Confocal Volume Research Articles

Dynamic light scattering unveils stochastic degradation in large-pore mesoporous silica nanoparticles

Mesoporous Silica Nanoparticles (MSNs) have been increasingly investigated as versatile drug delivery carriers. A particular challenge for the systemic use of MSNs lies in the control of their degradation, which has not been fully understood until now. We implemented standard dynamic light scattering (DLS) experiments and introduced a novel DLS technique in a confocal volume to track the dynamics of large-pore MSN degradation in situ. This unique DLS technique, which involves a small observation volume, was chosen for its ability to count particle by particle during the degradation process, a method that has not been commonly used in nanoparticle research. The experiments were performed in different media compositions at low particle concentrations, below the silica solubility limit. MSNs with large conical pores were prepared and studied as they offer the possibility to incorporate and release large-sized biomolecules. Large-pore MSNs followed a singular degradation mechanism following a stochastic-like behavior, a finding that challenges the common idea that all nanoparticles (NPs) degrade similarly and homogeneously over time. We showed that some NPs are observed intact over a prolonged period while most other NPs have already vanished or been transformed into swollen NPs. Thus, a heterogeneous degradation process occurs, while the total concentration of NPs undergoes an exponential decay. These large conical pores MSNs will be utilized as reliable biomolecule nanocarriers by predicting the factors underlying the NP hydrolytic stability.

Small multimodal thermometry with detonation-created multi-color centers in detonation nanodiamond

Detonation nanodiamond (DND) is the smallest class of diamond nanocrystal capable of hosting various color centers with a size akin to molecular pores. Their negatively charged nitrogen-vacancy center (NV−) is a versatile tool for sensing a wide range of physical and even chemical parameters at the nanoscale. The NV− is, therefore, attracting interest as the smallest quantum sensor in biological research. Nonetheless, recent NV− enhancement in DND has yet to yield sufficient fluorescence per particle, leading to efforts to incorporate other group-IV color centers into DND. An example is adding a silicon dopant to the explosive mixture to create negatively charged silicon-vacancy centers (SiV−). In this paper, we report on efficient observation (∼50% of randomly selected spots) of the characteristic optically detected magnetic resonance (ODMR) NV− signal in silicon-doped DND (Si-DND) subjected to boiling acid surface cleaning. The NV− concentration is estimated by continuous-wave electron spin resonance spectroscopy to be 0.35 ppm without the NV− enrichment process. A temperature sensitivity of 0.36K/Hz in an NV− ensemble inside an aggregate of Si-DND is achieved via the ODMR-based technique. Transmission electron microscopy survey reveals that the Si-DNDs core sizes are ∼11.2 nm, the smallest among the nanodiamond’s temperature sensitivity studies. Furthermore, temperature sensing using both SiV− (all-optical technique) and NV− (ODMR-based technique) in the same confocal volume is demonstrated, showing Si-DND’s multimodal temperature sensing capability. The results of the study thereby pave a path for multi-color and multimodal biosensors and for decoupling the detected electrical field and temperature effects on the NV− center.

Exploring Rotational Diffusion with Plasmonic Coupling.

Measuring the orientation dynamics of nanoparticles and nonfluorescent molecules in real time with optical methods is still a challenge in nanoscience and biochemistry. Here, we examine optoplasmonic sensing taking the rotational diffusion of plasmonic nanorods as an experimental model. Our detection method is based on monitoring the dark-field scattering of a relatively large sensor gold nanorod (GNR) (40 nm in diameter and 112 nm in length) as smaller plasmonic nanorods cross its near field. We observe the rotational motion of single small gold nanorods (three samples with about 5 nm in diameter and 15.5, 19.1, and 24.6 nm in length) in real time with a time resolution around 50 ns. Plasmonic coupling enhances the signal of the diffusing gold nanorods, which are 1 order of magnitude smaller in volume (about 300 nm3) than those used in our previous rotational diffusion experiments. We find a better angular sensitivity with plasmonic coupling in comparison to the free diffusion in the confocal volume. Yet, the angle sensitivity we find with plasmonic coupling is reduced compared to the sensitivity expected from simulations at fixed positions due to the simultaneous translational and rotational diffusion of the small nanorods. To get a reliable plasmonic sensor with the full angular sensitivity, it will be necessary to construct a plasmonic assembly with positions and orientations nearly fixed around the optimum geometry.

