Multicolor Imaging Research Articles

Lysosome-targeted silicon quantum dots theranostics for simultaneous fluorescent imaging and photodynamic therapy

As an emerging treatment method, photodynamic therapy (PDT) has attracted considerable interest due to the characteristics of non-invasiveness, repeatable treatment, high spatiotemporal resolution and few side effects. However, the life span (<40 ns) and diffusion distance (<20 nm) of reactive oxygen species such as singlet oxygen (1O2) in tumor cells are extremely short, which has seriously limited therapeutic efficacy of PDT. The enrichment site of photosensitizers in cancer cells is usually the first site of PDT action, which will not only affect the biological signaling pathway of cancer cell death, but also is closely related to the final therapeutic effect. Therefore, the design and preparation of photosensitizers targeting specific subcellular organelles can directly break the biological function of the organelle and trigger the corresponding cell death signaling pathway, which can significantly improve the efficacy of PDT. Herein, a lysosome-targeted silicon quantum dots (L-Si QDs) was first made by diethylene glycol-mediated synthetic route as a multicolor fluorescent imaging reagents and a new photosensitizer. The as-prepared L-Si QDs exhibit bright fluorescence with excellent pH stability and time stability, excitation-dependent emission, and good biocompatibility. Furthermore, the results of cell experiments showed that L-Si QDs was accumulated in lysosomes after being taken up by cancer cells, and can efficiently produce 1O2 upon 635 nm laser irradiation, which can damage lysosomes, up-regulate cleavage caspase-3, increase Bax release, down-regulate Bcl-2 and induce cell apoptosis finally. This study significantly broadens the biomedical applications of silicon quantum dots and provides excellent nanomaterials candidates for tumor phototherapy.

Volumetric live-cell autofluorescence imaging using Fourier light-field microscopy.

This study introduces a rapid, volumetric live-cell imaging technique for visualizing autofluorescent sub-cellular structures and their dynamics by employing high-resolution Fourier light-field microscopy. We demonstrated this method by capturing lysosomal autofluorescence in fibroblasts and HeLa cells. Additionally, we conducted multicolor imaging to simultaneously observe lysosomal autofluorescence and fluorescently-labeled organelles such as lysosomes and mitochondria. We further analyzed the data to quantify the interactions between lysosomes and mitochondria. This research lays the foundation for future exploration of native cellular states and functions in three-dimensional environments, effectively reducing photodamage and eliminating the necessity for exogenous labels.

Double emission CDs modified with rhodamine B for ratiometric sensing detection and bioimaging

In this paper, rhodamine B (RhB) was modified on carbon dots (CDs) by hydrothermal method, and a double emission ratiometric fluorescent probe (RhB-CDs) with red luminescence was successfully constructed. The synthesized RhB-CDs had exceptional optical properties and exhibit two emission peaks (480 nm and 590 nm) at 350 nm excitation. It could be used for ratiometric detection of MnO4− and Cr2O72−, and the fluorescent color would also change (red to orange) when the pH changed from 6 to 7, showing great application potential in the field of sensing and detection. Moreover, the ratio of the emission peak fluorescence intensity had a linear relationship with the water content of alcohols, which was successfully used to detect actual water samples. In addition, the synthesized RhB-CDs had low cytotoxicity and were successfully used for multicolor cell imaging.

Graphene-Based Physically Unclonable Functions with Dual Source of Randomness.

There is growing interest in systems with randomized responses for generating physically unclonable functions (PUFs) in anticounterfeiting and authentication applications. Atomic-level control over its thickness and unique Raman spectrum make graphene an attractive material for PUF applications. Herein, we report graphene PUFs that emerge from two independent stochastic processes. Randomized variations in the shape and number of graphene adlayers were achieved by exploiting and improving the mechanistic understanding of the chemical vapor deposition of graphene. The randomized positioning of the graphene domains was then facilitated by dewetting the polymer film, followed by oxygen plasma etching. This approach yielded surfaces with randomly positioned and shaped graphene islands with varied numbers of layers and, therefore, Raman spectra. Raman mapping of surfaces resulted in multicolor images with a high encoding capacity. Advanced feature-matching algorithms were employed for the authentication of multicolor images. The use of two independent stochastic processes on a two-dimensional nanomaterial platform enables the creation of unique and complex surfaces that excessively challenge clonability.

Multi-color two-photon scanning structured illumination microscopy imaging of live cells.

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.

