Multicolor Long-Term Single-Particle Tracking Using 10 nm Upconverting Nanoparticles.

Single-particle tracking (SPT) is a powerful technique to unveil molecular behaviors crucial to the understanding of many biological processes, but it is limited by factors such as probe photostability and spectral orthogonality. To overcome these limitations, we develop upconverting nanoparticles (UCNPs), which are photostable over several hours at the single-particle level, enabling long-term multicolor SPT. We investigate the brightness of core-shell UCNPs as a function of inert shell thickness to minimize particle size while maintaining sufficient signal for SPT. We explore different rare-earth dopants to optimize for the brightest probes and find that UCNPs doped with 2% Tm3+/30% Yb3+, 10% Er3+/90% Yb3+, and 15% Tm3+/85% Yb3+ represent the optimal probes for blue, green, and near-infrared emission, respectively. The multiplexed 10 nm probes enable three-color single-particle tracking on live HeLa cells for tens of minutes using a single, near-infrared excitation source. These photostable and multiplexed probes open new avenues for numerous biological applications.

Fast and Artifact-Free Excitation Multiplexing Using Synchronized Image Scanning.

Multicolor fluorescence microscopy is an essential tool to visualize structures and dynamics in the life and materials sciences. However, the near-simultaneous acquisition of labels differing in excitation spectrum is difficult and renders such measurements prone to artifacts. We present a simple strategy to provide quasi-simultaneous fluorescence imaging with multiple excitation wavelengths by using an optical element to displace the sample image on the sensor at a rate that is much faster than the image acquisition rate and synchronizing this with the illumination. The emission elicited by the different wavelengths can then be encoded into the point-spread function of the imaging or visualized as multiple distinct images. In doing so, our approach can eliminate or mitigate artifacts caused by temporal aliasing in conventional sequential imaging. We demonstrate the use of our system to uncover hidden emissive states in single quantum dots and for the imaging of Ca2+ signaling in neurons.

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes.

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pKa , off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

[Horseshoe retinal tear: factors that determine the risk of developing rhegmatogenous retinal detachment]. / Klapannyi razryv setchatki: faktory, opredelyayushchie risk razvitiya regmatogennoi otsloiki setchatki.

Horseshoe (flap) retinal tears are the leading cause of rhegmatogenous retinal detachment (RRD). Identification of the most significant predictors of RRD in patients with a horseshoe tear will enable the development of an optimal treatment strategy. PURPOSE: This study aimed to determine the main risk factors for RRD development based on the analysis of the condition of vitreoretinal interface in the area of horseshoe tears, both isolated and those that resulted in retinal detachment. MATERIAL AND METHODS: A total of 88 patients with horseshoe retinal tears (43 patients with RRD due to the horseshoe tear and 45 with isolated horseshoe tears) were included in the study. All patients underwent wide-field multispectral laser scanning and optical coherence tomography to determine the shape of the horseshoe tear and the extent of vitreoretinal adhesion (VRA). Cluster analysis was used to differentiate horseshoe tears by shape. Spearman's correlation analysis was used to identify the relationship between the shape of the horseshoe tear and localization of VRA. RESULTS: Spearman's correlation analysis revealed a strong negative correlation between the length-to-width ratio of the horseshoe tear and the extent of VRA. Cluster analysis helped determine four shapes of horseshoe tears, each corresponding to a certain localization of VRA. Analysis of RRD risk, depending on the characteristics of the horseshoe tear, showed that the most significant risk factor for the development of RRD is the presence of a horseshoe tear with width greater than its length, which is characterized by a larger VRA area. CONCLUSION: The study established that the larger the horseshoe tear width and the smaller its length, the larger the VRA area and, consequently, the higher the risk of RRD development. Horseshoe retinal tears with a length-to-width ratio of less than 1/1 are the most dangerous in terms of RRD risk, which is important to consider when selecting the treatment tactics.

Multiplexed PSF Engineering for Three-Dimensional Multicolor Particle Tracking.

