Calibration 161 

To obtain the relationship between the measured fluorescence intensity and the solution temperature, the calibration process was performed using the setup illustrated in Figure 7.1. 7.2.4.1 Camera sensitivity calibration

In the LIF technique, the solution temperature is estimated from the fluorescence intensity recorded by the camera. The response of the camera is therefore critical in accurately estimating the temperature of the solution.

A cuvette filled with the Kiton Red solution with a concentration of 1 mg L-1 was illuminated by the iBeam laser (488 nm). The fluorescence from KR solution was then imaged by the colour camera. The camera response to the fluorescence was calibrated by calculating the image intensity at various excitation levels controlled by adjusting the output power of the laser ( ). Experiments were performed under different objectives. A spatially and temporally

162 averaged image intensity was calculated over a rectangular region (300 × 400 pixels) in the middle of the image and over 10 images recorded in a sequence.

The averaged image intensity is normalized as follows:

7.13 where is the background noise of the camera, determined by calculating the image intensity without laser excitation. Due to the low noise of the camera, was found to be around 100 out of 65535. is the saturated greyscale value (65535) for the 16 bits images.

Figure 7.3: Camera calibration showing the normalized image intensity as a function of the laser

output power.

Figure 7.3 shows the normalized intensity as a function of the laser output power under different objectives. Only the intensity of the red channel was calculated as Kiton Red occupied mainly the red channel. The straight lines are linear fits of the experimental data. Clearly, the relationship between the emission intensity incident on the sensor (which is proportional to the laser output) and the resultant image intensity recorded is linear for > 0.02 W, as indicated by the good agreement between the data and the fitting lines. Therefore, the pco.edge camera has a linear intensity response over most of its operating range. It can be also seen that for the same dye concentration, the signal is strongest by using the 20× objective, while it is very low under the 4× objective. Therefore, the dye concentration might need to be increased if using the 4× objective. It should be noted that no signal was detected

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.04 0.08 0.12 0.16 0.2 In IL(W) 4× 10× 20×

163 for < 0.02 W. One reason is that the fluorescence of the dye is too low to be detected by the camera under very low excitation. Another reason is the uncertainty in the laser power calibration which converts the current into power. The laser power calibration curve provided by the supplier was calibrated without fiber connection. However, the laser power was reduced after connecting to the fiber in the present experiments, due to the coupling efficient of the fiber. The offset was being corrected before taking ratios of the fluorescent signals during temperature measurement experiments.

7.2.4.2 Effect of concentration

The variation of intensity ratio against temperature was studied in a cuvette (45 mm × 12.5 mm × 12.5 mm) where the temperature was kept uniform. A transparent thermofoil heater was attached on the bottom of the cuvette and the temperature was controlled by adjusting the power supply connected to the heater. A K-type thermocouple of 0.5 mm diameter was inserted into the cuvette to monitor the solution temperature. For each experiment, the measurement was taken when the fluid temperature became steady and the standard deviation of the temperature measured by the thermocouple over 5 min was within 0.2 °C. The calibrations were carried out at different concentration ratios, CR = CKR/CRRh110, using the colour camera with and without the colour enhancement filter. The 4× objective was used in the calibration because it was to be used in later experiments to get a large observation view. The concentrations of the fluorescent dyes were higher than the values used in camera calibration procedure to provide sufficient signal. A stock solution with concentration of 10 mg L-1 for each dye was prepared using distilled water. The concentration of KR was kept constant and the Rh110 solution was diluted to get a CR from 1 to 40.

A sequence of 10 images of the emissions from KR and Rh110 was recorded at a frame rate of 10 Hz and an exposure time of 50 ms. Figure 7.4 shows representative colour images of the fluorescing mixture at CR = 5 under different temperatures. The first row (Figure 7.4 (a) to (c)) and the second row (Figure 7.4 (d) to (e)) were recorded without and with colour enhancement filter XB30, respectively. As can be seen, for both cases, the image becomes greener and darker with increasing temperature. This is because the red channel intensity which is dominated by KR decreases due to the negative temperature dependence of KR. The images recorded with filter XB30 are darker and greener than those taken without the filter because the filter blocks the intermediate wavelengths between green and red colours.

164

(a) (b) (c)

(d) (e) (f)

Figure 7.4: Colour images of fluorescing mixture (CR = 5, exposure time = 50 ms) at: (a) and (d):

25 °C; (b) and (e): 45 °C; (c) and (f): 70 °C; (a) to (c) were taken without XB30 and (d) to (f) were taken with XB30.

Figure 7.5: Variation of normalized intensity ratio with respect to temperature (solid lines: linear

fitting of the experimental data without XB30; dashed lines: linear fitting of the experimental data with XB30). 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 25 30 35 40 45 50 55 60 65 R( T) /R (T0 ) T (°C) CR = 1 CR = 1 CR = 10 CR = 10 CR = 20 CR = 20 CR = 40 CR = 40 With XB30 Without XB30

165 The intensities of the red and green channels were extracted from each image and the ratio was normalized by that at T0 = 21 °C, . The normalized intensity ratio as a function of temperature for each condition is plotted in Figure 7.5. It is shown that for the samples with the same CR, using the colour enhancement filter (XB30) gives a steeper slope and therefore a higher temperature sensitivity.

The overall sensitivity ln against CR is shown in Figure 7.6 with solid lines representing the values evaluated from Eq. (7.10). It is observed that the sensitivity decreases with increasing concentration ratio. The temperature sensitivity with the XB30 filter is much higher than that without it. The experimental data agree reasonably well with the theoretical values predicated by the model.

Figure 7.6: Variation of temperature sensitivity S with respect to concentration ratio CR. Solid lines

were evaluated from Eq. (7.10).

Funatani et al., (2004) noted that the temperature sensitivity was reduced by 10% when using a colour camera for the two-colour LIF technique compared to the LIF using two cameras. This is caused by the spectral conflicts and overlaps between colour channels. By introducing the colour enhancement filter in this study, the overall temperature sensitivity is improved by at least 40%. The maximum sensitivity of 2.2%/°C can be achieved at CR = 1 with the filter XB30 while 1.6%/°C is the maximum without using the filter. Therefore, the colour camera

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166 with a colour enhancement filter can be used for two-dye LIF measurement without sensitivity reduction.

It should be noted that, at a very high or low CR, the fluorescence mixture will become either too red or too green. The measurement will depend on the low signal to noise ratio (S/N) of the darker channel (Sakakibara and Adrian, 1999). This leads to the big disparities between the experimental data and theoretical values at CR = 1 and CR = 40 as shown in Figure 7.6. In order to get a maximized S/N ratio, CR ~ 20 was selected in this study so that the intensity of each channel fills the same level of the dynamic range of the camera.

7.2.4.3 Effect of geometry

The calibration was taken under four optical conditions for CR = 20: rectangular cuvette (45 mm × 12.5 mm × 12.5 mm) at depth D1 = 0.5 mm and D2 = 1.5 mm, circular tube with diameter d = 2 mm, and circular tube with diameter d = 0.98 mm. Figure 7.7 shows that the normalized intensity ratios under four conditions are almost superimposed, indicating that the temperature sensitivity is independent of the optical detection parameters, such as collection volume and optical path. It confirms that the effects of the channel geometry and optical path have been cancelled out by this two-dye ratiometric LIF technique.

Figure 7.7: Variation of normalized intensity ratio with respect to temperature.

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