2.1 Holographic Microscopy for the Study of Phase, Diffusive …
59
2.1 Holographic Microscopy for the Study of Phase,
Diffusive and Mirror Microobjects
For the first time, the use of a holographic recording of wave front with its consequent
reconstruction as a lensless method of microobjects studies was proposed by D. Gabor
[78–82]. Though his idea of images enlargement due to the difference of wavelengths
at the level of recording and during the reconstruction has not found wide application,
the holographic principle itself considerably broadened and added the possibilities
of classical microscopy approaches [83, 84]. Microscopy, using such possibilities
of holography and holographic interferometry as microobjects 3D image recovery
with its posterior processing, interferometric comparison of time-spaced microobject
states of any irregular shape [85, 86], has a number of advantages over the classical
interference microscopy.
As it is known, in the studies of medical and biological objects, in vivo microscopy
plays a great role. But, along with noninvasiveness, high sensitivity, precision, resolution, possibility to observe the microobjects under study with rather high magnification and so on what differs in vivo microscopy advantageously, this method
like any other method has some disadvantages. So, in optical microscopes a large
increase inevitably leads to a decrease in sharpness. It complicates the study and
photorecording of short-lived bulk structure, the sizes of which are beyond the limits
of depth of microscope objective sharpness. The possibilities of classical in vivo
microscopy are considerably limited by the object mobility. The objects with higher
mobility, such as muscle tissue and so on, are not suitable for the microscopical
study, because at insignificant contractions, the structures under consideration get
outside the field of view, especially when working with high magnification. The same
difficulty can be observed in cinemicrography. Moreover, in classical microscopy,
the difference in depth planes of the object under study (it can be a cell or cell population) is recorded, for example, photographically with the objective overfocusing
during each exposure. It is obvious that time consumed on overfocusing, microscope
adjustment and single-frame photography of microobject section limits and sometimes excludes the possibility of research operation. Firstly, it concerns the study of
fast processes or short-lived microobjects where there is a necessity to set volume
correlation between structural formations in the present moment (in the studies of
the process of cell or cell ensemble division or during microcirculation and so on).
Moreover, living organisms are used to be developing continuously. A morphologist, a physiologist or a cytochemist looking through the microscope in some cases
cannot be sure that focusing on the new preparation part they see it in the same
functional mode. Developing processes in some cases are so transient that they can
escape the researcher’s view because of the inertness of human visual analyzer. In
this connection, the number of objects suitable for the studies by the method of vital
microscopy is limited. The limitation of lifetime of the majority of microobjects
leads to the fact that in the classical microscopy considerable part of the studies is
connected with the estimation of microobjects quantity and geometrics in sections,
smears and so on [87]. At the same time when changing a living microobject on a
59
2.1 Holographic Microscopy for the Study of Phase,
Diffusive and Mirror Microobjects
For the first time, the use of a holographic recording of wave front with its consequent
reconstruction as a lensless method of microobjects studies was proposed by D. Gabor
[78–82]. Though his idea of images enlargement due to the difference of wavelengths
at the level of recording and during the reconstruction has not found wide application,
the holographic principle itself considerably broadened and added the possibilities
of classical microscopy approaches [83, 84]. Microscopy, using such possibilities
of holography and holographic interferometry as microobjects 3D image recovery
with its posterior processing, interferometric comparison of time-spaced microobject
states of any irregular shape [85, 86], has a number of advantages over the classical
interference microscopy.
As it is known, in the studies of medical and biological objects, in vivo microscopy
plays a great role. But, along with noninvasiveness, high sensitivity, precision, resolution, possibility to observe the microobjects under study with rather high magnification and so on what differs in vivo microscopy advantageously, this method
like any other method has some disadvantages. So, in optical microscopes a large
increase inevitably leads to a decrease in sharpness. It complicates the study and
photorecording of short-lived bulk structure, the sizes of which are beyond the limits
of depth of microscope objective sharpness. The possibilities of classical in vivo
microscopy are considerably limited by the object mobility. The objects with higher
mobility, such as muscle tissue and so on, are not suitable for the microscopical
study, because at insignificant contractions, the structures under consideration get
outside the field of view, especially when working with high magnification. The same
difficulty can be observed in cinemicrography. Moreover, in classical microscopy,
the difference in depth planes of the object under study (it can be a cell or cell population) is recorded, for example, photographically with the objective overfocusing
during each exposure. It is obvious that time consumed on overfocusing, microscope
adjustment and single-frame photography of microobject section limits and sometimes excludes the possibility of research operation. Firstly, it concerns the study of
fast processes or short-lived microobjects where there is a necessity to set volume
correlation between structural formations in the present moment (in the studies of
the process of cell or cell ensemble division or during microcirculation and so on).
Moreover, living organisms are used to be developing continuously. A morphologist, a physiologist or a cytochemist looking through the microscope in some cases
cannot be sure that focusing on the new preparation part they see it in the same
functional mode. Developing processes in some cases are so transient that they can
escape the researcher’s view because of the inertness of human visual analyzer. In
this connection, the number of objects suitable for the studies by the method of vital
microscopy is limited. The limitation of lifetime of the majority of microobjects
leads to the fact that in the classical microscopy considerable part of the studies is
connected with the estimation of microobjects quantity and geometrics in sections,
smears and so on [87]. At the same time when changing a living microobject on a