Optimization of crystal violet technique for enhanced fingerprint detection on various surfaces

The crystal violet (CV) staining technique represents a prevalent approach for the development of latent fingerprints, especially on adhesive tape surfaces. Nevertheless, the technique necessitates intricate formulations to augment its performance. In this investigation, an optimized CV staining protocol was developed, characterized by the absence of residual dye on the target substrates and the capability of facilitating fingerprint visualization under ambient light conditions. Four donors, comprising two males and two females, deposited natural fingerprints on various substrates, including glass microscope slides, aluminum foil, and 115 g glossy coated paper, without any specific guidelines. Fingerprints developed using cyanoacrylate fuming served as benchmarks and were contrasted with those generated through alternative methods: CV, ardrox, rhodamine 6G, powdering, and the optimized CV staining protocol. The fingerprint development experiment was replicated at seven distinct time intervals, encompassing 1 day, 1 week, 1, 3, 6, 9, and 12 months, resulting in a dataset of 420 fingerprints. The evaluation of fingerprint identifiability employed a scoring system established by the Home Office Centre for Applied Science and Technology. The results indicated that the optimized CV staining technique demonstrated superior performance, boasting a 92.9% rate of identifiable fingerprint development in contrast to other employed methodologies. Consequently, this optimized CV staining approach is recommended as an efficient, rapid, and straightforward critical dyeing method, applicable to a wide array of substrates in forensic investigations.


| INTRODUC TI ON
Fingerprints serve as indispensable components in forensic science, contributing vital evidence that aids in the resolution of criminal investigations.Latent fingerprints-those inadvertently deposited on diverse substrates at a crime scene-require specialized techniques for their morphological visualization.An array of methods for fingerprint development and analysis exist, including optical, chemical, dye-based, and instrumental techniques, each with unique advantages and limitations [1].The selection of an appropriate fingerprint development methodology is contingent upon the nature of the substrate involved, and each technique presents an assortment of merits and demerits, ranging from potential health risks to considerations of feasibility, cost, efficiency, and temporal factors.Advances in technology have engendered significant improvements, particularly when traditional methods fall short of the requisite standards.
Among the myriad of techniques for fingerprint development, cyanoacrylate (CA) fuming remains a staple; however, numerous dyeing techniques, including ardrox and rhodamine 6G, have been introduced to enhance contrast under controlled laboratory conditions.Despite their efficacy, these methods are not without limitations, such as reduced sensitivity dependent on surface structure, specialist exposure to varying light wavelengths, and potential degradation of biological materials during evidence processing [2].
Crystal violet (CV), also known by alternative nomenclatures such as methyl violet 10B, hexamethyl pararosaniline chloride, or basic violet 3 (CI 42555), functions dually: as a standalone technique for latent fingerprint development on adhesive substrates and as an auxiliary chromatic agent in conjunction with CA treatments on non-porous materials.The CV methodology hinges on the chemical interaction between the CV dye and the lipids and fatty acids present in fingerprints, finding particular utility in detecting fingerprints on adhesive tapes [3].The technique has proven efficacious in scenarios where traditional powders and fluorescent dyes are ineffective, particularly on surfaces treated with or without CA fuming [4].Moreover, CV manifests fluorescent properties when combined with dyes such as rhodamine 6G, ardrox, and basic yellow, particularly when exposed to the bluegreen region (∼400-600 nm) of the electromagnetic spectrum [3,5].The properties of CV can be modulated through varying formulations; for example, phenol inclusion has been reported to enhance fingerprint visibility on adhesive tape surfaces [6], while a 10% HCl solution effectively amplified fingerprint clarity and definition [7].
This study examines a novel CV formulation designed to leave no dye residues on target substrates, thereby obviating the need for subsequent washing procedures.This optimized CV staining protocol was assessed for its efficacy in developing fingerprints on various substrates, including glass, semiporous glossy paper, and metallic surfaces, within specified temporal frameworks.
Given that optimized CV is not directly applicable to adhesive substrates, this investigation focused on comparing its performance with traditional CV, particularly when used in tandem with CA applications.Comparative efficacy was also evaluated against established dyeing techniques, including powdering, ardrox, and rhodamine 6G.

| Reagents and chemicals
Formation procedures for all fingerprint staining reagents, except the optimized CV method used in this study, were followed [8,9].

| Crystal violet staining method
A working solution was prepared by dissolving 1 g of crystal violet (Meck, Germany) in 1000 mL of distilled water.