Determination of the confocal volume of a Raman microscope for probing for single-molecule SERS applications

The confocal volume in Raman spectroscopy refers to the sample region from which scattered photons are collected by a confocal Raman spectrometer, defined by the cone-shaped light beam generated by the microscope’s objective lens in the confocal configuration. The confocal volume is characterized by the confocal height and confocal area, which determine the range of heights and lateral positions, respectively, from which the spectrometer effectively collects scattered photons. In the present study, the confocal height and area were calculated for different configurations of a confocal Raman spectrometer to obtain the confocal volume using a Si wafer sample. Understanding the confocal volume was essential to optimize Raman microscopy experiments and estimate the number of molecules generating surface-enhanced Raman scattering spectroscopy spectra in a single-molecule regime.

(Invited) Confocal Raman Microscopy in the Study of Ion Insertion Processes and Material Dimensional Change

Confocal Raman microscopy is being applied to quantify chemical composition at electrode-solution interfaces and within electrode-supported films under electrochemical potential control. Experimental strategies (i.e., cell designs and data acquisition and processing techniques) that are enabling sensitive in-situ spectral measurements will be discussed with focus on redox transformations at electrode-solution interfaces and within electrode-supported polymer membranes and thin films. Emphasis will be on the ability to investigate ion insertion/de-insertion processes and measure material dimensional (e.g., swelling/de-swelling) changes as the applied potential is varied (in-situ). The microscope system being utilized employs a high numerical aperture oil immersion objective to achieve (i) diffraction-limited focusing and probe volume characteristics and (ii) high efficiency in collecting Raman scattered radiation from molecules of interest. With the objective mounted to an inverted microscope frame, the electrochemical cell is positioned above the objective on the microscope stage. A glass microscope coverslip attached to the bottom of the electrochemical cell serves as the optical window. One electrochemical cell platform has been constructed around an optically transparent indium tin oxide (ITO) coated coverslip that serves as both the cell working electrode and optical window. The objective brings excitation radiation through the coverslip and to a focus a few micrometers above the ITO surface. Species diffusing into the confocal probe volume region are detected and quantified based on their Raman spectral band frequencies and intensities. A second electrochemical cell platform has a thin layer design and enables use of conventional disk working electrodes. The performance of this cell together with a spectral difference technique that allows detection of transformations within redox polymers supported on carbon electrodes will be discussed. The platform is being applied to detect swelling/de-swelling in electrode-supported redox polymers in response to changes in material redox state. Molecular composition (i.e., the mix of polymer framework, solvent and mobile ions) within the confocal probe volume is measured quantitatively from Raman spectra. Tracking the frequency and intensity of spectral bands as electrode potential is varied enables voltage-induced swelling and de-swelling to be correlated to changes in the composition of specific types of mobile species (solvent and ions). Similar application of the strategy to investigate material dimensional change with variation in atmospheric composition will be highlighted.

Comparison of two Phaeodactylum tricornutum ecotypes under nitrogen starvation and resupply reveals distinct lipid accumulation strategies but a common degradation process.

