characterization of a setup to test the impact of high-amplitude pressure waves on living cells - black acrylic sheet
The effects of pressure waves on cells may provide several possible applications in biology and medicine, including direct killing of tumors, drug delivery, or gene transfer.
In this study, we describe the physical properties of mechanical pressure waves that produce nanoseconds of laser pulses in the settings.
Cell culture conditions defined.
Systematically characterize the system on relevant length and time scales (
Micron and nano seconds)
We use Photon Doppler speedometer (PDV)
And obtain the velocity distribution of cell culture containers when the pressure wave is outdated.
These distributions, as input to numerical pressure wave simulations, help to further quantify the pressure conditions on cellular length scales.
At the biological level, we demonstrate the killing of malignant tumor cells and quantify the stress threshold of cell destruction through experiments.
Apply black acrylic gloss varnish to the bottom of each microtiter plate (C.
Kreul GmbH, hallendov, Germany)
At least 48 hours of treatment.
Subsequently, the 5-inch mm-thick transparent silicone board is bonded to the varnish surface with the ACRIFIX 192 polymerization adhesive (
The pressure wave is generated by illumination with a single 6ns ns laser pulse with a wavelength of 1064 nm (Brilliant B Q-
Switched Nd: YAG laser from Erlangen Quantel, Germany).
Laser pulse energy with Q-
Switch delay device.
We used the meter (Thorlabs S370C)
Determine the laser power at the time of 10 hz constant laser pulse generation to calculate the pulse energy of the sample position at different delay times.
In order to record the stress wave in the water, we used Miller-
Needle water listener (Dr.
Miller instruments, Oberursel, Germany)
Installed on an electric translation platform with two-way repeatability of 1. 6u2005μm (see ).
The water listener signal is recorded with a tektds 5054 digital fluorescent oscilloscope and converted to a pressure distribution using the sensitivity given by the manufacturer.
The reflection of the bottom hole pressure wave is accompanied by the acceleration of the surface.
As mentioned above, the pressure of the reflected wave can be derived from the surface velocity profile.
In order to test the velocity distribution at the bottom of the well, we used the Photon Doppler velocity measurement method (PDV)
PDV is an optical technology, based on the Doppler Displacement of light reflected from a moving object, allowing non-
Speed invasive measurement with high time resolution.
To enhance the PDV signal, a reflective chromium/silver coating or aluminum foil is covered at the bottom.
The two programs produce the same result.
The drawing of the PDV settings used to measure the surface speed profile is shown in.
Light of Cw (Continuous wave)
Fiber Laser (
Lighting Laser for short)
A sample with a maximum output power of 2 w is directed to the sample surface by an optical probe.
The wavelength of the laser can be adjusted around the center wavelength = 1557 kHz nm, and the corresponding frequency is/= 200hz (
Where is the speed of light).
The optical probe is basically composed of lenses that focus the light of the laser on the surface of the sample.
A small part of the light with surface diffusion or special reflection is collected by the same optical probe used for lighting, and separated from the lighting light by a circulator.
The reflected light of the Doppler shift frequency is directed to a high-bandwidth optical detector in which it is mixed with the light of the second cw laser (
Called reference laser)
The maximum output power is 40 mw and the frequency is equal to the frequency of the illuminated laser.
Both lasers have a small bandwidth of less than 1 khz, which is critical for accurate speed measurement.
The frequency of the Doppler Displacement light reflected from the moving surface is given by the detector, and the ultra-poor mixing of the magnetic field of the Doppler Displacement light and the light of the reference laser results in a beat signal field with a frequency equal to the frequency difference of the superimposed light: record the beat signal with a high bandwidth digital oscilloscope.
In order to calculate the velocity distribution, the time evolution of beat frequency must be determined.
For this reason, the short-term Fourier transform (STFT)
An algorithm similar to the Strand description is applied to extract the beat frequency from the measured signal.
The application of STFT means that the achievable speed resolution depends on the width of the STFT window function, and therefore on the time resolution.
For the Gaussian window function, the relationship between the standard deviation of the window function and the standard deviation of the speed is: for the measurement given, the STFT window size = 32.
8 ns was selected with a speed resolution of = 1. 9u2005m/s.
Numerical simulation of the pressure wave transmitted in water by standard speed
Verlet algorithm implemented in open source molecular dynamics (MD)
Code LAMMPS for simulation.
The pressure wave follows the specified velocity distribution (
See above and discuss in detail)
As the initial condition for water particles that constitute the frontier of propagation pressure.
The water particles are rough-grained (CG)
By mapping 4 HO molecules to a single (CG)
According to the method described by Steinhauser, water particles. .
All CG water particles are related to dpmde (
Loss-consuming particle dynamics under equal energy
A local thermostat that combines the speed of particles with internal energy storage.
The DPDE is a rough-
The granulation simulation technology of MD implemented in LAMMPS has recently been applied to biological systems.
It simulates the complex particle dynamics of a fully detailed system (e. g. a fluid)
By assigning an internal energy variable and a heat capacity to each particle,-
Th particles are given by =.
Using this CG method, the pressure generated by the fluid at different locations can be measured in the length range, which is equivalent to the size of a typical eukariotic cell, this is in the use of needle water listeners due to the limitations discussed in detail above.
CG particles that make up the pressure front are assigned initial velocity profiles and then start to pass their momentum, I . E. e.
Their kinetic energy goes to the neighboring particles, thus spreading the pressure frontier through the fluid. A velocity-
Verlet time integration scheme with time step delta = 4.
Use the reference density of 89 fs and 1000 m/s/And = 1498 m/s. Equation (1)
Finally, the pressure generated is allowed to be calculated by averaging the particle velocity at the corresponding position.
The result is displayed in (red lines)
It is very consistent with the measurement results of the experimental water listener.
Weyerbrock, neurosurgery, Medical Center, University of Freiburg, Germany.
U87 is officially cultivated (Gibco)
Supplement 10% fetal calf serum (FCS)
1% antibiotics and antibiotics (Gibco).
For pressure wave experiments, microtiter plates were prepared as described above.
Cells were cultured in these plates for 2 to 3 days before the experiment. Tests with non-prepared (transparent)
The microtiter plate shows about cells.
80% meet after this time.
When 96 well plates are used, one laser pulse 15 holes are exposed per hole.
19 wells on the same board are used as unprocessed references.
In the experiment of 48 orifice plates, 6 wells were treated, and 16 wells were used as reference for untreated.
Digest the cell layer with TrypLE (Gibco)
Mix before there is no centrifugal cell count.
For cell counting with a blood cell meter, we used a mixture of 1: 1 sample liquid with Taiwan Pan blue.
Analyze the culture in the same way.