On the glow of cremated remains: long-lived green photo-luminescence of heat-treated human bones.

The long-lived green luminescence of human bone (that has been heated to 600 °C for a short duration) is attributed to a carbon quantum dot material (derived from collagen) encapsulated and protected by an inorganic matrix (derived from bone apatite) and is more intense in dense rigid and crystalline parts of (healthy) human bones. The strong collagen-apatite interaction results (upon decomposition) in a protective inorganic environment of the luminescent centers allowing long-lived triplet-based emission of a carbon (quantum) dot-like material at room temperature, as well as resilience against oxidation between 550 and 650 °C. The graphitic black phase (obtained upon heating around 400 °C) is a precursor to the luminescent carbon-based material, that is strongly interacting with the crystalline inorganic matrix. Human bone samples that have been heated to 600 °C were subjected to steady-state and time-resolved spectroscopy. Excitation-emission matrix (EEM) luminescence spectroscopy revealed a broad range of excitation and emission wavelengths, indicating a heterogeneous system with a broad density of emissive states. The effect of low temperature on the heat-treated bone was studied with Cryogenic Steady State Luminescence Spectroscopy. Cooling the bone to 80 K leads to a slight increase in total emission intensity as well as an intensity increase towards to red part of the spectrum, incompatible with a defect state model displaying luminescent charge recombination in the inorganic matrix. Time-resolved spectroscopy with an Optical Multichannel Analyzer (OMA) and Time Correlated Single Photon Counting (TCSPC) of these samples showed that the decay could be fitted with a multi-exponential decay model as well as with second-order decay kinetics. Confocal Microscopy revealed distinct (plywood type) structures in the bone and high intensity-fast decay areas as well as a spatially heterogeneous distribution of green and (fewer) red emissive species. The use of the ATTO 565 dye aided in bone-structure visualization by chemical adsorption. Conceptually our data interpretation corresponds to previous reports from the material science field on luminescent powders.

Subminute thermal damage to cell types present in the skin.

INTRODUCTION: Psoriasis is characterized by an increase in the proliferation of keratinocytes and nerve fiber activity, contributing to the typical skin lesions. Pulsed Dye Laser (PDL) treatment is effective for the treatment of psoriatic lesions but its mechanism remains unclear. One hypothesis is that PDL causes thermal damage by the diffusion of heat to neighboring structures in lesional skin. There is limited information on the thermal sensitivity of these neighboring skin cells when exposed to hyperthermia for durations lasting less than a minute. Our study aimed to investigate the cell-specific responses to heat using sub-minute exposure times and moderate to ablative hyperthermia. MATERIALS AND METHODS: Cultured human endothelial cells, smooth muscle cells, neuronal cells, and keratinocytes were exposed to various time (2-20 sec) and temperature (45-70 °C) combinations. Cell viability was assessed by measuring intracellular ATP content 24 h after thermal exposure and this data was used to calculate fit parameters for the Arrhenius model and CEM43 calculations. RESULTS: Our results show significant differences in cell survival between cell types (p < 0.0001). Especially within the range of 50-60 °C, survival of neuronal cells and keratinocytes was significantly less than that of endothelial and smooth muscle cells. No statistically significant difference was found in the lethal dose (LT50) of thermal energy between neuronal cells and keratinocytes. However, CEM43 calculations showed significant differences between all four cell types. CONCLUSION: The results imply that there is a cell-type-dependent sensitivity to thermal damage which suggests that neuronal cells and keratinocytes are particularly susceptible to diffusing heat from laser treatment. Damage to these cells may aid in modulating the neuro-inflammatory pathways in psoriasis. These data provide insight into the potential mechanisms of PDL therapy for psoriasis and advance our understanding of how thermal effects may play a role in its effectiveness.

Modeling pulsed dye laser treatment of psoriatic plaques by combining numerical methods and image-derived lesion morphologies.