Three-dimensional spatiotemporal tracking of microscopic particles in multiple colors is a challenging optical imaging task. Existing approaches require a trade-off between photon efficiency, field of view, mechanical complexity, spectral specificity, and speed. Here, we introduce multiplexed point-spread-function engineering that achieves photon-efficient, 3D multicolor particle tracking over a large field of view. This is accomplished by first chromatically splitting the emission path of a microscope to different channels, engineering the point-spread function of each, and then recombining them onto the same region of the camera. We demonstrate our technique for simultaneously tracking five types of emitters in vitro as well as colocalization of DNA loci in live yeast cells.

Sensitivity and specificity of MultiColor imaging in detecting proliferative diabetic retinopathy.

PURPOSE: To evaluate the accuracy of MultiColor imaging (MC) compared to fluorescein angiography (FA) in detecting proliferative diabetic retinopathy (PDR) and associated diabetic retinopathy features. METHODS: Fifty-nine eyes from 38 PDR patients were included. MC images were reviewed by 2 independent masked graders. A qualitative analysis based on the following features was performed: neovascular complexes (NVC), disc neovascularization (NVD), neovascularization elsewhere (NVE), microaneurysm (MA), intraretinal hemorrhage (IRH), vitreous hemorrhage (VH), preretinal hemorrhage (PRH), fibrosis, hard exudates (HE), epiretinal membrane (ERM), diabetic macular edema (DME), ischemia and laser spots (LS). Measures of diagnostic accuracy compared to FA were determined. RESULTS: The sensitivity for the detection of NVC using MC was 95.1%, with a specificity of 40.0%, positive predictive value (PPV) of 92.9% and negative predictive value (NPV) of 50.0%. Sensitivity and specificity were higher in detecting NVD (88.9% and 76.9%) while NVE registered higher PPV (88.9%). MC was highly sensitive in detecting IRH, HE, ERM and LS (100%), MA (98.0%) and fibrosis (95.5%). Highest specificity was found for VH (100.0%), DME (100.0%), PRH (98.1%) and LS (89.5%). The area under the receiver-operating characteristic analysis of MC was excellent in NVD (0.83, 95% confidence interval (CI), 0.71-0.95, p < 0.001), IRH (0.89, 95% CI 0.74-1.00, p < 0.001), VH (0.81, 95% CI 0.60-1.00, p = 0.005) and PRH (0.89, 95% CI 0.68-1.00, p = 0.004) and outstanding in LS detection (0.95, 95% CI 0.87-1.00, p < 0.001). These results are likely due to the contrast and quality of the MC since better discrimination is enabled by the green wavelength. CONCLUSION: MC is useful in evaluation of PDR patients and can complement noninvasive imaging. MC detected some PDR features more accurately than FA such as NVD, IRH, VH, PRH, and LS.

Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT.

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore's response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as "frequency multiplexed DNA-PAINT," or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

Multicolor imaging in macular telangiectasia-a comparison with fundus autofluorescence.

PURPOSE: To describe various clinical features of idiopathic juxtafoveal retinal telangiectasis group 2A or idiopathic macular telangiectasia type 2 (MacTel) on multicolor imaging (MCI) and compare imaging findings of MacTel on MCI with fundus autofluorescence (FAF). METHODS: Patients with a clinical diagnosis of MacTel based on Gass and Blodi's classification were included. FAF and MCI images were graded qualitatively for stage of disease, margins of involvement, hyperautofluorescence on FAF (corresponding retinal atrophy on MCI), and detection of crystals. FAF and MCI were graded quantitatively for the area and number of quadrants involved, hypoautofluorescene on FAF (corresponding intraretinal pigment hyperplasia or retinal pigment epithelium [RPE] atrophy on MCI), and foci of right-angled venules. RESULTS: Seventy-eight eyes of forty five patients were included with both imaging modalities showing no difference with respect to staging of non-proliferative MacTel. Retinal crystals were recognized on MCI but not on FAF. Neurosensory retinal atrophy and  subretinal neovascular membranes were detected using MCI with 92.3 and 83.3% sensitivity, respectively. Intraretinal pigmented hyperplasia was more accurately detected (70.1 vs 58.4%) compared with RPE atrophy on MCI. MCI showed larger area of involvement, higher number of quadrants involved (p < 0.001), and better delineation of margins (p = 0.002) compared with FAF. A higher mean number of vessel dipping foci was noted on MCI in comparison with FAF (3.34 vs 3.1). CONCLUSION: Various parameters were more easily defined using MCI compared with FAF which qualifies MCI as an enface depth-resolved imaging adjunct to conventional multimodal imaging in MacTel. The ability to detect enface as well as cross-sectional imaging features makes MCI a valuable tool in MacTel.