Phaeodactylum tricornutum is a model species frequently used to study lipid metabolism in diatoms. When exposed to a nutrient limitation or starvation, diatoms are known to accumulate neutral lipids in cytoplasmic lipid droplets (LDs). Those lipids are produced partly de novo and partly from the recycle of plastid membrane lipids. Under a nitrogen resupply, the accumulated lipids are catabolized, a phenomenon about which only a few data are available. Various strains of P. tricornutum have been isolated around the world that may differ in lipid accumulation patterns. To get further information on this topic, two genetically distant ecotypes of P. tricornutum (Pt1 and Pt4) have been cultivated under nitrogen deprivation during 11 days followed by a resupply period of 3 days. The importance of cytoplasmic LDs relative to the plastid was assessed by a combination of confocal laser scanning microscopy and cell volume estimation using bright field microscopy pictures. We observed that in addition to a basal population of small LDs (0.005 μm3 to 0.7 μm3) present in both strains all along the experiment, Pt4 cells immediately produced two large LDs (up to 12 μm3 after 11 days) while Pt1 cells progressively produced a higher number of smaller LDs (up to 7 μm3 after 11 days). In this work we showed that, in addition to intracellular available space, lipid accumulation may be limited by the pre-starvation size of the plastid as a source of membrane lipids to be recycled. After resupplying nitrogen and for both ecotypes, a fragmentation of the largest LDs was observed as well as a possible migration of LDs to the vacuoles that would suggest an autophagic degradation. Altogether, our results deepen the understanding of LDs dynamics and open research avenues for a better knowledge of lipid degradation indiatoms.

In Situ Confocal Raman Microscopy of Redox Polymer Films on Bulk Electrode Supports.

A spectroelectrochemical cell is described that enables confocal Raman microscopy studies of electrode-supported films. The confocal probe volume (∼1 μm3) was treated as a fixed-volume reservoir for the observation of potential-induced changes in chemical composition at microscopic locations within an ∼20 μm thickness layer of a redox polymer cast onto a 3 mm diameter carbon disk electrode. Using a Raman system with high collection efficiency and wavelength reproducibility, spectral subtraction achieved excellent rejection of background interferences, opening opportunities for measuring within micrometer-scale thickness redox films on widely available, low-cost, and conventional carbon disk electrodes. The cell performance and spectral difference technique are demonstrated in experiments that detect transformations of redox-active molecules exchanged into electrode-supported ionomer membranes. The in situ measurements were sensitive to changes in the film oxidation state and swelling/deswelling of the polymer framework in response to the uptake and discharge of charge-compensating electrolyte ions. The studies lay a foundation for confocal Raman microscopy as a quantitative in situ probe of processes within electrode-immobilized redox polymers under development for a range of applications, including electrosynthesis, energy conversion, and chemical sensing.

Opto-mechanical material characterization via dual-geometry Brillouin confocal microscopy

Brillouin scattering is a light-matter interaction that induces a frequency shift in the scattered photons. Such shift directly depends on the optical and mechanical properties of the material itself. In the last decade, Brillouin frequency shift has become a reliable proxy for mechanical stiffness in biological samples such as cells and tissues, with the subsequent development of Brillouin confocal microscopy; however, such correlation does not apply to samples with high degree of inhomogeneity. Recently, we showed that a dual geometry Brillouin microscopy enables the direct measurement of refractive index and speed of sound. We now report an improved dual-geometry microscopy technique that allows measurement of optoacoustical properties within a confocal volume. This goal has been achieved through the measurement of frequency, linewidth, and intensity of Brillouin components of the scattered light spectrum. Finally, we proved our three-dimensional imaging capability by mapping, with micron-scale resolution, the refractive index, density, and viscoelastic properties of a liquid-liquid phase separation system. This technique can be applied to a multitude of biological heterogenous scenarios such as nucleolus characterization, high concentration lipid sites, dynamic protein aggregates and more.

3D Spectroscopic Tracking of Individual Brownian Nanoparticles during Galvanic Exchange.

Monitoring chemical reactions in solutions at the scale of individual entities is challenging: single-particle detection requires small confocal volumes, which are hardly compatible with Brownian motion, particularly when long integration times are necessary. Here, we propose a real-time (10 Hz) holography-based nm-precision 3D tracking of single moving nanoparticles. Using this localization, the confocal collection volume is dynamically adjusted to follow the moving nanoparticle and allow continuous spectroscopic monitoring. This concept is applied to study galvanic exchange in freely moving colloidal silver nanoparticles with gold ions generated in situ. While the Brownian trajectory reveals particle size, spectral shifts dynamically reveal composition changes and transformation kinetics at the single-object level, pointing at different transformation kinetics for free and tethered particles.

