a mounting alternative for a camera to record the histology image for filing
and image processing. All image processing techniques discussed in the previous
chapters are used to improve the quality of the captured images. A representative
image of an optical microscope with camera attached is shown in Figure 18.2. A histology image of an aneurysm in heart muscle captured by an optical microscope is
shown in Figure 18.3.
The demand for increased detail and resolution has led to the development of
several other imaging techniques, mainly initially based on the principle of optical microscopy. Next, we discuss one of these technologies called fluorescent
microscopy.
18.3 FLUORESCENT MICROSCOPY
The phenomenon of fluorescence was discovered by the end of nineteenth century
by the British scientist George G. Stokes (1819–1903). Stokes observed that several
chromophores emit light after illumination. Specifically, he noted that the emitted
light from some biological samples after illumination has a longer wavelength than
the irradiation source. This observation led to a new generation of microscopes that
allow imaging of biological tissues based on the fluoresce emission of the objects in
the sample.
Several biological molecules and objects, such as pigments, resins, and vitamins, exhibit a phenomenon known as autofluorescence. Other tissues may not be