Optical Imaging in Biology: Basics and Applications
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Other types of spectrometers are the band-sequential spectrometers where
grayscale images can be obtained by scanning the emitted light through several wavelengths. An image stack is obtained which can be subjected to analysis. A popular
type of spectrometer that uses this technique is the Fourier Transform Imaging spectrometers (Fig. 2). FTIR spectrometers are generally used to probe samples in the
infrared region. The underlying principle of these is based on the fact that when
light interferes with itself after the introduction of a path difference, the constituent
spectral information can be extracted from the interferogram. The multispectral light
will generate an interferogram that is the sum of their respective cosine waves. The
presence and intensities of the constituents can be given by an inverse Fourier transform. A Michelson interferometer is often used in this type of arrangement, and by
rotating the beam or the interferometer at low angles, the optical path difference is
created. The beam coming from the collection objective is divided into two beams
and are then recombined to obtain the interference pattern. By fast Fourier transformation, the entire spectrum is recovered from the interferogram. There are other
spectrometers such as the single-shot spectrometers which can enable imaging of
fast biological processes which is not possible with other scanning spectrometers. In
such spectrometers, a hologram is generated, and a disperser is used to distribute the
diffraction pattern on a 2-dimensional CCD array. The spectral content is calculated
or reconstructed using an algebraic reconstruction algorithm; therefore, the inherent
spatial information can be obtained along with the spectral data from just one image
at a very short exposure time.
4 Fluorescence Imaging
The emission of light when molecules revert to the ground state from an electronically excited state through any physical, mechanical, or chemical mechanism is
called luminescence. Photoluminescence can be defined as luminescence generated
under the influence of ultraviolet or visible radiation. This can be broadly divided into
two categories, fluorescence, and phosphorescence. Both of these processes involve
the spontaneous emission of a longer wavelength of light. The main difference is that
in fluorescence the signal stops immediately in the absence of the excitation source.
Phosphorescence involves an afterglow with durations ranging from fractions of a
second up to hours even in the absence of excitation light. Sir George G. Stokes
first described fluorescence in 1852 based on his observation that mineral ‘fluorspar’
produces light upon excitation with ultraviolet radiation. This was later validated
thoroughly across different specimens such as crystals, resins, chlorophyll, vitamins,
and inorganic compounds. Autofluorescence is another variant that can be described
as a natural phenomenon of emitting light without any excitation. The most common
examples from biological systems are nicotinamide adenine dinucleotide phosphate
hydrogen (NADPH) and Flavins. Also, often the proteins containing aromatic amino
acids such as tryptophan, tyrosine, and phenylalanine produce autofluorescence. The
other type involves the production of fluorescence signals due to the incorporation of
643
Other types of spectrometers are the band-sequential spectrometers where
grayscale images can be obtained by scanning the emitted light through several wavelengths. An image stack is obtained which can be subjected to analysis. A popular
type of spectrometer that uses this technique is the Fourier Transform Imaging spectrometers (Fig. 2). FTIR spectrometers are generally used to probe samples in the
infrared region. The underlying principle of these is based on the fact that when
light interferes with itself after the introduction of a path difference, the constituent
spectral information can be extracted from the interferogram. The multispectral light
will generate an interferogram that is the sum of their respective cosine waves. The
presence and intensities of the constituents can be given by an inverse Fourier transform. A Michelson interferometer is often used in this type of arrangement, and by
rotating the beam or the interferometer at low angles, the optical path difference is
created. The beam coming from the collection objective is divided into two beams
and are then recombined to obtain the interference pattern. By fast Fourier transformation, the entire spectrum is recovered from the interferogram. There are other
spectrometers such as the single-shot spectrometers which can enable imaging of
fast biological processes which is not possible with other scanning spectrometers. In
such spectrometers, a hologram is generated, and a disperser is used to distribute the
diffraction pattern on a 2-dimensional CCD array. The spectral content is calculated
or reconstructed using an algebraic reconstruction algorithm; therefore, the inherent
spatial information can be obtained along with the spectral data from just one image
at a very short exposure time.
4 Fluorescence Imaging
The emission of light when molecules revert to the ground state from an electronically excited state through any physical, mechanical, or chemical mechanism is
called luminescence. Photoluminescence can be defined as luminescence generated
under the influence of ultraviolet or visible radiation. This can be broadly divided into
two categories, fluorescence, and phosphorescence. Both of these processes involve
the spontaneous emission of a longer wavelength of light. The main difference is that
in fluorescence the signal stops immediately in the absence of the excitation source.
Phosphorescence involves an afterglow with durations ranging from fractions of a
second up to hours even in the absence of excitation light. Sir George G. Stokes
first described fluorescence in 1852 based on his observation that mineral ‘fluorspar’
produces light upon excitation with ultraviolet radiation. This was later validated
thoroughly across different specimens such as crystals, resins, chlorophyll, vitamins,
and inorganic compounds. Autofluorescence is another variant that can be described
as a natural phenomenon of emitting light without any excitation. The most common
examples from biological systems are nicotinamide adenine dinucleotide phosphate
hydrogen (NADPH) and Flavins. Also, often the proteins containing aromatic amino
acids such as tryptophan, tyrosine, and phenylalanine produce autofluorescence. The
other type involves the production of fluorescence signals due to the incorporation of