Plasmonics for advance single-molecule fluorescence spectroscopy and imaging in biology

The elucidation of complex biological processes often requires monitoring the dynamics and spatial organization of multiple distinct proteins organized on the sub-micron scale. This length scale is well below the diffraction limit of light, and as such not accessible by classical optical techniques. Further, the high molecular concentrations found in living cells, typically in the micro- to mili-molar range, preclude single-molecule detection in confocal volumes, essential to quantify affinity constants and protein-protein reaction rates in their physiological environment. To push the boundaries of the current state of the art in single-molecule fluorescence imaging and spectroscopy, plasmonic materials offer encouraging perspectives. From thin metallic films to complex nano-antenna structures, the near-field electromagnetic coupling between the electronic transitions of single emitters and plasmon resonances can be exploited to expand the toolbox of single-molecule based fluorescence imaging and spectroscopy approaches. Here, we review two of the most current and promising approaches to study biological processes with unattainable level of detail. On one side, we discuss how the reduction of the fluorescence lifetime of a molecule as it approaches a thin metallic film can be exploited to decode axial information with nanoscale precision. On the other, we review how the tremendous progress on the design of plasmonic antennas that can amplify and confine optical fields at the nanoscale, powered a revolution in fluorescence correlation spectroscopy. Besides method development, we also focus in describing the most interesting biological application of both technologies.

Imaging and detection of long-lived fluorescence probes in presence of highly emissive and scattering background.

Optical biomedical imaging and diagnostics is a rapidly growing field that provides both structural and functional information with uses ranging from fundamental to practical clinical applications. Nevertheless, imaging/visualizing fluorescence objects with high spatial resolution in a highly scattering and emissive biological medium continues to be a significant challenge. A fundamental limiting factor for imaging technologies is the signal-to-background ratio (SBR). For a long time to improve the SBR, we tried to improve the brightness of fluorescence probes. Many novel fluorophores with improved brightness (almost reaching the theoretical limit), redshifted emission, highly improved photostability, and biocompatibility greatly helped advance fluorescence detection and imaging. However, autofluorescence, scattering of excitation light, and Raman scattering remain fundamental limiting problems that drastically limit detection sensitivity. Similarly, significant efforts were focused on reducing the background. High-quality sample purification eliminates the majority of autofluorescence background and in a limited confocal volume allows detection to reach the ultimate sensitivity to a single molecule. However, detection and imaging in physiological conditions does not allow for any sample (cells or tissue) purification, forcing us to face a fundamental limitation. A significant improvement in limiting background can be achieved when fluorophores with a long fluorescence lifetime are used, and time-gated detection is applied. However, all long-lived fluorophores present low brightness, limiting the potential improvement. We recently proposed to utilize multipulse excitation (burst of pulses) to enhance the relative signal of long-lived fluorophores and significantly improve the SBR. Herein, we present results obtained with multipulse excitation and compare them with standard single-pulse excitation. Subtraction of images obtained with a single pulse from those obtained with pulse burst (differential image) highly limits background and instrumental noise resulting in more specific/sensitive detection and allows to achieve greater imaging depth in highly scattering media, including skin and tissue.

Computational Proposal for Tracking Multiple Molecules in a Multifocus Confocal Setup.

Tracking single molecules continues to provide new insights into the fundamental rules governing biological function. Despite continued technical advances in fluorescent and non-fluorescent labeling as well as data analysis, direct observations of trajectories and interactions of multiple molecules in dense environments remain aspirational goals. While confocal methods provide a means to deduce dynamical parameters with high temporal resolution, such as diffusion coefficients, they do so at the expense of spatial resolution. Indeed, on account of a confocal volume’s symmetry, typically only distances from the center of the confocal spot can be deduced. Motivated by the need for true three dimensional high speed tracking in densely labeled environments, we propose a computational tool for tracking many fluorescent molecules traversing multiple, closely spaced, confocal measurement volumes providing independent observations. Various realizations of this multiple confocal volumes strategy have previously been used for long term, large area, tracking of one fluorescent molecule in three dimensions. What is more, we achieve tracking by directly using single photon arrival times to inform our likelihood and exploit Hamiltonian Monte Carlo to efficiently sample trajectories from our posterior within a Bayesian nonparametric paradigm. A nonparametric paradigm here is warranted as the number of molecules present are, themselves, a priori unknown. Taken together, we provide a computational framework to infer trajectories of multiple molecules at once, below the diffraction limit (the width of a confocal spot), in three dimensions at sub-millisecond or faster time scales.

