Evaluation of the microbial wet-vacuum system (M-Vac®) for DNA sampling from rough, porous substrates, and its compatibility with fully automated platforms.

DNA retrieval methods traditionally used during forensic evidence recovery including swabbing and tape lifting, can have limited effectiveness when used on porous, rough substrates such as bricks and carpet. This is possibly due to the DNA material being dispersed and unreachable for surface sampling techniques. In this evaluation we investigated the effectiveness of the Microbial Wet-Vacuum System (M-Vac®; M-Vac® Systems, Inc., Sandy, UT), as it has been reported to retrieve greater amounts of DNA material from challenging exhibits. A four-stage evaluation was conducted, starting with seeding carpet and brick substrates with a known donor's saliva in two dilutions and comparing the DNA recovery of tape lifting, swabbing, and the M-Vac®. A victim struggle scenario on carpet was then mimicked to compare trace DNA recovery by each method. Two mock scenarios were also conducted; a shirt was submerged in a creek bed for a period of five days to sample for the wearer's DNA, and a car boot was sampled to assess the possibility of recovering a victim's DNA amongst background DNA from the usual car occupants. Finally, the compatibility of the M-Vac® sampling process was optimised for the fully automated DNA lysis and extraction platforms used in the NSW (Australia) jurisdiction by comparing filter subsampling methods. The results from the study were mixed. For bricks, none of the collection methods were effective in retrieving DNA. On carpet, the M-Vac® retrieved the greatest quantities of DNA from the saliva-seeded samples, however, tape lifts outperformed all methods for 'touch' DNA recovery. The M-Vac® retrieved the greatest amount of DNA from the t-shirt recovered from a creek bed as it was able to retrieve the embedded DNA. The final mock case car boot scenario resulted in greater victim DNA recovery from tape lifts, with the M-Vac® more likely to recover mixtures too weak and/or complex to be interpreted. Finally, operational considerations regarding the compatibility of the M-Vac® system with fully automated DNA lysis and extraction are discussed. Considering the substantial time and cost to deploy the M-Vac®, it is recommended to be utilised in casework only after swabbing and tape lifting methods have failed to yield sufficient DNA material, where the substrate properties would likely benefit from the M-Vac's® niche capabilities for retrieving embedded DNA, and low levels of background DNA may be anticipated.

Generative Adversarial Networks in Digital Pathology and Histopathological Image Processing: A Review.

Digital pathology is gaining prominence among the researchers with developments in advanced imaging modalities and new technologies. Generative adversarial networks (GANs) are a recent development in the field of artificial intelligence and since their inception, have boosted considerable interest in digital pathology. GANs and their extensions have opened several ways to tackle many challenging histopathological image processing problems such as color normalization, virtual staining, ink removal, image enhancement, automatic feature extraction, segmentation of nuclei, domain adaptation and data augmentation. This paper reviews recent advances in histopathological image processing using GANs with special emphasis on the future perspectives related to the use of such a technique. The papers included in this review were retrieved by conducting a keyword search on Google Scholar and manually selecting the papers on the subject of H&E stained digital pathology images for histopathological image processing. In the first part, we describe recent literature that use GANs in various image preprocessing tasks such as stain normalization, virtual staining, image enhancement, ink removal, and data augmentation. In the second part, we describe literature that use GANs for image analysis, such as nuclei detection, segmentation, and feature extraction. This review illustrates the role of GANs in digital pathology with the objective to trigger new research on the application of generative models in future research in digital pathology informatics.

Chick Embryo Experimental Platform for Micrometastases Research in a 3D Tissue Engineering Model: Cancer Biology, Drug Development, and Nanotechnology Applications.

Colonization of distant organs by tumor cells is a critical step of cancer progression. The initial avascular stage of this process (micrometastasis) remains almost inaccessible to study due to the lack of relevant experimental approaches. Herein, we introduce an in vitro/in vivo model of organ-specific micrometastases of triple-negative breast cancer (TNBC) that is fully implemented in a cost-efficient chick embryo (CE) experimental platform. The model was built as three-dimensional (3D) tissue engineering constructs (TECs) combining human MDA-MB-231 cells and decellularized CE organ-specific scaffolds. TNBC cells colonized CE organ-specific scaffolds in 2-3 weeks, forming tissue-like structures. The feasibility of this methodology for basic cancer research, drug development, and nanomedicine was demonstrated on a model of hepatic micrometastasis of TNBC. We revealed that MDA-MB-231 differentially colonize parenchymal and stromal compartments of the liver-specific extracellular matrix (LS-ECM) and become more resistant to the treatment with molecular doxorubicin (Dox) and Dox-loaded mesoporous silica nanoparticles than in monolayer cultures. When grafted on CE chorioallantoic membrane, LS-ECM-based TECs induced angiogenic switch. These findings may have important implications for the diagnosis and treatment of TNBC. The methodology established here is scalable and adaptable for pharmacological testing and cancer biology research of various metastatic and primary tumors.