Whole-Cell, 3D, and Multicolor STED Imaging with Exchangeable Fluorophores.

We demonstrate stimulated emission depletion (STED) microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multicolor, and live-cell STED microscopy.

Leek-derived codoped carbon dots as efficient fluorescent probes for dichlorvos sensitive detection and cell multicolor imaging.

A biomass nitrogen and sulfur codoped carbon dots (NS-Cdots) was prepared by a simple and clean hydrothermal method using leek, and was employed as efficient fluorescent probes for sensitive detection of organophosphorus pesticides (OPs). The leek-derived NS-Cdots emitted blue fluorescence, but was quenched by H2O2. Due to acetylcholinesterase/choline oxidase-based cascade enzymatic reaction that produces H2O2 and the inhibition effect of OPs on acetylcholinesterase activity, a NS-Cdots-based fluorescence "off-on" method to detect OPs-dichlorvos (DDVP) was developed. More sensitivity and wider linear detection range were achieved from 1.0 × 10-9 to 1.0 × 10-3 M (limit of detection = 5.0 × 10-10 M). This developed method was applied to the detection of DDVP in Chinese cabbage successfully. The average recoveries were in the range of 96.0~104.0% with a relative standard deviation of less than 3.3%. In addition, the NS-Cdots fluorescent probes were also employed successfully in multicolor imaging of living cells, manifesting that the NS-Cdots fluorescent probes have great application potential in agricultural and biomedical fields. Graphical Abstract.

Protein-Specific, Multicolor and 3D STED Imaging in Cells with DNA-Labeled Antibodies.

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.

Multi-parametric imaging of cell heterogeneity in apoptosis analysis.

Apoptosis is a multistep process of programmed cell death where different morphological and molecular events occur simultaneously and/or consequently. Recent progress in programmed cell death analysis uncovered large heterogeneity in response of individual cells to the apoptotic stimuli. Analysis of the complex and dynamic process of apoptosis requires a capacity to quantitate multiparametric data obtained from multicolor labeling and/or fluorescent reporters of live cells in conjunction with morphological analysis. Modern methods of multiparametric apoptosis study include but are not limited to fluorescent microscopy, flow cytometry and imaging flow cytometry. In the current review we discuss the image-based evaluation of apoptosis on the single-cell and population level by imaging flow cytometry in parallel with other techniques. The advantage of imaging flow cytometry is its ability to interrogate multiparametric morphometric and fluorescence quantitative data in statistically robust manner. Here we describe the current status and future perspectives of this emerging field, as well as some challenges and limitations. We also highlight a number of assays and multicolor labeling probes, utilizing both microscopy and different variants of imaging cytometry, including commonly based assays and novel developments in the field.

Comparison of conventional color fundus photography and multicolor imaging in choroidal or retinal lesions.