Automated bone cell classification for confocal laser scanning microscopy volumes

Manual cell classification in microscopy images is a time-consuming process that heavily relies on the subjective perception of the investigator. Identifying bone cells introduces additional difficulties with irregular geometries, and in some culture conditions, the presence of bone mineral. As fluorescence-based lineage tracing becomes more common, classifying cell types based upon cell color can further increase subjectivity. Our goal is to develop and validate a fully automated cell classification algorithm that can (i) objectively identify cells in flattened volumetric image stacks from three-dimensional (3D) bone cell cultures and (ii) classify the cells (osteoblast-lineage) based on the color of their cell bodies. The algorithm used here was developed in MATLAB 2019a and validated by comparing code outputs to manual labeling for eleven images. The precision, recall, and F1 scores were higher than 0.75 for all cell classifications, with the majority being greater than 0.80. No significant differences were found between the manually labelled and automated cell counts or cell classifications. Analysis time for a single image averaged seventeen seconds compared to more than ten minutes for manual labeling. This demonstrates that the program offers a fast, repeatable, and accurate way to classify bone cells by fluorescence in confocal microscopy image data sets. This process can be expanded to improve investigation of other pre-clinical models and histological sections of pathological tissues where color or fluorescence-based differences are used for cell identification.

Measuring Photophysical Transition Rates with Fluorescence Correlation Spectroscopy and Antibunching.

We present a new method that combines fluorescence correlation spectroscopy (FCS) on the microsecond time scale with fluorescence antibunching measurements on the nanosecond time scale for measuring photophysical rate constants of fluorescent molecules. The antibunching measurements allow us to quantify the average excitation rate of fluorescent molecules within the confocal detection volume of the FCS measurement setup. Knowledge of this value allows us then to quantify, in an absolute manner, the intersystem crossing rate and triplet state lifetime from the microsecond temporal decay of the FCS curves. We present a theoretical analysis of the method and estimate the maximum bias caused by the averaging of all quantities (excitation rate and photophysical rates) over the confocal detection volume, and we show that this bias is smaller than 5% in most cases. We apply the method for measuring the photophysical rate constants of the widely used dyes Rhodamine 110 and ATTO 655.

Abstract PR004: In vivo tracking of clonal dynamics during UV-induced skin carcinogenesis

