## 3 Results  
### Automated Optical Density and Fluorescence Calibration Procedure in Chi.Bio  
Even if the manual calibration protocols developed above need only be carried out once, or in cases of re-calibration either to compensate sensors' aging drifts or to improve the calibration in some measurement range, they are time-consuming, as they require a large number of dilutions. To overcome this limitation, we implemented an automated protocol embedded in the Chi.Bio software. This protocol carries out the dilutions, takes the measurements for each dilution, manages the data acquisition and comparison with the reference values obtained with a plate reader for the same dilutions, and finally fits the model parameters. For the calibration of OD, only an ordinary culture is required if the reference spectrophotometer has not changed, so that the conversion to particles does not change either. Notice that silica beads are expensive. Therefore, the restriction of their use to one initial calibration in a reference spectrophotometer using small volumes is a desirable feature. For the calibration of fluorescence, the dilutions are carried out using fluorescein, so the results are independent of any particular culture. Since sensor measurements were collected at several points while deriving the OD and MEFL mathematical models (Section 2.3), these measurements were later reused as markers for the automatic calibration implemented in the Chi.Bio platform. Subsequently, the Chi.Bio pumping system was used to automate the inflow of media and the outflow of the mixture as a dilution strategy. This automatization uses a series of point values (markers), within the operational digital range of the Chi.Bio spectrophotometer, to enable or disable the pumps in order to maintain a constant working volume. This dilutionstrategy was adopted to account for the limited accuracy of the pumps when handling small liquid volumes, as required in classical manual stepwise dilution protocols. As shown in Figure 2, the process begins with Chi.Bio containing a highly concentrated culture or fluorescein sample, followed by a continuous addition of fresh media to dilute the sample until a digital output value (marker), in the operational digital range, is reached. At that point, four consecutive digital values of the current sample are taken using the Chi.Bio spectrophotometer, and their average is calculated to improve measurement accuracy (Figure 2B). Subsequently, an extracted volume of the same current culture is taken from the Chi.Bio vial to measure its corresponding OD value or fluorescein concentration value in Cytation 3 (Figure 2C). Both the Chi.Bio averaged digital output and its corresponding OD or fluorescein concentration value measured by Cytation 3 are entered through the user interface (Figure S3: OD calibration interface; Figure S5: Fluorescence calibration interface in the Supplementary Materials) specifically developed for the OD or fluorescence calibration protocol (Figure 2D). This process is repeated \(n\) times, according to the number of dilution steps specified by the user. At the end of the calibration process, the algorithm uses the models of OD (Equation (1)) or MEFL (Equation (3)) to re-estimate its corresponding parameters with the data obtained by each dilution in the step \(n\) (Figure 2E). The resulting calibration curves demonstrated high accuracy and consistency, with \(R^{2}\) values of 0.999 for OD and MEFL, an RMSE value of 0.035 for OD, and a normalized RMSE value of 0.035 for MEFL as shown in the following tables: Table S1: OD curve fitting parameters. The comparison between OD and fluorescence values obtained from the automated Chi.Bio calibration and those measured with Cytation 3 confirmed strong agreement and robustness across multiple experimental measured data. This result is further supported by the acceptable absolute error calculated between measurements from both instruments, as detailed in the following: Table S2 Comparison measurements between reference OD values and Chi.Bio OD values.