OBJECTIVES: Knowledge of the physical effects of pulsed dye laser (PDL) treatment of psoriatic lesions is essential in unraveling the remedial mechanisms of this treatment and hence also in maximizing in its disease-modifying potential. Therefore, the main objective of this study was to provide estimates of these physical effects (for laser wavelengths of 585 and 595 nm), with the aim of identifying pathogenic processes that may be affected by these conditions. METHODS: We modeled the laser light propagation and subsequent photothermal heating by numerically solving the transient diffusion and heat equations simultaneously. To this end, we used the finite element method in conjunction with an image-derived psoriatic lesion morphology (which was defined by segmenting blood vessels from a confocal microscopy image of a fluorescently labeled section of a 3 mm punch biopsy of a psoriatic lesion). The resulting predictions of the generated temperature field within the lesion were then used to assess the possibility of stalling or arresting some suspected pathogenic processes. RESULTS: According to our results, it is conceivable that perivascular nerves are thermally denatured, as almost all locations that reach 60°C were found to be within 18 µm (at 585 nm) and 11 µm (at 595 nm) of a blood vessel wall. Furthermore, activation of TRPV1 and TRPV2 channels in perivascular neuronal and immune cells is highly likely, since a critical temperature of 43°C is generated at locations within up to 350 µm of a vessel wall (at both wavelengths) and sustained for up to 700 ms (at 585 nm) and 40 ms (at 595 nm), while a critical temperature of 52°C is reached by locations within 80 µm (at 585 nm) and 30 µm (at 595 nm) of a vessel wall and sustained for up to 100 ms (at 585 nm) and 30 ms (at 595 nm). Finally, we found that the blood vessel coagulation-inducing temperature of 70°C is sustained in the vascular epithelium for up to 19 and 5 ms at 585 and 595 nm, respectively, rendering partial or total loss of vascular functionality a distinct possibility. CONCLUSIONS: The presented approach constitutes a useful tool to provide realistic estimates of the photothermal effects of PDL treatment of psoriatic plaques (as well as other selective photothermolysis-based treatments), yielding information that is essential in guiding future experimental studies toward unraveling the remedial mechanisms of these treatments.

Home-Use Hyaluronic Acid Jet Injectors: Unreliable and Unsafe.

BACKGROUND: Needle-free hyaluronic acid (HA) jet injectors are gaining popularity for rejuvenation treatment. The devices are widely available online and are used for self-injection or in beauty salons by nonphysicians. However, little is known about their performance and safety. OBJECTIVE: To explore the injection efficiency and cutaneous biodistribution patterns administered with home-use compared with medical jet injectors and to assess safety aspects. MATERIALS AND METHODS: The authors injected HA into ex vivo human skin with 4 home-use and 2 medical injectors. The intracutaneous dose of HA was calculated, and the cutaneous biodistribution of HA was assessed using a 3-dimensional Fluorescent Imaging Cryomicrotome System (3D-FICS). Safety aspects were evaluated based on the presence of a manual, CE (conformité européenne) mark, and sterility. RESULTS: The intracutaneous dose delivered by the home-use injectors was markedly lower compared with the medical injectors. 3D imaging for home-use injectors showed superficial epidermal distribution with low distribution volumes. For medical injectors, volumes were substantially larger and mainly middermal. All evaluated safety aspects were lacking. CONCLUSION: Results of this study suggest that the specific combinations of home-use injectors and HA used in this study are unreliable and unsafe, which casts doubts on the performance of these treatments in general.

Temperature-specific spectral shift of luminescing thermally altered human remains.

Human bone has shown to have luminescent properties that remain throughout the phases of cremation, with the exception of fully carbonized bone, when excited with a narrow band light source. During this research, an alternate light source (420-470nm, peak at 445nm) was used to visualize and investigate latent details relevant for forensic investigations of human remains recovered at fire scenes. As fire is a destructive force, it induces a vast variety of physical and chemical alterations to all components of the bone, making the subsequent analysis and interpretation of burned human remains challenging. A spectral shift in emission bandwidth, from green to red, was previously observed when the exposure temperature increased from 700 to 800 °C. This spectral shift was reproduced on a total of 10 human forearms, divided into 20 segments, by burning at 700 °C and 900 °C in an ashing furnace. The shift of emission bandwidth caused only by an increase in temperature was furthermore investigated by colorimetric analysis, proving the spectral shift to be significant. By easily quantifying the spectral shift, substantiation is provided for the use of this technique in practice to improve the interpretation of heat induced changes of bone.

Towards Onsite Age Estimation of Semen Stains Using Fluorescence Spectroscopy.