Abstract Background: The impact of chronic UV exposure on clonal dynamics and genomic diversity remains unclear. Our central hypothesis is that skin cancer is induced not by solely accumulation of somatic mutations, but rather a combination of mutations and disruption of the spatial and temporal constraints imposed by the skin’s 3-D architecture. Here we characterize clonal dynamics and transcriptional signatures during skin carcinogenesis using multicolor lineage tracing. Methods: We generated a K14Cre-ERT2 Confetti mice with inducible fluorophore (flr) expression. Mice were UV-irradiated for 3 months. Clones were 3-D digitized using confocal microscopy (z-stacks) and clone volumes estimated computationally. scRNAseq was used to compare UV-exposed (EXP) vs. non-exposed (NON) epidermis vs. skin tumors. Results: We generated 914 serial images of the EXP/NON skin over the course of 6 months following initiation of UV. We analyzed 16,135 clones from the EXP and 21,506 clones from the NON skin. We classified clone sizes into 3 classes represented by the small (<50,000 μm3), medium (50,000-500,000 μm3), and large “goliath” (> 500,000 μm3). The median size of clones does not differ between UV treatments and does not change with time. However, clones from EXP samples have significantly greater mean size than NON ones. Their mean sizes differed by some 1.5-fold, with an over 6-fold increase in variance, resulting in the sizes distribution to be highly skewed towards large clones with a long, narrow tail. Goliath clones are rarely present in the NON skin; however, they increase in number dramatically by months 3-4, plateauing between months 5-6. Using 3 ecological metrics (clone size, clone numbers, and coefficient of variation) we see phase shifts, which primarily distinguish months 1 & 2 from months 3 & 4. scRNAseq of EXP/NON epidermis and tumors revealed differential representation of 16 clusters, the majority of which could be mapped to previously defined keratinocyte populations. We observe dynamic changes to these clusters when progressing from normal skin to chronically exposed skin, and then to tumors. EXP clusters were associated with expression of cystatins (Scfa 3, BC100530), and alarmins/proliferative keratins (Krt16, Krt6a), which have been associated with skin injury. Clusters expressing cystatins and alarmins also increased in tumors. Flr-expressing keratinocytes harvested from large clones in EXP epidermis exhibited altered keratinocyte differentiation (downregulation of Krt77, Loricrin and Nfkbia, upregulation of cystatin), inflammation (downregulation of Nfkbia), and upregulation of metabolic regulators (carbonic anhydrase II and retinol transport (Rbp1)). Genes differentially expressed in exposed skin and retained in tumors may be required for carcinogenesis, while those expressed only in exposed skin likely required for adaptive responses to UV. Our findings have important implications for understanding cancer through an eco-evolutionary framework and designing novel approaches to cancer prevention. Citation Format: Stanislav Avdieiev, Leticia Tordesillas, Omar Chavez Chiang, Zhihua Chen, Luiza Silva Simoes, Y. Ann Chen, Noemi Andor, Robert Gatenby, Elsa R. Flores, Joel S. Brown, Kenneth Y. Tsai. In vivo tracking of clonal dynamics during UV-induced skin carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR004.

Optical nanotopography of fluorescent surfaces by axial position modulation.

We present an optical method that combines confocal microscopy with position modulation to perform axial tracking and topographic imaging of fluorescent surfaces. Using a remote focusing system, the confocal observation volume is oscillated in the axial direction. The resulting modulation of the detected signal is used as a feedback to precisely control the distance to an object of interest. The accuracy of this method is theoretically analyzed and the axial-locking accuracy is experimentally evaluated. Topographic imaging is demonstrated on fluorescently coated beads and fixed cells. This microscope allows for nanometric topography or tracking of dynamic fluorescent surfaces.

Adapting confocal Raman microscopy for in situ studies of redox transformations at electrode-electrolyte interfaces

Confocal Raman microscopy was applied to quantify redox species present within the diffusion layer adjacent to an electrode surface under potentiostatic control. A glass microscope coverslip with a thin indium tin oxide (ITO) coating served as both the working electrode and optical window for a microscope-stage mountable spectroelectrochemical cell. A high numerical aperture objective mounted in an inverted microscope frame just below the stage brought excitation radiation through the coverslip window and to a tight focus a few micrometers above the ITO film surface. Species diffusing into the confocal probe volume defined by the excitation beam focus and the collected light region were detected, identified and quantified based on their Raman scattering frequencies and intensities. In measurements that interrogated the interconversion of ferrocyanide and ferricyanide ions as a function of applied voltage, least-squares regression analysis of spectral datasets predicted the formal potential and relative surface concentrations of the ions in good agreement with the expected Nernstian response. Preliminary studies of methyl viologen reduction at an ITO film/Nafion membrane interface were conducted and show the possibility for estimation of mass transport coefficients of redox species within ionic polymer materials.

Confocal Raman Microscopy Enables Label-Free, Quantitative, and Structurally Informative Detection of DNA Hybridization at Porous Silica Surfaces.

