By controlling and increasing the environmental humidity to slow the evaporation rate, the consistency of the evaporation flux significantly improved compared to conditions with uncontrolled humidity, as outlined in Figure and. Various levels of controlled humidity were evaluated, with the highest SNR observed at 60% relative humidity for 3-min drying time (Figure and). However, there was no significant difference in SNR between 40% and 60% relative humidity for the same drying time. This suggests that the minimum humidity required to achieve SNR above 15 was 40%. At extreme humidity levels, such as 20% or 80%, the SNRs did not surpass 15. This outcome was likely due to the insufficient moisture at low humidity to establish an effective evaporation flux, or excessive moisture at high humidity, which hindered evaporation and prevented proper drying. 40% and 60% relative humidities were assessed for consistency at the 2-min and 3-min dry times to determine ideal environmental humidity (Table ). The intra-assay %CVs of two wells for the 2-min dry time at 40% relative humidity were 4.60% and 16.45%, with the inter-assay %CV of 12.27%. In comparison, when increasing the dry time to 3-min, the intra-assay %CVs were reported as 16.17% and 7.41%, while the inter-assay %CV increased to 15.34%. The results showed that at 40% relative humidity, a 2-min drying time provided more consistency; however, with %CV values exceeding 10%, this condition was not suitable for achieving the industrial standards of repeatability and reproducibility. In comparison, 60% relative humidity at 3-min was able to achieve lower intra-assay %CVs of 9.25% and 11.13%, with an inter-assay %CV of 9.94%. This was also evident in the fluorescence images (Figure and Figure ), where 60% relative humidity had the nicest shaped squares and least visible background noise. The higher consistency, despite the longer dry time, makes 60% relative humidity at 3-min dry time the most repeatable and reproducible for industry applications. The longer drying time was due to the more balanced humidity environment, which created a uniform evaporation flux that slowed down compared to 40% relative humidity. Thus, 60% relative humidity and 3-min dry time was chosen as the optimized factors.