Materials and methods

Diamond Dye™ spraying process optimisation

In order to assess the movement of cells from their original deposition location during DD spraying, optimisation was undertaken prior to the persistence study. Glass slides (Livingstone, Australia) were cleaned with 1% hypochlorite, 70% ethanol and deionised water followed by exposure to the UV light for irradiation for 15 min [27]. Saliva was deposited in 5 µl amounts onto 63 glass slides inside the deposit area indicated with a 11 mm diameter hollow ring dispense-a-label sticky dot (Avery, Australia) that was attached to the underside of the slide (Fig. 1). The saliva was then left to dry for approximately 24 h.

Fig. 1: A slide with the 11 mm ring on the underside (A) and a representation of the cells counted outside of the circle that has been superimposed on the outline where the ring had been (whole slide not shown) (B)

The solution of Diamond Nucleic Acid Dye™ (Promega) was prepared in a 1 in 500 dilution of 10,000X stock solution in 70% ethanol and de-ionised water. Image collection was undertaken using DinoLIGHT (EDGE AM4115T-GFBW, AnMo Electronics Corporation) with an excitation of 480 nm and an emission filter of 510 nm at 50x magnification. Three different application methods were used to apply DD on glass slides: a small perfume bottle (single spray) sprayed once and twice, and a Voilamart HS08 mini air compressor (Voilamart™) that allows for continuous application sprayed twice over (in a back-and-forth motion) the surface for approximately 2 s (total time).

To determine the potential impact of the application distance from the target area on the visualisation of cells and/or their displacement from the deposit area, each of the three application methods were tested three times at each of the following spraying distances: 3 cm, 6 cm, 9 cm, 12 cm, 15 cm and 20 cm. Stained slides were air-dried for approximately 30 min and then imaged. The number of cells outside of the deposit circle (Fig. 1B) was then counted and compared across the different applications and distances to determine which application and distance combination produced the lowest movement of cellular material during DD application.

Persistence of touch on different substrates

The persistence of touch cells was tested on six surfaces: glass slides, plastic transparency film (Nobo, Australia), aluminium (black), melamine (black), leather (textured) (black) and cotton (dark blue). All materials were of a dark colour where possible, with the exception of glass and plastic, which were imaged with the black microscope base as a background to aid cell visualisation. All surfaces were UV treated (BLX Bio-Link Crosslinker, Vilber) on both sides for 30 min period of time prior to cell deposition. For four of the five substrates (4 cm x 22 cm), excluding glass slides, the deposits were prepared by depositing a thumbprint of the dominant hand inside the five 11 mm hollow sticky rings that were attached on top of the substrate surface. The deposit rings were approximately 4 cm apart (Fig. 2). For the glass slides, an individual glass was used for each deposit.

Fig. 2: A visual description of the set up for the creation of five repeats of deposits on each of the substrates, excluding glass where individual slides were used per repeat

Prior to deposition, a single participant washed their hands and waited 15 min. During the wait period, donors were instructed to not eat, drink, wear gloves or use hand sanitiser but could carry on their normal office activities. This process was repeated before each subsequent deposit.

The deposits were stained with DD using a continuous spray application (approx. 2 s) 20 cm away from the surface (determined from results of 3.1) utilising Voilamart air compressor.

Samples were accessed during each of the 20 tested time intervals over 12 months using ImageJ software (see Section "ImageJ Settings"). The tested timeframes post application of DD included immediate, 15 min, 30 min, 1 h, 2 h, 6 h, 24 h, 30 h, 48 h, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months and 12 months. Additionally, at the 6 and 12 months’ time points, the DD was re-applied to each surface, and assessed after 30 min, to test if any apparent loss of cells and fluorescence is due to cell loss or reduction in fluorescence intensities. After 12 months of storage (and post 2nd re-spraying), the substrates were sampled using the wet-dry swabbing method for DNA quantification (see Section "Sample processing"). The latter was done to allow the comparison of DD cell abundance with retrieved DNA quantities. The samples were kept in cardboard boxes in a room that did not have windows and were not removed from this room at any time. The samples were taken out only during imaging (one at a time) and then immediately returned to the storage boxes until the next tested time frame.