Characterization of DNA at solid/liquid interfaces remains a challenge because most surface-sensitive techniques are unable to provide quantitative insight into the base content, length, or structure. Surface-enhanced Raman scattering measurements of DNA hybridization on plasmonic-metal substrates have been used to overcome small Raman-scattering cross-sections; however, surface-enhanced Raman spectroscopy measurements are not generally quantitative due to the fall-off in the scattering signal with the decay of the electric field enhancement from the surface, which also limits the length of oligonucleotides that can be investigated. In this work, we introduce an experimental methodology in which confocal Raman microscopy is used to characterize hybridization reactions of ssDNA immobilized at the solid/liquid interface of porous silica particles. By focusing the femtoliter confocal probe volume within a single porous particle, signal enhancement arises from the ∼1500-times greater surface area detected compared to a planar substrate. Because the porous support is a purely dielectric material, the scattering signal is independent of the proximity of the oligonucleotide to the silica surface. With this technique, we characterize a 19-mer capture strand and determine its hybridization efficiency with 9-mer and 16-mer target sequences from the scattering of a structurally insensitive phosphate-stretching mode. Changes in polarizability and frequency of scattering from DNA bases were observed, which are consistent with Watson-Crick base pairing. Quantification of base content from their duplex scattering intensities allows us to discriminate between hybridization of two target strands of equivalent length but with different recognition sequences. A duplex having a single-nucleotide polymorphism could be distinguished from hybridization of a fully complementary strand based on differences in base content and duplex conformation.

Non-invasive intradermal imaging of cystine crystals in cystinosis.

Development of noninvasive methodology to reproducibly measure tissue cystine crystal load to assess disease status and guide clinical care in cystinosis, an inherited lysosomal storage disorder characterized by widespread cystine crystal accumulation. To develop an unbiased and semi-automated imaging methodology to quantify dermal cystine crystal accumulation in patients to correlate with disease status. 101 participants, 70 patients and 31 healthy controls, were enrolled at the University of California, San Diego, Cystinosis Clinics, Rady Children's Hospital, San Diego and at the annual Cystinosis Research Foundation family conference for an ongoing prospective longitudinal cohort study of cystinosis patients with potential yearly follow-up. Intradermal reflectance confocal microscopy (RCM) imaging, blood collection via standard venipuncture, medical record collection, and occasional skin punch biopsies. The primary outcome was to establish an automated measure of normalized confocal crystal volume (nCCV) for each subject. Secondary analysis examined the association of nCCV with various clinical indicators to assess nCCV's possible predictive potential. Over 2 years, 57 patients diagnosed with cystinosis (median [range] age: 15.1 yrs [0.8, 54]; 41.4% female) were intradermally assessed by RCM to produce 84 image stacks. 27 healthy individuals (38.7 yrs [10, 85]; 53.1% female) were also imaged providing 37 control image stacks. Automated 2D crystal area quantification revealed that patients had significantly elevated crystal accumulation within the superficial dermis. 3D volumetric analysis of this region was significantly higher in patients compared to healthy controls (mean [SD]: 1934.0 μm3 [1169.1] for patients vs. 363.1 μm3 [194.3] for controls, P<0.001). Medical outcome data was collected from 43 patients with infantile cystinosis (media [range] age: 11 yrs [0.8, 54]; 51% female). nCCV was positively associated with hypothyroidism (OR = 19.68, 95% CI: [1.60, 242.46], P = 0.02) and stage of chronic kidney disease (slope estimate = 0.53, 95%CI: [0.05, 1.00], P = 0.03). This study used non-invasive RCM imaging to develop an intradermal cystine crystal quantification method. Results showed that cystinosis patients had increased nCCV compared to healthy controls. Level of patient nCCV correlated with several clinical outcomes suggesting nCCV may be used as a potential new biomarker for cystinosis to monitor long-term disease control and medication compliance.

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AbstractThe dynamics of two different fluorescent dyes labelling DNA and cationic liposomes is monitored through Fluorescence Cross‐Correlation Spectroscopy (FCCS). Measuring the extent of correlation between the flickering of the two dyes as they diffuse in and out of overlapping confocal volumes, the association of DNA and liposomes to form lipoplexes is quantified. This was achieved analysing the correlation with non 1:1 stoichiometric sample description.Further details can be found in the article by Ana I. Gómez‐Varela, Ricardo Gaspar, Adelaide Miranda, Juliane L. Assis, Rafael H.F. Valverde, Marcelo Einicker‐Lamas, Bruno F. B. Silva, and Pieter A.A. De Beule (e202000200). image