Rational Surface Design of Upconversion Nanoparticles with Polyethylenimine Coating for Biomedical Applications: Better Safe than Brighter?

Upconversion nanoparticles (UCNPs) coated with polyethylenimine (PEI) are popular background-free optical contrast probes and efficient drug and gene delivery agents attracting attention in science, industry, and medicine. Their unique optical properties are especially useful for subsurface nanotheranostics applications, in particular, in skin. However, high cytotoxicity of PEI limits safe use of UCNP@PEI, and this represents a major barrier for clinical translation of UCNP@PEI-based technologies. Our study aims to address this problem by exploring additional surface modifications to UCNP@PEI to create less toxic and functional nanotheranostic materials. We designed and synthesized six types of layered polymer coatings that envelop the original UCNP@PEI surface, five of which reduced the cytotoxicity to human skin keratinocytes under acute (24 h) and subacute (120 h) exposure. In parallel, we examined the photoluminescence spectra and lifetime of the surface-modified UCNP@PEI. To quantify their brightness, we developed original methodology to precisely measure the colloidal concentration to normalize the photoluminescence signal using a nondigesting mass spectrometry protocol. Our results, specified for the individual coatings, show that, despite decreasing the cytotoxicity, the external polymer coatings of UCNP@PEI quench the upconversion photoluminescence in biologically relevant aqueous environments. This trade-off between cytotoxicity and brightness for surface-coated UCNPs emphasizes the need for the combined assessment of the viability of normal cells exposed to the nanoparticles and the photophysical properties of postmodification UCNPs. We present an optimized methodology for rational surface design of UCNP@PEI in biologically relevant conditions, which is essential to facilitate the translation of such nanoparticles to the clinical applications.

Sensitive Cytokine Assay Based on Optical Fiber Allowing Localized and Spatially Resolved Detection of Interleukin-6.

We demonstrated a cytokine detection device based on gold nanoparticle modified silica optical fiber for the monitoring of locally variable cytokine interleukin-6 (IL-6) concentrations using a sandwich immunoassay scheme. The fiber is designed to be introduced into an intrathecal catheter with micrometer-sized holes drilled along its length to enable fluid exchange between the outside and inside of the catheter. An exposed optical fiber (diameter 125 µm) modified with a layer of gold nanoparticles was functionalized with the IL-6 capture antibody to form the sensing interface. The immunocapture device was incubated with a cytokine solution to capture the analyte. The device was then exposed to the IL-6 detection antibody which was loaded on the fluorescently labeled magnetic nanoparticles, making it possible to quantify the cytokine concentration based on the intensity of fluorescence. A reliable method for quantifying the fluorescent signal on a 3D structure was developed. This device was applied to the detection of cytokine IL-6 with the low limit of detection of 1 pg mL-1 in a sample volume of 1 µL. The device has the linear detection range of 1-400 pg mL-1 and spatial resolution on the order of 200-450 µm, and it is capable of detecting localized IL-6 secreted by live BV2 cells following their liposaccharide stimulation. This biological detection system is suitable for monitoring multiple health conditions.

Quantitative blood flow velocity imaging using laser speckle flowmetry.

Laser speckle flowmetry suffers from a debated quantification of the inverse relation between decorrelation time (τc) and blood flow velocity (V), i.e. 1/τc = αV. Using a modified microcirculation imager (integrated sidestream dark field - laser speckle contrast imaging [SDF-LSCI]), we experimentally investigate on the influence of the optical properties of scatterers on α in vitro and in vivo. We found a good agreement to theoretical predictions within certain limits for scatterer size and multiple scattering. We present a practical model-based scaling factor to correct for multiple scattering in microcirculatory vessels. Our results show that SDF-LSCI offers a quantitative measure of flow velocity in addition to vessel morphology, enabling the quantification of the clinically relevant blood flow, velocity and tissue perfusion.

