146
2 Holographic Microscopy of Phase and Diffuse Objects …
For expansion and collimation of the reference beam, a telescopic attachment
was used with magnification of 20×. Balancing of propagation difference between
the reference and the object beams was conducted with the delay line made by the
mirrors 4 and 5. The intensities of the beams were regulated by the neutral filters 14
and 23 and correlated as 1:1 during recording.
The convergence angle of the reference and the object beams was ~30° that corresponds to spatial frequencies of the bearing interference pattern in the hologram plane
of ~600 mm
−1 . For hologram recording, the photolayers PE-2 and LOI-2 were used
with further processing in the developers FMG and GP-2, respectively, with washing
in distilled water and drying in ethanol. Hologram fixing was not performed. The
best diffraction efficiency was reached at the exposure time of ~15 s.
Holographic interferograms of the muscle fibers under study were recorded with
the photocamera 18 (on the film of “Micrat 300” type) or photomultiplier tube (PMT)
22. The camera deformation character was studied beforehand in which the gaseous
pressure was created. For this purpose, wave front was recorded on the hologram,
which passed through the camera, under pressure p = 0. Then, the hologram was
reconstructed by the reference beam, and the interference pattern was observed in real
time in the finite width bands under the summation of wave fronts reconstructed from
the hologram and passing through the camera the pressure of which was increasing.
The experiments showed that “membrane” deformations were not observed for
optical windows in the pressure interval p = 0–4 atm. Meanwhile, the active volume
of the camera was slightly growing as a result of the distance increase between the
optical windows approximately by 10 μm. Such camera deformations correspond to
additional phase incursion ϕ = const and can have significant errors as a result of
measurements.
While processing the method of interference study of single muscle fibers under
hyperbary, differential holographic interferometry methods were tested both of the
double-exposed interferometry as well as of the real-time one. In this case, comparison of wave fronts was conducted which passed through the same fiber under two
different types of pressure.
However, the experiment showed that it is difficult to share the contribution to
the alterations of the muscle fiber interferogram caused directly by the pressure and
by uncontrolled shift of the fiber between the exposures. The first exposure was
conducted under the camera pressure p = 0, then the camera was moved with the
help of a microscrew in the horizontal plane by 100–200 μm, and the reference beam
inclination was changed by the turn of the wedge. Then, pressure was pumped, and
the second exposure was conducted. When such a hologram is lighted with such a
beam, the microobject interferogram is formed in the bands of finite width, and the
first image corresponds to the absence of pressure, and the second one—to microobject under hyperbary. For simplification of interferogram processing procedure, the
reference bands were oriented perpendicular to the axis of the muscle fiber.
The series of holographic interferograms of single muscle fibers obtained in the
bands of finite and infinite width is presented in Fig. 2.41. The local inclination of the
interference band y(x) at point x in the interferogram makes it possible to estimate
phase incursion ϕ(x) carried in by the fiber
2 Holographic Microscopy of Phase and Diffuse Objects …
For expansion and collimation of the reference beam, a telescopic attachment
was used with magnification of 20×. Balancing of propagation difference between
the reference and the object beams was conducted with the delay line made by the
mirrors 4 and 5. The intensities of the beams were regulated by the neutral filters 14
and 23 and correlated as 1:1 during recording.
The convergence angle of the reference and the object beams was ~30° that corresponds to spatial frequencies of the bearing interference pattern in the hologram plane
of ~600 mm
−1 . For hologram recording, the photolayers PE-2 and LOI-2 were used
with further processing in the developers FMG and GP-2, respectively, with washing
in distilled water and drying in ethanol. Hologram fixing was not performed. The
best diffraction efficiency was reached at the exposure time of ~15 s.
Holographic interferograms of the muscle fibers under study were recorded with
the photocamera 18 (on the film of “Micrat 300” type) or photomultiplier tube (PMT)
22. The camera deformation character was studied beforehand in which the gaseous
pressure was created. For this purpose, wave front was recorded on the hologram,
which passed through the camera, under pressure p = 0. Then, the hologram was
reconstructed by the reference beam, and the interference pattern was observed in real
time in the finite width bands under the summation of wave fronts reconstructed from
the hologram and passing through the camera the pressure of which was increasing.
The experiments showed that “membrane” deformations were not observed for
optical windows in the pressure interval p = 0–4 atm. Meanwhile, the active volume
of the camera was slightly growing as a result of the distance increase between the
optical windows approximately by 10 μm. Such camera deformations correspond to
additional phase incursion ϕ = const and can have significant errors as a result of
measurements.
While processing the method of interference study of single muscle fibers under
hyperbary, differential holographic interferometry methods were tested both of the
double-exposed interferometry as well as of the real-time one. In this case, comparison of wave fronts was conducted which passed through the same fiber under two
different types of pressure.
However, the experiment showed that it is difficult to share the contribution to
the alterations of the muscle fiber interferogram caused directly by the pressure and
by uncontrolled shift of the fiber between the exposures. The first exposure was
conducted under the camera pressure p = 0, then the camera was moved with the
help of a microscrew in the horizontal plane by 100–200 μm, and the reference beam
inclination was changed by the turn of the wedge. Then, pressure was pumped, and
the second exposure was conducted. When such a hologram is lighted with such a
beam, the microobject interferogram is formed in the bands of finite width, and the
first image corresponds to the absence of pressure, and the second one—to microobject under hyperbary. For simplification of interferogram processing procedure, the
reference bands were oriented perpendicular to the axis of the muscle fiber.
The series of holographic interferograms of single muscle fibers obtained in the
bands of finite and infinite width is presented in Fig. 2.41. The local inclination of the
interference band y(x) at point x in the interferogram makes it possible to estimate
phase incursion ϕ(x) carried in by the fiber