OPTICAL PROPERTIES OF COLLOIDAL SOLUTIONS OF CARBON DOTS

In this work, optical properties of carbon dots (CDs) chemically synthesized by solvothermal carbonization of a mixture of urea and anhydrous citric acid were studied. Size distribution of CDs with a typical chemical structure was estimated from dynamic light scattering. Absorption, fluorescence excitation/emission spectra as well as characteristic fluorescence lifetimes were measured. An energy diagram of photostimulated electronic transitions in this type of multicolor fluorescent carbon nanoparticles was also proposed. Temperature dependence of the PL/PLE-maps of CDs colloids in the range of 5‒75 ° C was measured and it demonstrates the possibility of their use for thermometry purposes. We also showed a possibility of CDs using for multicolor cell fluorescence imaging.

Real-time correction of chromatic aberration in optical fluorescence microscopy

Multi-color fluorescence imaging is a powerful tool for studying the spatial relationships and interactions among sub-cellular structures in biological specimens. However, if improperly corrected, geometrical distortions caused by mechanical drift, refractive index mismatch, or chromatic aberration can lead to lower image resolution. In this paper, we present an extension of the image processing framework of Scipion by integrating a protocol called OFM Corrector, which corrects geometrical distortions in real-time using a B-spline-based elastic continuous registration technique. Our proposal provides a simple strategy to overcome chromatic aberration by digitally re-aligning color channels in multi-color fluorescence microscopy images, even in 3D or time. Our method relies on a geometrical calibration, which we do with fluorescent beads excited by different wavelengths of light and subsequently registered to get the elastic warp as a reference to correct chromatic shift. Our software is freely available with a user-friendly GUI and can be broadly used for various biological imaging problems. The paper presents a valuable tool for researchers working in light microscopy facilities.

Development of Highly Dense Material-Specific Fluorophore Labeling Method on Silicon-Based Semiconductor Materials for Three-Dimensional Multicolor Super-Resolution Fluorescence Imaging

Development of Highly Dense Material-Specific Fluorophore Labeling Method on Silicon-Based Semiconductor Materials for Three-Dimensional Multicolor Super-Resolution Fluorescence Imaging

FluoroPi Device With SmartProbes: A Frugal Point-of-Care System for Fluorescent Detection of Bacteria From a Pre-Clinical Model of Microbial Keratitis.

Rapid and accurate diagnosis of microbial keratitis (MK) could greatly improve patient outcomes. Here, we present the development of a rapid, accessible multicolour fluorescence imaging device (FluoroPi) and evaluate its performance in combination with fluorescent optical reporters (SmartProbes) to distinguish bacterial Gram status. Furthermore, we show feasibility by imaging samples obtained by corneal scrape and minimally invasive corneal impression membrane (CIM) from ex vivo porcine corneal MK models. FluoroPi was built using a Raspberry Pi single-board computer and camera, light-emitting-diodes (LEDs), and filters for white-light and fluorescent imaging, with excitation and detection of bacterial optical SmartProbes: Gram-negative, NBD-PMX (exmax 488 nm); Gram positive, Merocy-Van (exmax 590 nm). We evaluated FluoroPi with bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) isolated from ex vivo porcine corneal models of MK by scrape (needle) and CIM with the SmartProbes. FluoroPi provides <1 µm resolution and was able to readily distinguish bacteria isolated from ex vivo models of MK from tissue debris when combined with SmartProbes, retrieved by both scrape and CIM. Single bacteria could be resolved within the field of view, with limits of detection demonstrated as 103 to 104 CFU/mL. Sample preparation prior to imaging was minimal (wash-free), and imaging and postprocessing with FluoroPi were straightforward, confirming ease of use. FluoroPi coupled with SmartProbes provides effective, low-cost bacterial imaging, delineating Gram-negative and Gram-positive bacteria directly sampled from a preclinical model of MK. This study provides a crucial stepping stone toward clinical translation of a rapid, minimally invasive diagnostic approach for MK.

Pseudoheterodyne interferometry for multicolor near-field imaging.

We report the development and characterization of a detection technique for scattering-type scanning near-field optical microscopy (s-SNOM) that enables near-field amplitude and phase imaging at two or more wavelengths simultaneously. To this end, we introduce multispectral pseudoheterodyne (PSH) interferometry, where infrared lasers are combined to form a beam with a discrete spectrum of laser lines and a time-multiplexing scheme is employed to allow for the use of a single infrared detector. We first describe and validate the implementation of multispectral PSH into a commercial s-SNOM instrument. We then demonstrate its application for the real-time correction of the negative phase contrast (NPC), which provides reliable imaging of weak IR absorption at the nanoscale. We anticipate that multispectral PSH could improve data throughput, reduce effects of sample and interferometer drift, and help to establish multicolor s-SNOM imaging as a regular imaging modality, which could be particularly interesting as new infrared light sources become available.

