84
2 Holographic Microscopy of Phase and Diffuse Objects …
The number of RNA varies depending on the cell activity level: biosynthesis level,
ATP content, possibly on the level of axoplasmic transport. RNA content shifts in
function neurons can serve as an indicator of their activity changes.
Cytospectrophotometry methods [197], which use light absorption by cell
substance, are of the utmost interest for lifetime studies. But, absorption band of
main cell components—proteins and nucleic acids—is outside of visible region (260–
290 nm). The spectra of separate substances can be blocked what complicates their
identification. Interesting works [198] on lifetime spectrometry of nerve cells have
appeared recently. Using absorption spectra of some pigments, the dynamic of some
redox reactions of huge mollusks neurons was studied.
To visualize different substances in the nerve tissue preparations, they are subject
to color using the reactions of specific bonding of some dyes. Using histochemical
methods, nucleic acids, proteins, fats and ferments distribution were studied [199].
In this respect, more interesting was the use of lifetime dyes, for example, methylene
blue, acridine orange, neutral red, Janus green, which under limited disturbing action
allow seeing separate structures and its dynamic in living cells.
The majority of nerve cells under lifetime estimation appear to be devoided of
dyed structures, but they can differ by refractive index distribution. It became the
basis for a wide use of phase-contrast and interference microscopy in nerve tissue
lifetime studies [200].
Cell structures, for example, membrane structures, being ordered possess the
quality of anisotropy that makes it possible to use polarization devices during
the analysis of their structure [201]. These methods by strengthening the image
contrast allow receiving the information about lifetime cell structure. Using interference microscopy, quantitative analysis was carried out of integral content of dry
substance—proteins, cytoplasm fats and DNA of the nucleus [202].
Till now, we talked about rather slow structural changes of nerve tissue, which
show only the general process of metabolism in function neurons. At the same time,
there appeared considerable number of the works [184–189] dedicated to the direct
analysis of optical changes in excited systems, in particular, in nerve fibers. The
changes recorded graphically in these works are extremely small (ΔF/F ~ 10
−5 –
10
−6 ) that unfortunately excludes their direct observation in an object image. So,
G. N. Berestovskii [203] showed the true change in huge squid axon of light scattering, optical transmission and birefringence. For example, the authors observed
high decrease of birefringence by 0.005 ± 0.002% during the first millisecond after
the stimulation and then slow return to the initial level. The increase of light scattering
was recorded [204] in huge squid axon under the angle of 90° relative to the direction
of transmitted light during action potential. During the record of small-angle light
scattering (10°–30°), converse effect of light scattering decrease is observed (ΔF/F
~ 10
−6 ). Detailed experimental analysis of this firstly detected non-electric process
during the soldering [205] allowed determining that light scattering changes after the
excluding of axoplasm as well as under axon perfusion that testifies the localization
of the observed changes in membrane and near membrane region. Evidently, depolarization causes the amplification of protein hydration and increase of their radial
orientation. Membrane and near membrane protein structures thickness increases
2 Holographic Microscopy of Phase and Diffuse Objects …
The number of RNA varies depending on the cell activity level: biosynthesis level,
ATP content, possibly on the level of axoplasmic transport. RNA content shifts in
function neurons can serve as an indicator of their activity changes.
Cytospectrophotometry methods [197], which use light absorption by cell
substance, are of the utmost interest for lifetime studies. But, absorption band of
main cell components—proteins and nucleic acids—is outside of visible region (260–
290 nm). The spectra of separate substances can be blocked what complicates their
identification. Interesting works [198] on lifetime spectrometry of nerve cells have
appeared recently. Using absorption spectra of some pigments, the dynamic of some
redox reactions of huge mollusks neurons was studied.
To visualize different substances in the nerve tissue preparations, they are subject
to color using the reactions of specific bonding of some dyes. Using histochemical
methods, nucleic acids, proteins, fats and ferments distribution were studied [199].
In this respect, more interesting was the use of lifetime dyes, for example, methylene
blue, acridine orange, neutral red, Janus green, which under limited disturbing action
allow seeing separate structures and its dynamic in living cells.
The majority of nerve cells under lifetime estimation appear to be devoided of
dyed structures, but they can differ by refractive index distribution. It became the
basis for a wide use of phase-contrast and interference microscopy in nerve tissue
lifetime studies [200].
Cell structures, for example, membrane structures, being ordered possess the
quality of anisotropy that makes it possible to use polarization devices during
the analysis of their structure [201]. These methods by strengthening the image
contrast allow receiving the information about lifetime cell structure. Using interference microscopy, quantitative analysis was carried out of integral content of dry
substance—proteins, cytoplasm fats and DNA of the nucleus [202].
Till now, we talked about rather slow structural changes of nerve tissue, which
show only the general process of metabolism in function neurons. At the same time,
there appeared considerable number of the works [184–189] dedicated to the direct
analysis of optical changes in excited systems, in particular, in nerve fibers. The
changes recorded graphically in these works are extremely small (ΔF/F ~ 10
−5 –
10
−6 ) that unfortunately excludes their direct observation in an object image. So,
G. N. Berestovskii [203] showed the true change in huge squid axon of light scattering, optical transmission and birefringence. For example, the authors observed
high decrease of birefringence by 0.005 ± 0.002% during the first millisecond after
the stimulation and then slow return to the initial level. The increase of light scattering
was recorded [204] in huge squid axon under the angle of 90° relative to the direction
of transmitted light during action potential. During the record of small-angle light
scattering (10°–30°), converse effect of light scattering decrease is observed (ΔF/F
~ 10
−6 ). Detailed experimental analysis of this firstly detected non-electric process
during the soldering [205] allowed determining that light scattering changes after the
excluding of axoplasm as well as under axon perfusion that testifies the localization
of the observed changes in membrane and near membrane region. Evidently, depolarization causes the amplification of protein hydration and increase of their radial
orientation. Membrane and near membrane protein structures thickness increases