2.2 Holographic Study of Structural and Functional Characteristics …
113
Surviving preparation of a myelinated nerve fiber was made by splitting of a frog
sciatic nerve with tungsten needles. Depending on the experiment requirements, the
fiber was put into Ringer physiological solution (n = 1.3337), silicon (n = 1.4057)
and vaseline (n = 1.4805) oil. The functional state of the nerve fiber, used in the
experiments, was estimated according to its ability of stable potential generation
in response to electric irritation produced by right-angled current pulses with the
duration of 0.1 ms and frequency of 1 Hz. When double-exposed holographic interferograms in bands with infinite width were ready, the first exposure corresponded to
preparation addition into the object beam. During the second exposure, the object was
absent. Interferograms in infinite width bands were formed by glass wedge injection
of 3° and 5° into the object beam (band period was 0.4–0.2 μm). For differential interferogram recording, two stages of the same object separated by some time interval
were recorded on one hologram.
To study structure nerve fiber alterations in rhythmic stimulation regime, the
stroboholographic method was used (see Fig. 2.14b). In this case, a stroboscope with
a system of synchronization was placed between the continuous wave laser and the
beam splitter. The character of hologram recording is accumulative. For microobjects
like nerve fiber, the properties of which are reversible for excitation frequencies of
100 Hz, it is possible to record holograms with a set of laser synchronized pulses of
low intensiveness with a certain excitation phase.
To register holographic interferograms in different excitation phases, a stroboscope with a synchronizing system was used, which included a delay unit. Laser
radiation after the stroboscope had the duration of 1 ms with the frequency of 100
Hz. A delay line was launched simultaneously with lightning the object through a
photodiode. The form and the duration of the beam pulse were controlled by photodiode FA-27A and double-beam oscillograph 15 (see Fig. 2.14c). The delayed pulse
controlled the electrical stimulator in such a way that the action potential took place
in the microscope visual field simultaneously with the hologram recording beam.
The electrical stimulator occasionally sent a stimulating pike-shaped pulse onto the
nerve fiber.
Delay time alterations allow scanning excited nerve fiber phases. To control
synchronization, the nerve pulse with the help of microelectrodes and the amplifier as well as the photodiode signal was shifted to the oscillograph. Nevertheless,
holographic interferograms for detection of certain nerve fiber excitation phases did
not allow detecting obvious structure-functional alterations in it (the desired signal
was within the measurement margin of error). Experiment conditions optimization
and interferogram recording according to the maximal phase incursion on the nerve
fiber can be implemented using an automated system, which can help to reduce laboriousness of interferogram processing and deciphering, enhances accuracy and gaging
speed, specifically nerve fiber refractive index distribution, nerve fiber deformation
during excitation pulse propagation, neuron pulsation, etc. Increase of interference
bands quality (with contrast and possibility of band center detection) can be reached
using coherent noises averaging device.
Real-time hologram recoding was the most effective during this stage of the
study (see Fig. 2.15). Lens 90× (oil immersion) was used for image recording of
113
Surviving preparation of a myelinated nerve fiber was made by splitting of a frog
sciatic nerve with tungsten needles. Depending on the experiment requirements, the
fiber was put into Ringer physiological solution (n = 1.3337), silicon (n = 1.4057)
and vaseline (n = 1.4805) oil. The functional state of the nerve fiber, used in the
experiments, was estimated according to its ability of stable potential generation
in response to electric irritation produced by right-angled current pulses with the
duration of 0.1 ms and frequency of 1 Hz. When double-exposed holographic interferograms in bands with infinite width were ready, the first exposure corresponded to
preparation addition into the object beam. During the second exposure, the object was
absent. Interferograms in infinite width bands were formed by glass wedge injection
of 3° and 5° into the object beam (band period was 0.4–0.2 μm). For differential interferogram recording, two stages of the same object separated by some time interval
were recorded on one hologram.
To study structure nerve fiber alterations in rhythmic stimulation regime, the
stroboholographic method was used (see Fig. 2.14b). In this case, a stroboscope with
a system of synchronization was placed between the continuous wave laser and the
beam splitter. The character of hologram recording is accumulative. For microobjects
like nerve fiber, the properties of which are reversible for excitation frequencies of
100 Hz, it is possible to record holograms with a set of laser synchronized pulses of
low intensiveness with a certain excitation phase.
To register holographic interferograms in different excitation phases, a stroboscope with a synchronizing system was used, which included a delay unit. Laser
radiation after the stroboscope had the duration of 1 ms with the frequency of 100
Hz. A delay line was launched simultaneously with lightning the object through a
photodiode. The form and the duration of the beam pulse were controlled by photodiode FA-27A and double-beam oscillograph 15 (see Fig. 2.14c). The delayed pulse
controlled the electrical stimulator in such a way that the action potential took place
in the microscope visual field simultaneously with the hologram recording beam.
The electrical stimulator occasionally sent a stimulating pike-shaped pulse onto the
nerve fiber.
Delay time alterations allow scanning excited nerve fiber phases. To control
synchronization, the nerve pulse with the help of microelectrodes and the amplifier as well as the photodiode signal was shifted to the oscillograph. Nevertheless,
holographic interferograms for detection of certain nerve fiber excitation phases did
not allow detecting obvious structure-functional alterations in it (the desired signal
was within the measurement margin of error). Experiment conditions optimization
and interferogram recording according to the maximal phase incursion on the nerve
fiber can be implemented using an automated system, which can help to reduce laboriousness of interferogram processing and deciphering, enhances accuracy and gaging
speed, specifically nerve fiber refractive index distribution, nerve fiber deformation
during excitation pulse propagation, neuron pulsation, etc. Increase of interference
bands quality (with contrast and possibility of band center detection) can be reached
using coherent noises averaging device.
Real-time hologram recoding was the most effective during this stage of the
study (see Fig. 2.15). Lens 90× (oil immersion) was used for image recording of