| Development of natural latent fingerprints
The experimental design incorporated natural fingerprints from four volunteer donors, comprising two males and two females, within the age range of 30-40 years.Informed consent forms were duly obtained from all participants prior to the initiation of the study.To simulate real-world forensic scenarios, the donors were not provided with any specific guidelines; instead, latent fingerprints were naturally developed through the course of routine activities without any preparatory measures.These fingerprints were deposited on non-porous substrates: specifically, microscope slides (Isolab, Germany) for glass surfaces, aluminum foil (Koroplast, Turkey) for metallic surfaces, and 115 g glossy coated paper (Koza, Turkey) for semi-porous surfaces.The duration of fingerprint-to-surface contact was standardized at 10 s.
Within the framework of this investigation, the aim was to conduct a comparative analysis of fingerprints developed through five distinct staining methodologies.To this end, individual fingerprints were used in their entirety for each respective staining technique, thereby eliminating the necessity to subdivide the fingerprints for separate analyses.The study was conducted over a series of seven predefined time intervals: 24 h, 7 days, 1, 3, 6, 9, and 12 months.
Each experimental iteration involved the development of 60 natural fingerprints, facilitating a comparative analysis across four donors, three substrate types, and five staining techniques.Consequently, the study incorporated a total of 420 latent natural fingerprints, as illustrated in Figure 1.

| Development of latent fingerprints
Nonporous substrates, including metal and glass, were positioned within a fuming chamber (CyanoSafe-Sirchie, USA) in a manner that precluded direct contact between surfaces.For uniform vapor distribution, liquid cyanoacrylate (Evobond 502 super glue, Taiwan) was dispensed into a receptacle and subsequently placed on a heating table within the chamber.An average of 1 g of CA was utilized for a chamber volume of 0.5 m 3 .The heating plate's temperature was calibrated to 120°C, and ambient humidity was maintained at 80% to fa- on the substrate following an approximate 12-h period [2].Various staining methods, including CV, ardrox, rhodamine 6G, fingerprint dusting, and the optimized CV dyeing technique, were employed to augment the visibility of fingerprints initially developed through CA fuming.

| Dusting method
Either black or white powder (Sirchie, USA) was applied to nonporous substrates using a standard-sized fiberglass brush (Sirchie, USA) featuring a plastic handle.During application, care was exercised to minimize physical friction generated by the brush's fiberglass filaments.Post-development, excess powder residues were removed using the same fiberglass brush, and the outcomes were subsequently documented through photography.
Fluorescence staining can be performed via dipping, spraying, or immersion; however, immersion's advantages make it the most widely used method for forensic applications [12].For samples subjected to CA treatment, immersion in a rhodamine 6G solution followed.The treated surfaces were gently rinsed under running water and then allowed to air-dry in a vertically oriented position.
Fingerprints developed via CA fuming and subsequently dyed with rhodamine 6G were illuminated using a Lumatec Superlite All CA-treated samples underwent immersion in an ardrox solution.These samples were rinsed under running water, avoiding the application of pressure.The samples were positioned vertically in an appropriate setting to facilitate surface drying.
Fingerprints developed via CA fuming and subsequently subjected to ardrox fluorescent dyeing were illuminated using a Lumatec Superlite S400 multi-wavelength light source, covering a wavelength spectrum ranging from 415 to 470 nm.Optimal absorbance was attained using a yellow filter under light with a wavelength of 415 nm.Following this, the developed fingerprints were captured photographically.
Samples treated with CA were also exposed to an optimized CV solution prepared according to specific concentration ratios.
All sample surfaces acquired a uniform purple hue post-application.
These samples were positioned vertically in a fume hood to expedite surface drying.Due to the unique formulation of the optimized CV solution, any unbound dye was naturally removed, leaving behind only the CA-induced purple residues.The contrast between the fingerprint patterns and the background surface reached maximal levels.The optimized CV solution stained and developed latent fingerprints, yielding a dark purple outcome.Photographic records were made upon the visualization of the fingerprints.

| Fingerprint analysis
All developed fingerprints were captured using a high-resolution imaging setup comprising a Nikon D7200 Camera equipped with a Sigma 105 mm F/2.8 EX DG OS HSM Macro Lens.Image sharpening was conducted using Adobe Photoshop CS6.For the purpose of fingerprint identification, the study employed the scoring scale developed by the Home Office Centre for Applied Science and Technology (CAST), as outlined in Table S1 [13].To quantitatively assess the level of identifiability of the developed fingerprints, a minimum of 12 distinct fingerprint characteristics were sought, corresponding to scores of 3 and 4 on the quantitative fingerprint rating scale (Table S1).Fingerprint identification was executed in compliance with the ACE-V methodology-analysis, comparison, evaluation, and verification-as stipulated by the International Association for Identification, Standardization II Committee Report [14][15][16].Two fingerprint experts independently conducted the identification and grading of the developed fingerprints.The study demonstrated a high degree of precision in its scoring methodologies, as evidenced by the consistency between expert assessments.TA B L E 1 Summary of the total number and corresponding percentages of successfully developed fingerprints categorized by dyeing method (n = 84).