The age estimation of biological traces is one of the holy grails in forensic investigations. We developed a method for the age estimation of semen stains using fluorescence spectroscopy in conjunction with a stoichiometric ageing model. The model describes the degradation and generation rate of proteins and fluorescent oxidation products (FOX) over time. The previously used fluorimeter is a large benchtop device and requires system optimization for forensic applications. In situ applications have the advantage that measurements can be performed directly at the crime scene, without additional sampling or storage steps. Therefore, a portable fiber-based fluorimeter was developed, consisting of two optimized light-emitting diodes (LEDs) and two spectrometers to allow the fluorescence protein and FOX measurements. The handheld fiber can be used without touching the traces, avoiding the destruction or contamination of the trace. In this study, we have measured the ageing kinetics of semen stains over time using both our portable fluorimeter and a laboratory benchtop fluorimeter and compared their accuracies for the age estimation of semen stains. Successful age estimation was possible up to 11 days, with a mean absolute error of 1.0 days and 0.9 days for the portable and the benchtop fluorimeters, respectively. These results demonstrate the potential of using the portable fluorimeter for in situ applications.

Mechanical or thermal damage: differentiating between underlying mechanisms as a cause of bone fractures.

To investigate the differences between pre- and post-fire fractures, 30 human forearm bones were subjected to either blunt-force impact, burning, or both. Bones, covered in soft tissue and wrapped in clothing, were burned in a reconstructed house fire. The burning context and dynamics led to differential burning, that was equal amongst the three groups. To evaluate the damage to the bones, a data collection sheet was developed based on the current literature on fracture features. To analyze the relation between exposure temperature and fracture class and occurrence, color was measured to estimate the exposure temperature. Observable and measurable changes on bone, and fracture surfaces, were macro- and microscopically analyzed. Many features overlapped between the three groups, and thus are not usable for differentiation. Fractures caused by blunt force impact (post-mortem, pre-fire) showed a rough fracture surface with smoothness in curved/slanted regions near the margin after burning, while heat-induced bone fractures showed a smooth fracture surface. The margins and surface of bone fractures that originated after the fire (indirect heat-induced) were evenly discolored, whereas heat-induced bone fractures showed uneven discoloration of the fracture margin and surface. Although there were generally more heat-induced fractures in the 450-700 °C range, no other distinctive trend was observed between exposure temperature and class of fractures.

Phosphorescence of thermally altered human bone.

Bone has photoluminescent characteristics that can aid the analysis of thermally altered human skeletal remains as part of the forensic anthropological investigation. Photoluminescence stands collectively for fluorescence and phosphorescence. Because the difference in lifetime between fluorescence and phosphorescence is usually in the range of nano- to microseconds, it is only possible to visually determine whether bone phosphoresces when the lifetime is long enough to be observed. For this study, a distinction was made between long-decay and short-decay phosphorescence. So far, it was unknown whether (thermally altered) human bone emits long-decay phosphorescence after being illuminated and, thus, whether phosphorescence contributes to the observed photoluminescence. If so, whether the observable phosphorescence is dependent on temperature, exposure duration, surrounding medium, bone type, skeletal element, and excitation light and could aid the temperature estimation of heated bone fragments. In this study, bone samples were subjected to heat in the range of from room temperature to 900 °C for various durations in either air or adipose as surrounding medium. In addition, different skeletal elements of a human cadaver were recollected after cremation in a crematorium. Both sample collections were illuminated with light of different bandwidths and visually inspected for phosphorescence and photoluminescence. The samples were scored by means of a scoring index for the intensity of long-decay phosphorescence and photographically documented. The results show that thermally altered human bone fragments do phosphoresce. The observed phosphorescence is more dependent on temperature than on exposure duration, surrounding medium or skeletal element. Of the used wavelength bands, ultraviolet light provided the most temperature-related information, showing changes in both phosphorescence intensity and emission spectrum. Long-decay phosphorescence and fluorescence with short-decay phosphorescence coincide; however, there are also temperature-dependent differences. It is therefore concluded that phosphorescence contributes to the observable photoluminescence and that the visibly observable phosphorescent characteristics can aid the temperature estimation of cremated human skeletal fragments.

Estimating the Time of Deposition of Semen Traces using Fluorescence Protein-Lipid Oxidation Signatures.

In the forensic field, knowledge about the time of deposition of semen traces is extremely valuable to law enforcement agencies to assess the relevance of the traces and the validity of witness testimonies. However, currently, no method exists that is able to estimate the time of deposition of semen stains, due to the complex chemistry of the constituents and variation in degradation patterns. Here, we demonstrate a non-contact age estimation method to assess the time of deposition of semen stains using fluorescence spectroscopy. Protein-lipid oxidation reactions were monitored in semen stains over time using protein fluorescence and fluorescent oxidation product signatures to reveal distinctive aging patterns. On the basis of the relative amounts of these fluorescent products and the rate at which they are converted, successful age estimation was achieved up to a value of 16 days, with a median absolute error of 1.7 days. We demonstrate here a new tool that can fill the current gap in intelligence about the age of semen traces that has been challenging the forensic community worldwide.

