60
2 Holographic Microscopy of Phase and Diffuse Objects …
histologic specimen, considerable part of the information on the object of investigation is distorted and lost. Such a situation leads, for example, to attempts of using
electronics and computer technologies for the synthesis of 3D images by the averaging of multiple photographs of sections of several homogeneous objects focused
on different levels of morphogenesis. Such techniques are too bulky, complicate the
work of the experimenter and exclude considerable part of structural changes.
In due course, the necessity of developing fixation and visualization histologic
methods was detected by the absence of the possibility to process and study living
specimens immediately and also by the necessity of their dead storage (up to several
years). On the other hand, to date photographic recording of sections and smears is
almost the only way to create disease classifiers and atlases of microobjects.
In its turn, the holographic method of recording and time reconstruction of full
object optical information enables to solve the problems concerning microobjects
certification and also the studies on their recognition in a new way. Living microobjects holograms producing can help when studying rare and typical diseases and
allow classifying these unique pathologies. Taking into consideration the dynamics
of the aspect of the disease and the possibilities of transformation of shape and
sizes of micro objects (red cells, lymphocytes, nerve and muscle fibers and so on),
holographic recording can assist the medical diagnostics. The use of holographic
recording method in microscopy enables to reveal weak movement region in the
microobject quite easily (for example, cytoplasmic flow in the cell [88] or the process
of plasmolysis of the onion epidermis cells). If the motion is not an oscillating periodic process than on the level of the hologram recording, the moving part in the object
does not practically influence its formation and during the reconstruction has smaller
contrast than its fixed parts. Though in this case the quantitative determination of
speed of movement is difficult, velocity profile can be easily determined.
The first works in this area appeared in 1966 when R. F. Wanlighten and Kh.
Osterberg informed about their holographic microscope [89]. During the depth of
field tuning up to 40 μm, the microobjects with the size up to 2 μm could be seen
on the reconstructed image. A hologram preserved the information about 3D object.
G. Ellis [90] went further. He used prior processing of the reconstructed image,
and due to this, he could show phase-contrast, dark field and interference effects.
Diatoms with the size about 100 μm have been chosen as objects of study.
G. Knoh has not only demonstrated the possibilities of holography, but also studied
moving plankton (size is about 1 mm) in movement using for the “fixation” pulsed
ruby laser operated in a free-running lasing mode [91].
K. Snow and R. Wandervaken described holographic microscopes operated in
reflected and transmitted light. Phase as well as diffusive specimens could be chosen
as objects of study. And these microscopes were interference and operated in real
time [92].
The first domestic work in this area appeared in 1972 [93] and was dedicated to the
study of onion epidermis cells. At that time not only qualitative, but also quantitative
data were produced, which were recorded from interferograms. Cell topogram was
produced, and two-dimensional distribution of optical thickness was built. The path
between two neighboring interference fringes was 4 μm. Maximum thickness of
2 Holographic Microscopy of Phase and Diffuse Objects …
histologic specimen, considerable part of the information on the object of investigation is distorted and lost. Such a situation leads, for example, to attempts of using
electronics and computer technologies for the synthesis of 3D images by the averaging of multiple photographs of sections of several homogeneous objects focused
on different levels of morphogenesis. Such techniques are too bulky, complicate the
work of the experimenter and exclude considerable part of structural changes.
In due course, the necessity of developing fixation and visualization histologic
methods was detected by the absence of the possibility to process and study living
specimens immediately and also by the necessity of their dead storage (up to several
years). On the other hand, to date photographic recording of sections and smears is
almost the only way to create disease classifiers and atlases of microobjects.
In its turn, the holographic method of recording and time reconstruction of full
object optical information enables to solve the problems concerning microobjects
certification and also the studies on their recognition in a new way. Living microobjects holograms producing can help when studying rare and typical diseases and
allow classifying these unique pathologies. Taking into consideration the dynamics
of the aspect of the disease and the possibilities of transformation of shape and
sizes of micro objects (red cells, lymphocytes, nerve and muscle fibers and so on),
holographic recording can assist the medical diagnostics. The use of holographic
recording method in microscopy enables to reveal weak movement region in the
microobject quite easily (for example, cytoplasmic flow in the cell [88] or the process
of plasmolysis of the onion epidermis cells). If the motion is not an oscillating periodic process than on the level of the hologram recording, the moving part in the object
does not practically influence its formation and during the reconstruction has smaller
contrast than its fixed parts. Though in this case the quantitative determination of
speed of movement is difficult, velocity profile can be easily determined.
The first works in this area appeared in 1966 when R. F. Wanlighten and Kh.
Osterberg informed about their holographic microscope [89]. During the depth of
field tuning up to 40 μm, the microobjects with the size up to 2 μm could be seen
on the reconstructed image. A hologram preserved the information about 3D object.
G. Ellis [90] went further. He used prior processing of the reconstructed image,
and due to this, he could show phase-contrast, dark field and interference effects.
Diatoms with the size about 100 μm have been chosen as objects of study.
G. Knoh has not only demonstrated the possibilities of holography, but also studied
moving plankton (size is about 1 mm) in movement using for the “fixation” pulsed
ruby laser operated in a free-running lasing mode [91].
K. Snow and R. Wandervaken described holographic microscopes operated in
reflected and transmitted light. Phase as well as diffusive specimens could be chosen
as objects of study. And these microscopes were interference and operated in real
time [92].
The first domestic work in this area appeared in 1972 [93] and was dedicated to the
study of onion epidermis cells. At that time not only qualitative, but also quantitative
data were produced, which were recorded from interferograms. Cell topogram was
produced, and two-dimensional distribution of optical thickness was built. The path
between two neighboring interference fringes was 4 μm. Maximum thickness of