PURPOSE: Our purpose was to compare the characteristics of the retinal and choroidal lesions including choroidal nevus, choroidal melanoma and congenital hypertrophy of the retina pigment epithelium using conventional color fundus photography (CFP) and multicolor imaging (MCI). METHODS: The paired images of patients with retinal or choroidal lesions were assessed for the visibility of lesion's border, halo and drusen using a grading scale (0-2). The area of the lesion was measured on both imaging modalities. The same grading was also done on the individual color channels of MCI for a further evaluation. RESULTS: Thirty-three eyes of 33 patients were included. There were no significant differences in the mean border, drusen and halo visibility scores between the two imaging modalities (p = 0.12, p = 0.70, p = 0.35). However, the mean area of the lesion was significantly smaller on MCI than that on CFP (14.9±3.3 versus 18.7±3.4 mm2, p = 0.01). CONCLUSION: The appearance of choroidal and/ or retinal lesions on MCI may be different than that on CFP. Though MCI can provide similar information with CFP for the features of retinal and/ or choroidal lesions including border, halo and drusen; the infrared light reflection on MCI underestimates the extent of the choroidal lesion by 33%.

Performance characteristics of multicolor versus blue light and infrared imaging in the identification of reticular pseudodrusen.

PURPOSE: To describe the appearance of reticular pseudodrusen on multicolor imaging and to evaluate its diagnostic accuracy as compared with the two modalities that may be considered the current reference standard, blue light and infrared imaging. METHODS: Retrospective study in which all multicolor images (constructed from images acquired at 486 nm-blue, 518 nm-green and 815 nm-infrared) of 45 consecutive patients visited in a single center was reviewed. Inclusion criteria involved the presence of >1 reticular pseudodrusen on a 30° × 30° image centered on the fovea as seen with the blue light channel derived from the multicolor imaging. Three experienced observers, masked to each other's results with other imaging modalities, independently classified the number of reticular pseudodrusen with each modality. RESULTS: The median interobserver agreement (kappa) was 0.58 using blue light; 0.65 using infrared; and 0.64 using multicolor images. Multicolor and infrared modalities identified a higher number of reticular pseudodrusen than blue light modality in all fields for all observers (p < 0.0001). Results were not different when multicolor and infrared were compared (p ≥ 0.27). CONCLUSIONS: These results suggest that multicolor and infrared are more sensitive and reproducible than blue light in the identification of RPD. Multicolor did not appear to add a significant value to infrared in the evaluation of RDP. Clinicians using infrared do not need to incorporate multicolor for the identification and quantification of RPD.

Perfluorocarbon-Based 19 F†MRI Nanoprobes for In†Vivo Multicolor Imaging.

In vivo multicolor imaging is important for monitoring multiple biomolecular or cellular processes in biology. 19 F magnetic resonance imaging (MRI) is an emerging in vivo imaging technique because it can non-invasively visualize 19 F nuclei without endogenous background signals. Therefore, 19 F MRI probes capable of multicolor imaging are in high demand. Herein, we report five types of perfluorocarbon-encapsulated silica nanoparticles that show 19 F NMR peaks with different chemical shifts. Three of the nanoprobes, which show spectrally distinct 19 F NMR peaks with sufficient sensitivity, were selected for in vivo multicolor 19 F MRI. The nanoprobes exhibited 19 F MRI signals with three colors in a living mouse. Our in vivo multicolor system could be utilized for evaluating the effect of surface functional groups on the hepatic uptake in a mouse. This novel multicolor imaging technology will be a practical tool for elucidating in vivo biomolecular networks by 19 F MRI.

Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes.

In multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near-infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850-1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post-acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Multiplexed imaging of intracellular protein networks.

Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.

DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery.

We present a facile approach to make aptamer-conjugated FRET (fluorescent resonance energy transfer) nanoflowers (NFs) through rolling circle replication for multiplexed cellular imaging and traceable targeted drug delivery. The NFs can exhibit multi-fluorescence emissions by a single-wavelength excitation as a result of the DNA matrix covalently incorporated with three dye molecules able to perform FRET. Compared with the conventional DNA nanostructure assembly, NF assembly is independent of template sequences, avoiding the otherwise complicated design of DNA building blocks assembled into nanostructures by base-pairing. The NFs were uniform and exhibited high fluorescence intensity and excellent photostability. Combined with the ability of traceable targeted drug delivery, these colorful DNA NFs provide a novel system for applications in multiplex fluorescent cellular imaging, effective screening of drugs, and therapeutic protocol development.