Correction for the Hematocrit Bias in Dried Blood Spot Analysis Using a Nondestructive, Single-Wavelength Reflectance-Based Hematocrit Prediction Method.

The hematocrit (Hct) effect is one of the most important hurdles currently preventing more widespread implementation of quantitative dried blood spot (DBS) analysis in a routine context. Indeed, the Hct may affect both the accuracy of DBS methods as well as the interpretation of DBS-based results. We previously developed a method to determine the Hct of a DBS based on its hemoglobin content using noncontact diffuse reflectance spectroscopy. Despite the ease with which the analysis can be performed (i.e., mere scanning of the DBS) and the good results that were obtained, the method did require a complicated algorithm to derive the total hemoglobin content from the DBS's reflectance spectrum. As the total hemoglobin was calculated as the sum of oxyhemoglobin, methemoglobin, and hemichrome, the three main hemoglobin derivatives formed in DBS upon aging, the reflectance spectrum needed to be unmixed to determine the quantity of each of these derivatives. We now simplified the method by only using the reflectance at a single wavelength, located at a quasi-isosbestic point in the reflectance curve. At this wavelength, assuming 1-to-1 stoichiometry of the aging reaction, the reflectance is insensitive to the hemoglobin degradation and only scales with the total amount of hemoglobin and, hence, the Hct. This simplified method was successfully validated. At each quality control level as well as at the limits of quantitation (i.e., 0.20 and 0.67) bias, intra- and interday imprecision were within 10%. Method reproducibility was excellent based on incurred sample reanalysis and surpassed the reproducibility of the original method. Furthermore, the influence of the volume spotted, the measurement location within the spot, as well as storage time and temperature were evaluated, showing no relevant impact of these parameters. Application to 233 patient samples revealed a good correlation between the Hct determined on whole blood and the predicted Hct determined on venous DBS. The bias obtained with Bland and Altman analysis was -0.015 and the limits of agreement were -0.061 and 0.031, indicating that the simplified, noncontact Hct prediction method even outperforms the original method. In addition, using caffeine as a model compound, it was demonstrated that this simplified Hct prediction method can effectively be used to implement a Hct-dependent correction factor to DBS-based results to alleviate the Hct bias.

A Novel, Nondestructive, Dried Blood Spot-Based Hematocrit Prediction Method Using Noncontact Diffuse Reflectance Spectroscopy.

Dried blood spot (DBS) sampling is recognized as a valuable alternative sampling strategy both in research and in clinical routine. Although many advantages are associated with DBS sampling, its more widespread use is hampered by several issues, of which the hematocrit effect on DBS-based quantitation remains undoubtedly the most widely discussed one. Previously, we developed a method to derive the approximate hematocrit from a nonvolumetrically applied DBS based on its potassium content. Although this method yielded good results and was straightforward to perform, it was also destructive and required sample preparation. Therefore, we now developed a nondestructive method which allows to predict the hematocrit of a DBS based on its hemoglobin content, measured via noncontact diffuse reflectance spectroscopy. The developed method was thoroughly validated. A linear calibration curve was established after log/log transformation. The bias, intraday and interday imprecision of quality controls at three hematocrit levels and at the lower and upper limit of quantitation (0.20 and 0.67, respectively) were less than 11%. In addition, the influence of storage and the volume spotted was evaluated, as well as DBS homogeneity. Application of the method to venous DBSs prepared from whole blood patient samples (n = 233) revealed a good correlation between the actual and the predicted hematocrit. Limits of agreement obtained after Bland and Altman analysis were -0.076 and +0.018. Incurred sample reanalysis demonstrated good method reproducibility. In conclusion, mere scanning of a DBS suffices to derive its approximate hematocrit, one of the most important variables in DBS analysis.

Fluorescence characteristics of human Barrett tissue specimens grafted on chick chorioallantoic membrane.

