Chapter 5: Discussions
5.2 Heating experiment
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electrodes. The calculation of the magnetic force generated by the electrodes in this thesis was much stronger than that used in other works.2,7 As an example compared to magnetic tweezers (permanent magnet) that generate 150 pN1 or 900 pN2 or 12 pN99 to move a 1.05 µm bead.
The distance of the particle movement to a predefined position achieved in this work, ranged at least four times to forty times longer than that of previous works many other methods do not provide the capability of fixing the particles in a position which enable the researcher to fix the particles at desired position. The other advantage of this work is offering multiple positioning points for fixing and trapping the particles inside the cell, due to 12 electrodes provided on the IC socket. In addition, particles could be manipulated at a speed of 3.03×10-5 (m s-1) between two electrodes which is 100 times faster than other literature values.2
The main advantages of the methods used here compared to previous works are a cell manipulation with wide flexibility in selecting various cell types, holding the cells in any desirable position, controlling the number of the injected particles in a short time, generating strong forces by means of current carrying wires, possibility of fixing the particles in multiple positions for analysis and triggering local effects into the living cell.
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10 12 14 16 18 20
0 5 10 15 20 25 30
T [K]
Voltage [V]
Resistance of capillary = 3.2 k
Figure 5.12: Calibration of TMC by applying different voltages (polynomial fit function used to find the best fit because of relation of temperature and voltage ( ))
In order to calibrate the TMC as a stable heat generator, the mass of the capillary can be calculated by two different methods. The first method is calculating the mass by the generated thermal energy in the TMC and in the second method mass the capillary mass is calculated based on its geometry and the properties of glass.
The generated thermal energy in TMC by applying 20 V DC is calculated by (eqn (16)) as below where is the slope of the TMC (R= 3.2 kΩ) temperature rise curve per time (Figure 4.11).
(16)
Where Cv is the heat capacity of glass (kJ kg-1˚C-1), m is the mass of the TMC (kg), V is the electrical voltage (V), I is the electric current (A) and t is the time (s)
The mass of the TMC by this total thermal energy method is calculated as:
.
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To implement the second method, the density of glass = multiplied by the volume of the capillary calculated from the length of capillary coated by Ta (L=26 mm), the inner diameter of ID=0.78 mm and outer an diameter of OD=1 mm is introduced in (eqn (17))
(17)
The mass value calculated by first method is much less than the second method, indicating that the heat generation is mostly limited to the TMC tip not the whole TMC body.
A critical point in fabricating a proper TMC is the Ta coating on two opposite sides of the TMC with sharp edges and making an appropriate connection the between Au wires and the Ta coat on the TMC. Another point is measuring the high resistance of the Ta coating in the range of megaohm (MΩ) due to oxidation of Ta on TMC after annealing. This issue was resolved by scratching the Ta coated surface in order to remove TaxO from the surface before connecting the Au wires.
5.2.2 Heat stress detection
In order to study the heat stress inside the cells, the formation of Reactive Oxygen Species (ROS) was detected. To monitor the heat stress induced ROS generation the cells were incubated in membrane permeable dye dichloro fluoresceindiacetate (DCF-DA). ROS oxidized DCF-DA to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Consequently, the ROS production because of heat stress stimulated metabolic disorder in the plant cell system was indicated by appearance of green fluorescence inside the cells. This result confirmed that DCF-DA could be readily used to study the increase of ROS in heated protoplasts.
In addition, TMCs allow measuring energy requirements and thresholds for triggering heat stress signaling in a single cell and studying the propagation of heat stress signaling from the effect site to the site of response.
20 V DC was applied to a TMC inserted into the A. thaliana cell for 10 minutes. During this period, the ROS indicating fluorescence reached its highest intensity. A control experiment was done with the same cell and the same
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conditions but without applying an electrical voltage to TMC. The control experiment indicate only small rise in fluorescence during 25 minutes of the monitoring period.
In (Figure 5.13) green dots represent the light intensity detected during the control experiment. It can be concluded that the intensity of florescent light due to heat stress is insignificant and therefore there is no ROS generation. The black dots denote the cell’s response during a 70 minutes experiment period after inserting the TMC and applying electric voltage. ROS indicating fluorescence increased over time, reached its maximum, and then reduced after terminating the electric voltage.
0 10 20 30 40 50 60 70
0 20 40 60
rel Intensity
Time[min]
Figure 5.13: Black dots indicate the light intensity, due to ROS generation in A. thaliana cell after applying heat stress and the green dots correspond to the control experiment, which shows slow
increasing in fluorescence within 25 minutes.
Two aspects were studied in the heat stress cell signaling experiment: first the effect of orthogonal distance of the TMC from the cell on the heat shock response and, second the effect of the heat value generated by the TMC which depends on the TMC resistance.
Regarding the heat shock response, several experiments were carried out by TMCs with various resistances to study the heat shock response of the cell against the heat in different orthogonal distances from capillary. (Figure 5.14) shows the results of heating experiment. These points are related to the highest intensity of each cell in presence the TMC curve and normalized (Figure 4.16). According to these data, lower resistance capillaries generate higher temperatures resulting in
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maximum intensity of the heat shock response. Highest ROS formation was achieved by a capillary with a resistance of R=3 kΩ.
0 50 100 150 200 250
20 30 40 50 60 70
80 R=5,5 k
R=3 k
R= 3,3 k
Normalized Intensity
Distance [µm]
Figure 5.14: Normalized fluorescent intensity of ROS generation versus orthogonal distances of TMCs from A. thaliana cell