| Statistical analysis
Crosstab test was utilized to ascertain the presence of statistically significant differences in correlation scores among fingerprint grades, which were derived from fingerprints developed through various methodologies.The Frequencies test was employed to identify the existence and prevalence of statistically significant disparities between the grades assigned to fingerprints.A p-value of less than 0.05 indicated statistical significance at a 95% confidence interval (95% CI).
Based on the outcomes derived from the frequencies test, the optimized CV method emerged as the most efficacious among the various fingerprint dyeing techniques employed in this study.In contrast, the dusting method yielded the lowest number of identifiable fingerprints, thus indicating its relative inefficiency compared to the other dyeing methodologies (Figure 2).In the context of identifiable fingerprints, denoted by scores of 3, glass and metal surfaces emerged as the most conducive substrates for fingerprint development.

The relationship between the effectiveness of various fingerprinting techniques and surface types is elucidated in
Utilizing the optimized CV dyeing method, the study observed a markedly enhanced image clarity-specifically in terms of contrastfor fingerprints derived from semi-porous surfaces as compared to those from glass and metal surfaces (Figure 3).
The optimized CV dyeing method yielded fingerprints with high contrast differences, resulting in identifiable prints.This approach demonstrated superior performance compared to other dyeing methods, as evidenced by Figure 4.
Depending on the ardrox dyeing method, the surface used, and the fingerprint development, decreases in image clarity were detected in the last months.The study found that the image clarity of fingerprints developed through CV, dusting, and fluorescence dyeing methods decreased faster than other methods on semi-porous surfaces.This can be observed in Figure 5.
The study also revealed a statistically significant decline over time in the number of fingerprints deemed suitable for identification Comparative assessment of fingerprints conducive to identification, categorized by development techniques and temporal parameters.
when developed using the CV method (p < 0.024) and the dusting method (p < 0.020), as indicated in Table 3.
No statistically significant difference was observed in the development of fingerprints obtained with optimized CV, ardrox, and rhodamine 6G dyeing methods (p < 0.05).

| DISCUSS ION
The CA fuming method has proven effective in developing white fingerprints on nonporous surfaces like metal and glass and on semiporous surfaces like glossy paper.However, its efficacy on light-colored materials is limited, a finding corroborated by previous research [12].Despite deploying various dyeing methods to augment the quality of low-contrast fingerprints, particular challenges persist.This study aimed to improve existing fingerprint development techniques by utilizing an optimized CV method designed to overcome the limitations of current dyeing protocols.
Fingerprints developed using the ardrox and rhodamine 6G fluorescence methods rely on manipulating light beams at varying wavelengths and filters [12].These procedures, while effective, print laboratories and simulated crime scenes.They concluded that crime scene exposure to dusting method components presents potential health hazards [19].However, it was emphasized that laboratory chemical exposure risks increase significantly in the absence of safety protocols (respirators, gloves, fume hoods, etc.) [20].Furthermore, the National Toxicology Program has reported that rhodamine 6G, a key component of the rhodamine 6G fluorescence dyeing method, may be carcinogenic [18].TA B L E 2 Comparative data on fingerprinting development techniques across different surface types, based on crosstab test results.wave UV rays and the rhodamine 6G dyeing method significantly reduced DNA yield [20,21].Conversely, the CV dyeing method did not adversely affect the quantity of DNA [22].Moreover, intense dyes such as ardrox and rhodamine 6G, commonly employed in fluorescent dyeing methods, were found to leave residues on the treated surfaces, thereby impeding effective fingerprint development [23].
Given the constraints surrounding fingerprint development, it became essential to clean chemically treated surfaces with distilled water.Though distilled water partially safeguards DNA integrity within latent fingerprints developed using CA vapor, it introduces contamination risks, compromising downstream biological testing and limiting the potential for multi-faceted analysis.Furthermore, the use of water in fluorescence dyeing methods contributes to environmental pollution [24].In contrast to traditional methods, the optimized CV method reduces water contamination risks due to its no-wash protocol, improving both the environmental impact and the potential for comprehensive fingerprint analysis [25].
The nature of the surface highly influences the selection of fingerprint development reagents.In this study, the CA fuming method, with its operational parameters set at 80% humidity and a fume temperature of 120°C, along with various dyeing methods, was applied uniformly across glass, metal, and semiporous glossy paper surfaces.
It was observed that glass and metal surfaces yielded a higher number of fingerprints suitable for identification.Fluorescent dyeing methods led to full-surface dye penetration in semiporous materials, diminishing the number of identifiable fingerprints.Consequently, semiporous surfaces were deemed unsuitable for fluorescent methods [2,26].Notably, the optimized CV dyeing technique demonstrated superior efficacy on semi-porous surfaces compared to other methods.