To improve (pre)malignant lesion identification in Barrett's esophagus (BE), recent research focuses on new developments in fluorescence imaging and spectroscopy to enhance tissue contrast. Our aim was to validate the chorioallantoic membrane (CAM) model as a preclinical tool to study the fluorescence characteristics such as autofluorescence and exogenously induced fluorescence of human Barrett's tissue. Therefore, esophageal biopsy specimens from Barrett's patients were freshly grafted onto the CAM of fertilized hen's eggs to simulate the in vivo situation. The BE biopsy specimens stayed between 1 and 9 days on the CAM to study the persistence of vitality. Fluorescence spectroscopy was performed using six excitation wavelengths (369, 395, 400, 405, 410, 416 nm). Obtained autofluorescence spectra were compared with in vivo spectra of an earlier study. Exogenous administration of 5-aminolevulinic-acid to the biopsy specimens was followed by fluorescence spectroscopy at several time points. Afterwards, the biopsy specimens were harvested and histologically evaluated. In total, 128 biopsy specimens obtained from 34 patients were grafted on the CAM. Biopsy specimens which stayed on average 1.7 days on the CAM were still vital. Autofluorescence spectra of the specimens correlated well with in vivo spectra. Administered 5-aminolevulinic-acid to the biopsy specimens showed conversion into protoporphyrin-IX. In conclusion, we showed that grafting freshly collected human BE biopsy specimens on the CAM is feasible. Our results suggest that the CAM model might be used to study the fluorescence behavior of human tissue specimens. Therefore, the CAM model might be a preclinical research tool for new photosensitizers.

Techniques that acquire donor profiling information from fingermarks - A review.

Fingermarks are among the most important types of evidence that can be encountered at the scene of a crime since the unique ridge pattern of a fingerprint can be used for individualization. But fingermarks contain more than the characteristic pattern of ridges and furrows, they are composed of a wide variety of different components that originate from endogenous and exogenous sources. The chemical composition can be used to obtain additional information from the donor of the fingermark, which in turn can be used to create a donor profile. Donor profiling can serve at least two purposes i) to enhance the evidential value of fingermarks and ii) to provide valuable tactical information during the crime scene investigation. Retrieving this additional information is not limited to fingermarks that have been used for individualization, but can also be applied on partial and/or distorted fingermarks. In this review we have summarized the types of information that can be obtained from fingermarks. Additionally, an overview is given of the techniques that are available addressing their unique characteristics and limitations. We expect that in the nearby future, donor profiling from contact traces, including fingermarks will be possible.

A literature review and novel theoretical approach on the optical properties of whole blood.

Optical property measurements on blood are influenced by a large variety of factors of both physical and methodological origin. The aim of this review is to list these factors of influence and to provide the reader with optical property spectra (250­2,500 nm) for whole blood that can be used in the practice of biomedical optics (tabulated in the appendix). Hereto, we perform a critical examination and selection of the available optical property spectra of blood in literature, from which we compile average spectra for the absorption coefficient (µ(a)), scattering coefficient (µ(s)) and scattering anisotropy (g). From this, we calculate the reduced scattering coefficient (µ(s)') and the effective attenuation coefficient (µ(eff)). In the compilation of µ(a) and µ(s), we incorporate the influences of absorption flattening and dependent scattering (i.e. spatial correlations between positions of red blood cells), respectively. For the influence of dependent scattering on µ(s), we present a novel, theoretically derived formula that can be used for practical rescaling of µ(s) to other haematocrits. Since the measurement of the scattering properties of blood has been proven to be challenging, we apply an alternative, theoretical approach to calculate spectra for µ(s) and g. Hereto, we combine Kramers­Kronig analysis with analytical scattering theory, extended with Percus­Yevick structure factors that take into account the effect of dependent scattering in whole blood. We argue that our calculated spectra may provide a better estimation for µ(s) and g (and hence µ(s)' and µ(eff)) than the compiled spectra from literature for wavelengths between 300 and 600 nm.

Immunolabeling and the compatibility with a variety of fingermark development techniques.

Much information can be obtained from the chemical composition of a fingermark, which can be helpful in crime scene investigation. Immunolabeling can be used to extract information about the donor of the fingermark and it can also act as a fingermark development tool in sequence with the standard fingermark development techniques. However, before immunolabeling can be used in forensic practice more information on the possibilities and limitations of this technique is required. In this study, our aim was to investigate if immunolabeling is compatible with standard development protocols (indanedione-zinc, indanedione-zinc followed by ninhydrin spraying, physical developer, cyanoacrylate fuming, cyanoacrylate followed by basic yellow staining, lumicyanoacrylate fuming and polycyanoacrylate fuming). Immunolabeling was carried out successfully on all developed fingermarks, whereby dermcidin was selected as antigen of interest. We can conclude that immunolabeling is compatible with a wide variety of different fingermark developers. This finding in combination with previous findings, makes immunolabeling an interesting technique, which can be of great value in the forensic field.

