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 x 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. (C) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE)

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.10 page(s

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, noninvasive and quantitative measurements of speckle decorrelation times related to microcirculatory flow. Using a multi exposure acquisition scheme, precise speckle decorrelation times were obtained. Applying SDFLSCI 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.15 page(s

Luminescent nanomaterials for molecular-specific cellular imaging

Imaging of molecular trafficking in cells and biological tissue aided by molecular-specific fluorescent labeling is very attractive, since it affords capturing the key processes in comprehensive biological context. Several shortcomings of the existing organic dye labeling technology, however, call for development of alternative molecular reporters, with improved photostability, reduced cytotoxicity, and an increased number of controllable surface moieties. Such alternative molecular reporters are represented by inorganic luminescent nanoparticles (NP) whose optical, physical, and chemical properties are discussed on the examples of luminescent nanodiamonds (LND) and upconversion nanoparticles (UCNP). The emission origins of these nanomaterials differ markedly. LND emission results from individual nitrogen-vacancy color-centers in a biocompatible nanodiamond host whose properties can be controlled via size and surface groups. Photophysics of UCNP is governed by the collective, nonlinear excitation transfer processes, resulting in conversion of longer-wavelength excitation to the shorter-wavelength emission. The emission/excitation spectral properties of UCNP falling within the biological tissue transparency window open new opportunities of almost complete suppression of the cell/tissue autofluorescence background. The developed surface of these nanoparticles represents a flexible platform populated with biocompatible surface moieties onto which cargo and targeting biomolecules can be firmly docked through a process called bioconjugation. These bioconjugated modules, e.g., nanodiamond-antibody, (quantum dot)-somatostatin, or (upconversion nanoparticle)-(mini-antibody) can gain admission into the cells by initiating the cell-specific, cell-recognized communication protocol. In this chapter, we aim to demonstrate the whole bottom-up bio-nano-optics approach for optical biological imaging capturing luminescent nanoparticle design, surface activation, and bioconjugation and the resultant bioconjugate module deployment in specific internalization in the cell.34 page(s

Blood circulation of nanoparticles in a chick embryo model

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Media 3: Quantitative laser speckle flowmetry of the in vivo microcirculation using sidestream dark field microscopy

Originally published in Biomedical Optics Express on 01 November 2013 (boe-4-11-2347

Systematic assessment of blood circulation time of functionalized upconversion nanoparticles in the chick embryo

Nanoparticle-based delivery of drugs and contrast agents holds great promise in cancer research, because of the increased delivery efficiency compared to ‘free’ drugs and dyes. A versatile platform to investigate nanotechnology is the chick embryo chorioallantoic membrane tumour model, due to its availability (easy, cheap) and accessibility (interventions, imaging). In our group, we developed this model using several tumour cell lines (e.g. breast cancer, colon cancer). In addition, we have synthesized in-house silica coated photoluminescent upconversion nanoparticles with several functional groups (COOH, NH₂, PEG). In this work we will present the systematic assessment of their in vivo blood circulation times. To this end, we injected chick embryos grown ex ovo with the functionalized UCNPs and obtained a small amount of blood at several time points after injection to create blood smears The UCNP signal from the blood smears was quantified using a modified inverted microscope imaging set-up. The results of this systematic study are valuable to optimize biochemistry protocols and guide nanomedicine advancement in the versatile chick embryo tumour model.7 page(s

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.6 page(s

Onion-like surface design of upconverting nanophosphors modified with polyethylenimine : shielding toxicity versus keeping brightness?

Background: Upconverting nanoparticles (UCNPs) represent a unique class of nanomaterials, able to convert infrared excitation light into long lifetime visible and infrared photoluminescence, within the "optical transparency window" of biological tissues. This makes UCNPs an attractive contrast agent for background-free bioimaging. However, assynthesized UCNPs are hydrophobic and need additional surface coating for stability in water-based solutions and further functionalization. Polyethylenimine (PEI), a polycationic amphiphilic polymer, is a well-known transfection agent for gene delivery and a popular material for UCNPs surface hydrophilization. Combining the functional properties of UCNPs and PEI is extremely useful for precise visualization of genetic manipulations and intracellular drug delivery. At the same time, PEI is toxic to cells, while the photoluminescent properties of UCNPs are very sensitive to surface chemistry and environment. Then, creation of hydrophilic, biocompatible and simultaneously bright UCNPs, modified by PEI (UCNP-PEI), is a challenging task. Objectives: To analyze the effects of multilayer shielding coatings on cytotoxicity, cellular uptake and photoluminescent properties of UCNP-PEI. Methods and results: UCNP-PEI were modified with additional two or three layers of various polymers and characterized by size, surface charge and photophysical properties. HaCaT keratinocytes were exposed to the particles for 24 or 120 h to study the cytotoxicity and cellular uptake. The results show that onion-like coatings of UCNP-PEI simultaneously decrease cytotoxicity and relative luminescence of the particles, depending on structure and method of formation of multilayer coating. Conclusions: Rational design of UCNP-PEI using extra coatings layers can help to keep acceptable levels of biocompatibility and photoluminescence intensity.5 page(s