| CON CLUS ION
cilitate fingerprint development.A 15-20 min fuming time facilitated the polymerization reaction between CA vapor and water-soluble fingerprint residue components, including lactic acid, ammonia, acetic acid, amines, alcohols, amino acids, alkanes, and proteins [2, 10, 11].This reaction sequence rendered the fingerprint patterns visible, culminating in the development of fingerprints characterized by a white hue.Fingerprint development was followed by chamber ventilation using prefilters and carbon filters to safely neutralize and expel residual toxic gases.The complete process duration was approximately 50 min.A secondary CA fuming process was implemented to enhance fingerprint visibility if the initial development was deemed suboptimal.The developed fingerprints were stabilized F I G U R E 1 Schematic diagram illustrating the study design for temporal and substrate-specific fingerprint development.
S400 multi-wavelength light source, operating within a wavelength range of 470-530 nm.Optimal absorbance was achieved by employing an orange filter under a light wavelength of 515 nm, after which the developed fingerprints were photographically documented.Surfaces subjected to CA treatment were immersed in a prepared CV solution.Subsequently, these surfaces were gently rinsed under cold running water without the exertion of pressure to remove excess CV solution.The CV dye-stained latent fingerprints during development, producing a dark purple coloration.Upon the emergence of visible fingerprints, photographic documentation was performed.
are labor-intensive and time-consuming.Existing research indicates that to mitigate the risk of photochemical and thermal damage to the skin and retina, the maximum exposure time to these light beams should not surpass 12 min, necessitating the use of protective eyewear and maintaining an optimal working distance of 0.5-1 m[17].When applied to prints developed via CA vapor, the optimized CV method achieves complete and uniform purple dyeing across the entire surface.Intriguingly, the dye on the reverse surface spontaneously dissipates within minutes, obviating the need for washing and maximizing the contrast differential between the background and the fingerprints.The resulting dark purple fingerprints are readily discernible without the requirement for additional light sources.The use of dyeing methods poses health risks to fingerprint experts due to the potential presence of carcinogenic compounds in the chemicals utilized.Due to concerns over trace element contamination and the potential for toxicological effects, heavy metal-based dusting powders have undergone reformulation.For instance, compounds containing lead and mercury, specifically, have been eliminated from contemporary forensic use.Multiple studies corroborate the health risks these powder formulations pose to experts[18].Barsan  and Bresler investigated potential health risks associated with chemical exposure during the fingerprint development techniques employed by FBI experts.Their research included air sampling from both finger-

Fingerprinting
Kumar's investigation delved into the impact of chemicals and varying light wavelengths on DNA yield in the context of fingerprint development.The study indicated that the employment of short-

Fingerprint
analysis constitutes a pivotal component in the adjudication of forensic investigations.This study leveraged an optimized CV dyeing technique to achieve environmentally sustainable results and secure fingerprints with elevated levels of image clarity across an array of surfaces and temporal intervals.Comparative analysis with traditional methodologies revealed substantive improvements.Notably, the developed fingerprints were discernible without requiring auxiliary light sources, enhancing the method's efficiency.In sum, the optimized CV dyeing technique emerges as a compelling, efficient, and cost-effective alternative for forensic laboratories in the framework of fingerprint dyeing.F I G U R E 3 Comparative fingerprint development at the 6-month mark across various surface types utilizing the optimized crystal violet method: (A) glossy paper surface, (B) aluminum foil surface, and (C) glass surface.F I G U R E 4 Longitudinal examination of fingerprint development on metallic (aluminum foil) substrates throughout the study (12-month duration), utilizing varied staining approaches for the same donor: (A) ardrox, (B) optimized crystal violet, (C) dusting method, (D) rhodamine 6G, (E) and crystal violet.

F I G U R E 5
Comparative fingerprint development at the 9-month interval across various fingerprinting methods on semiporous (glossy paper) surfaces: (A) Optimized crystal violet, (B) ardrox, (C) rhodamine 6G, (D) dusting method, and (E) crystal violet.TA B L E 3 Temporal variation in the number of fingerprints deemed suitable for identification.
Statistical analyses were conducted using IBM SPSS Statistics version 21.0, employing both Crosstab and Frequencies tests.The Abbreviation: CV, crystal violet.

Table 2 ,
which presents the results of the crosstab test.