Dynamic laser speckle (DLS) has been used in many applications in medicine and agriculture. This non-invasive optical technique, through the temporal variation of the speckle pattern, evaluates the activity of cells, bacteria, and other biological fluids , , , .
The influence of the relationship between the size of the speckle and the size of the pixel is an important aspect that must be considered in all these applications. It was verified that the signal obtained by this technique varies in frequency composition regarding the biological activity. Despite considering the well-known Nyquist theorem (sampling frequency) during the assembling of the signal, it is not enough since the configuration of the optical camera with its attached photographic objective and its numerical aperture (f-number) must also be guaranteed according to the frequency response .
Another important aspect is the stability of the laser illumination when using laser diodes or a He-Ne laser to ensure that the speckled grains fluctuate only due to biological changes and not because of changes in illumination. The experimental results show that the stability of the diode laser is greater than that of the He-Ne laser in all cases, breaking the paradigm of the stability of He-Ne devices .
Thus, it is necessary to analyse the effect of technical parameters that affect the perceived illumination level. In this sense, we studied the effect of sampling rate and, consequently, the time of exposure in a dynamic laser speckle analysis considering the absolute value of the differences index, the temporal speckle standard deviation index, and the temporal speckle mean index , , .
Knowing the effect of sampling rate over an index value, a researcher will be able to establish criteria to choose the appropriate sampling rate for a specific monitored phenomenon , , .
For this purpose, we show that, given a dynamic speckle test, there is an appropriate frequency band to the sampling rate. High sampling rate values may cause unintended consequences on the index value, such as the decrease of the excursion between two activity levels, given a determinate index value and illumination level. In turn, decreasing the sampling rate promoted by increasing the time of exposure will cause a reduction of the temporal speckle contrast and, consequently, limit the possibility of obtaining information from the sample.
To demonstrate the existence of this appropriate frequency band, we performed two types of tests; in the first, we analysed an ink drying process over time; in the second, we tested the activity state of a corn seed with three days of germination. In both cases, we used four different sampling rates and compared the behaviour of speckle indexes.
The next section describes how the speckle images were acquired and the setup used to analyse them. All theoretical definitions necessary to understand the analysis are presented in Section 3. The numerical results of the analysis are presented in Section 4, and an analysis of the results in Section 5. Finally, we present our conclusion in Section 6.