Lanthanide upconversion luminescence at the nanoscale: fundamentals and optical properties.

Upconversion photoluminescence is a nonlinear effect where multiple lower energy excitation photons produce higher energy emission photons. This fundamentally interesting process has many applications in biomedical imaging, light source and display technology, and solar energy harvesting. In this review we discuss the underlying physical principles and their modelling using rate equations. We discuss how the understanding of photophysical processes enabled a strategic influence over the optical properties of upconversion especially in rationally designed materials. We subsequently present an overview of recent experimental strategies to control and optimize the optical properties of upconversion nanoparticles, focussing on their emission spectral properties and brightness.

Targeted labeling of an early-stage tumor spheroid in a chorioallantoic membrane model with upconversion nanoparticles.

In vivo detection of cancer at an early-stage, i.e. smaller than 2 mm, is a challenge in biomedicine. In this work target labeling of an early-stage tumor spheroid (∼500 µm) is realized for the first time in a chick embryo chorioallantoic membrane (CAM) model with monoclonal antibody functionalized upconversion nanoparticles (UCNPs-mAb).

Quantitative laser speckle flowmetry of the in vivo microcirculation using sidestream dark field microscopy.

We present integrated Laser Speckle Contrast Imaging (LSCI) and Sidestream Dark Field (SDF) flowmetry to provide real-time, non-invasive and quantitative measurements of speckle decorrelation times related to microcirculatory flow. Using a multi exposure acquisition scheme, precise speckle decorrelation times were obtained. Applying SDF-LSCI in vitro and in vivo allows direct comparison between speckle contrast decorrelation and flow velocities, while imaging the phantom and microcirculation architecture. This resulted in a novel analysis approach that distinguishes decorrelation due to flow from other additive decorrelation sources.

Feasibility study of the optical imaging of a breast cancer lesion labeled with upconversion nanoparticle biocomplexes.

Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications.

Quantitative imaging of single upconversion nanoparticles in biological tissue.

The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs), enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (∼1 cm) depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 µm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement.

Background free imaging of upconversion nanoparticle distribution in human skin.

Widespread applications of nanotechnology materials have raised safety concerns due to their possible penetration through skin and concomitant uptake in the organism. This calls for systematic study of nanoparticle transport kinetics in skin, where high-resolution optical imaging approaches are often preferred. We report on application of emerging luminescence nanomaterial, called upconversion nanoparticles (UCNPs), to optical imaging in skin that results in complete suppression of background due to the excitation light back-scattering and biological tissue autofluorescence. Freshly excised intact and microneedle-treated human skin samples were topically coated with oil formulation of UCNPs and optically imaged. In the first case, 8- and 32-nm UCNPs stayed at the topmost layer of the intact skin, stratum corneum. In the second case, 8-nm nanoparticles were found localized at indentations made by the microneedle spreading in dermis very slowly (estimated diffusion coefficient, D(np) = 3-7 × 10(-12) cm(2) · s(-1)). The maximum possible UCNP-imaging contrast was attained by suppressing the background level to that of the electronic noise, which was estimated to be superior in comparison with the existing optical labels.

Age estimation of blood stains by hemoglobin derivative determination using reflectance spectroscopy.

Blood stains can be crucial in reconstructing crime events. However, no reliable methods are currently available to establish the age of a blood stain on the crime scene. We show that determining the fractions of three hemoglobin derivatives in a blood stain at various ages enables relating these time varying fractions to the age of the blood stain. Application of light transport theory allows addressing the spectroscopic changes in ageing blood stains to changes in chemical composition, i.e. the transition of oxy-hemoglobin into met-hemoglobin and hemichrome. We have found in 20 blood stains that the chemical composition of the blood stain with age, called hemoglobin reaction kinetics, under controlled circumstances, shows a distinct time-dependent behavior, with a unique combination of the three hemoglobin derivatives at all moments in time. Finally, we employed the hemoglobin reaction kinetics inversely to assess the age of 20 other blood stains studied, again over a time period of 0-60 days. We estimated an age of e.g. 55 days correct within an uncertainty margin of 14 days. In conclusion, we propose that the results obtained under controlled conditions demand further evaluation of their possible value for age determination of blood stains on crime scenes.