Oxidation monitoring by fluorescence spectroscopy reveals the age of fingermarks.

No forensic method exists that can reliably estimate the age of fingermarks found at a crime scene. Information on time passed since fingermark deposition is desired as it can be used to distinguish between crime related and unrelated fingermarks and to support or refute statements made by the fingermark donors. We introduce a non-contact method that can estimate the age of fingermarks. Fingermarks were approached as protein-lipid mixtures and an age-estimation model was build based on the expected protein and lipid oxidation reactions. Two measures of oxidation are required from the fingermark to estimate its age: 1) the relative amount of fluorescent oxidation products 2) the rate at which these products are formed. Fluorescence spectroscopy was used to obtain these measures. We tested the method on 44 fingermarks and were able to estimate the age of 55% of the male fingermarks, up to three weeks old with an uncertainty of 1.9 days.

Short communications: Exploring temporal fluorescent changes in the composition of human semen stains.

Semen traces are considered important pieces of evidence in forensic investigations, especially those involving sexsual offenses. Recently, our research group developed a fluorescence-based technique to accurately determine the age of semen traces. However, the specific compounds resonsible for the fluoresescent behaviour of ageing semens remain unknown. As such, in this exploratory study, the aim is to identify the components associated with the fluorescent behavior of ageing semen traces. In this investigation semen stains and various biofluorophores commonly found in body fluids were left to aged for 0, 2, 4, 7, 14 and 21 days. Subsequently, thin-layer chromatography (TLC) and ultra-performance liquid chromatography (UPLC) mass spectrometry were performed to identify the biofluorophores present in semen. Several contributors to the autofluorescence could be identified in semen stain, these include tryptophan, kynurenine, kynurenic acid, and norharman. The study sheds light on the.

The biodistribution of triamcinolone acetonide injections in severe keloids: an exploratory three-dimensional fluorescent cryomicrotome study.

Intralesional corticosteroid injections are a first-line treatment for keloids; yet clinical treatment results are highly variable and often suboptimal. Variation in triamcinolone acetonide (TAC) biodistribution may be an important reason for the variable effects of TAC treatment in keloids. In this exploratory study we investigated the biodistribution of TAC in keloids and normal skin using different drug delivery techniques. Fluorescent-labeled TAC suspension was administered into keloids and normal skin with a hypodermic needle and an electronic pneumatic jet injector. TAC biodistribution was represented by the fluorescent TAC volume and 3D biodistribution shape of TAC, using a 3D-Fluorescence-Imaging Cryomicrotome System. Twenty-one keloid and nine normal skin samples were analyzed. With needle injections, the mean fluorescent TAC volumes were 990 µl ± 479 in keloids and 872 µl ± 227 in normal skin. With the jet injector, the mean fluorescent TAC volumes were 401 µl ± 252 in keloids and 249 µl ± 67 in normal skin. 3D biodistribution shapes of TAC were highly variable in keloids and normal skin. In conclusion, TAC biodistribution in keloids is highly variable for both needle and jet injection. This may partly explain the variable treatment effects of intralesional TAC in keloids. Future research is needed to confirm this preliminary finding and to optimize drug delivery in keloids.

Specific fluorescent signatures for body fluid identification using fluorescence spectroscopy.

Non-invasive, rapid, on-site detection and identification of body fluids is highly desired in forensic investigations. The use of fluorescence-based methods for body fluid identification, have so far remain relatively unexplored. As such, the fluorescent properties of semen, serum, urine, saliva and fingermarks over time were investigated, by means of fluorescence spectroscopy, to identify specific fluorescent signatures for body fluid identification. The samples were excited at 81 different excitation wavelengths ranging from 200 to 600 nm and for each excitation wavelength the emission was recorded between 220 and 700 nm. Subsequently, the total emitted fluorescence intensities of specific fluorescent signatures in the UV-visible range were summed and principal component analysis was performed to cluster the body fluids. Three combinations of four principal components allowed specific clustering of the body fluids, except for fingermarks. Blind testing showed that 71.4% of the unknown samples could be correctly identified. This pilot study shows that the fluorescent behavior of ageing body fluids can be used as a new non-invasive tool for body fluid identification, which can improve the current guidelines for the detection of body fluids in forensic practice and provide the robustness of methods that rely on fluorescence.