ImageJ settings

ImageJ is an image processing program developed by the National Institute of Health and the Laboratory for Optical and Computations Instrumentation (LOCI, University of Wisconsin) [28]. Settings were chosen based on Goray et al. [19] with circularity set at 0.6-1; however, each surface type had a different pixel threshold due to the different background fluorescence observed. The threshold ranges were as follows: glass (35–48), plastic (44–70), melamine (63–75), aluminium (30–46), cotton (79–90) and leather (60–65). After the threshold was applied the despeckle function was used to further eliminate any background fluorescence.

Sample processing

DNA was collected from the deposits 12 months after deposition by wet and dry double swabbing of the surfaces using viscose swabs (Forensic Swab L, Sarstedt, Germany) that were combined for further analysis [29]. The wetting agent used was sterile water (LumaCina, Australia). DNA was extracted and quantified using PrepFiler™ (ThermoFisher) and Quantifiler Trio™ (Applied Biosystems), respectively, as per manufacturer recommendations. The total amount of DNA in the sample was calculated by multiplying the DNA concentration by the extract volume (50 µl).

Data analysis

Deposited cell number changes, at different time points, were calculated by estimating average percentage decrease or increase at each time point for each of the substrates. For this purpose, initially the total number of cells deposited was estimated by counting all the stained cells either immediately or up to 24 h post staining. While for many of the deposits the total maximum cell number was observed immediately after staining, for several deposits DD stain incorporation into the cells took a longer period of time, with increases in cell numbers observed between immediate and 24-hour periods.

Thus, for all deposits and replicates, the time point at which the highest number of cells were observed (from immediate to 24 h depending on surface and repeat), after DD application, was designated as the maximum cell number and assumed to have been the total cell number for that replicate. This maximum cell number was then used for further analysis (the time point with the highest cell number used in the calculations can be found in Supplementary data 1; highlighted in purple). From this maximum cell number deposit, it was then calculated if the cell numbers were increasing or decreasing at preceding and succeeding time frames. For example, for glass surface and replicate 4 (Supplementary data 1), the maximum cell count of 40 cells was observed at 24-hour time post deposit. This deposit was, therefore, assigned as maximum cell number and cell numbers prior to this deposit were assigned percent of the total deposit indicative of cell visualisation increase, which in this instance was from immediate to 24 h (i.e., at immediate time 34 cells were visualised and assigned 85% of the maximum). Conversely, after the maximum cell deposit time frame, continued persistence was estimated (e.g., for glass repeat 4, 35 cells were observed at 30 h post deposit which is 88% of the maximum deposit of 40 cells that shows a 12% loss of cell visualisation). The increases and decreases in cell visualisation were calculated for each time point as percent of cells observed compared to the maximum cell number. For several repeats, cell number increases were seen up to 24 h (where maximum cell count was observed). To standardise the assessment, cell persistence estimation was initiated at 30 h post deposit for all replicates (Supplementary data 1). From this timeframe, average cell persistence per substate type was assessed. First the percent difference between cell count assessed and that repeats cell count maximum, as described above, was calculated (described as “Percentage of cells continued to be visualised” in the Supplementary data 1). Then, the average for the 5 repeats for each set of variables was estimated (described as “Average percentage of cells of cells continuing to be visualised” in the Supplementary data 1).

The normality of the data was tested using a Shapiro-Wilk test with a significance level of < 0.05 (SPSS v.29). An ANOVA was used to determine if there were any differences between the spray devices with a significance level of p ≤ 0.05. (SPSS v.29). The Friedman test was conducted to assess differences in persistence between substrates (χ²(df) = 1, p ≤ 0.05 (SPSS v.29).