Multicolor emission-based nitrogen, sulfur and boron co-doped photoluminescent carbon dots for sequential sensing of Fe3+ and cysteine: RGB color sensor and live cell imaging

Herein, a simple hydrothermal synthesis is used to prepare multiple heteroatom-doped photoluminescent carbon dots (CDs) from thiourea (N and S source) and boric acid (B source) as precursors. The optical and physicochemical properties of the as-synthesized NSB-CDs were studied using UV–Vis, photoluminescence, TEM, FT-IR, XRD, Raman, and XPS analyses. The NSB-CDs exhibited excellent stability, high photostability, pH, and ionic strength tolerance; they retained their excellent stability independent of excitation. The NSB-CDs featured small sizes of approximately 3.2 ± 0.4 nm (range: 2.0–5.0 nm) as evidenced using TEM measurements. The NSB-CDs were used as a photoluminescent sensing platform to detect Fe3+ as well as cysteine (Cys) molecules. The competitive binding of Cys to Fe3+ resulted in NSB-CDs that retained their photoluminescence. For the rapid identification and quantification of Fe3+ and Cys, NSB-CDs were developed as a “switch-on” dual-function sensing platform. The linear detection range of Fe3+ was 0–20 μM (limit of detection [LOD]: 54.4 nM) and that of Cys was 0–50 μM (LOD: 4.9 nM). We also introduced a smartphone RGB analysis method for detecting low-concentration solutions based on digital images. The NSB-CDs showed no toxicity at 100 μg/mL. Photoluminescent probes for multicolor live-cell imaging can be used with NSB-CDs at this concentration, suggesting that NSB-CDs may be promising photoluminescent probes.

Spectrum-optimized direct image reconstruction of super-resolution structured illumination microscopy

Super-resolution structured illumination microscopy (SR-SIM) has become a widely used nanoscopy technique for rapid, long-term, and multi-color imaging of live cells. Precise but troublesome determination of the illumination pattern parameters is a prerequisite for Wiener-deconvolution-based SR-SIM image reconstruction. Here, we present a direct reconstruction SIM algorithm (direct-SIM) with an initial spatial-domain reconstruction followed by frequency-domain spectrum optimization. Without any prior knowledge of illumination patterns and bypassing the artifact-sensitive Wiener deconvolution procedures, resolution-doubled SR images could be reconstructed by direct-SIM free of common artifacts, even for the raw images with large pattern variance in the field of view (FOV). Direct-SIM can be applied to previously difficult scenarios such as very sparse samples, periodic samples, very small FOV imaging, and stitched large FOV imaging.

A multi-image compression and encryption scheme based on fractional chaotic map

In this paper, a visual security encryption scheme for multi-color images based on BP neural network and fractional chaotic map is proposed, which disguises secret images as a meaningful visual image. Firstly, three color images are compressed based on BP neural network. Then, according to the pseudo-random sequence generated by fractional chaotic map, the merged compressed images are scrambled by spiral transformation and diffused by XOR, in which the direction and degree of spiral transformation can be adjusted. In order to ensure the visual effect of the camouflage image, the lifting wavelet transform (LWT) is used to decompose the carrier image to obtain the coefficient matrix, and the cipher images are adjusted to a narrow range and embedded into the coefficient matrixes based on the pseudo-random sequence. Finally, visually secure image can be generated by inverse lifting wavelet transform. The reverse algorithm can restore the images by extraction, decryption and decompression. Experimental results verify that the proposed scheme has feasibility, robustness, anti-noise and clipping capability, and the PSNR value is no less than 31.4 under various attacks.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Pyridinic-N-rich carbon dots in IFE-based Turn-off Fluorometric detection of nerve agent Mimic- Diethyl chlorophosphate and multicolor cell imaging.

Fluorometric sensors for the detection of nerve agent mimics have received a lot of interest nowadays due to their high sensitivity and selectivity, ease of operation, and real-time monitoring. Pyridinic-N-rich carbon dots (NCDs) prepared through microwave-assisted pyrolysis of l-Malic acid and urea have been explored first time in this work as a novel turn-off fluorescent probe for the sensitive and selective detection of diethyl chlorophosphate (DCP), a nerve agent mimic. The as-prepared carbon dots contained a large amount of pyridinic nitrogen on their surface, which can modulate the photoluminescence properties of the NCDs. The blue emissive NCDs possessed both excitation wavelength-dependent and independent emission behavior. The detection of DCP was premised on quenching of the fluorescence emission intensity of NCDs in the presence of similar chemical reagents (e.g., trimethyl phosphate, triethyl phosphate, triethyl phosphonoacetate, triphenyl phosphate, diphenyl phosphate, tributyl phosphate). Fluorescence quenching of the NCDs in the presence of DCP has been attributed to the inner filter effect (IFE). From the linear Stern-Volmer plot (R2=0.9992), the limit of detection (LOD) was found to be 84μM for sensing DCP for the concentration ranging between 3 and 15mM. The biocompatibility of NCDs was assessed through cytotoxicity assay on MDA-MB-231 breast cancer cells. Fluorescence imaging demonstrated that NCDs have low cytotoxicity and can be employed successfully in cell imaging.

Multiscale attention network for retinal vein occlusion classification with multicolor image

AbstractRecently, automatic diagnostic approaches widely use various retinal images to classify ocular diseases. And retinal vein occlusion (RVO) is the second most common retinal vascular disease after diabetic retinopathy. In clinical practice, ophthalmologists are usually accustomed to resorting to images of one modality. But single‐modality images often ignore other modality‐specific information. To solve this problem, this paper uses a novel retinal imaging, the multicolor (MC) imaging, for RVO recognition. It can obtain four multiple modal images with different wavelengths to provide much richer information about retinal features. Since the MC images contain local and global pathologies at multiple scales, a multiscale attention structure is proposed to recognize RVO. In simple terms, this structure uses Resnet as the backbone network for feature extraction, with simultaneous input of images in four modalities. Then, the feature maps at different scales are fed into an attention module to fuse the global and local features, which combines two attention mechanisms, the channel attention mechanism and the spatial attention mechanism. The extensive experimental results demonstrate that our proposed framework achieves quite promising classification performance on the fundus diseases and normal images.

Review of Retinal Imaging Modalities for Hydroxychloroquine Retinopathy.

This review provides an overview of conventional and novel retinal imaging modalities for hydroxychloroquine (HCQ) retinopathy. HCQ retinopathy is a form of toxic retinopathy resulting from HCQ use for a variety of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Each imaging modality detects a different aspect of HCQ retinopathy and shows a unique complement of structural changes. Conventionally, spectral-domain optical coherence tomography (SD-OCT), which shows loss or attenuation of the outer retina and/or retinal pigment epithelium-Bruch's membrane complex, and fundus autofluorescence (FAF), which shows parafoveal or pericentral abnormalities, are used to assess HCQ retinopathy. Additionally, several variations of OCT (retinal and choroidal thickness measurements, choroidal vascularity index, widefield OCT, en face imaging, minimum intensity analysis, and artificial intelligence techniques) and FAF techniques (quantitative FAF, near-infrared FAF, fluorescence lifetime imaging ophthalmoscopy, and widefield FAF) have been applied to assess HCQ retinopathy. Other novel retinal imaging techniques that are being studied for early detection of HCQ retinopathy include OCT angiography, multicolour imaging, adaptive optics, and retromode imaging, although further testing is required for validation.

Improving the interference resistance of multi-color barcode images

A method of improving the interference resistance of multi-color barcodes is pro-posed, which is based on the application of two-level control of errors that occur when scanning a multi-color barcode image, using two multi-valued correcting codes: the BCH code - at the level of barcode patterns (lower level), and the Reed-Solomon code - at barcode symbol level (top level). The proposed method makes it possible to create multi-color barcodes with im-proved immunity characteristics, which ensure reliable reproduction of data during scanning of large-capacity multi-color barcode images in automatic identification sys-tems.

Flexible terahertz optics: light beam profile engineering via C-shaped metallic metasurface

The potential of terahertz (THz) imaging implementation in a large variety of applications requires compact, reliable, and relatively low-cost solutions in systems constituents. Here we focus on the development of passive optical elements and demonstrate flexible and reliable THz beam profile engineering for imaging aims via mechanical bending of a stainless steel-based C-shaped metasurface. The designed and laser-ablation technology fabricated metasurface provides compact THz focusing and enables THz light engineering as well as polarization control ability in THz imaging. Focusing, light profile engineering, and polarization control performances of the metasurfaces with different focal distances and C-shape designs are presented. Experimental data are well supported by simulations, using Finite Difference Time Domain (FDTD) method. THz images of different objects at ∼94 GHz using InP Gunn diode, bow-tie sensors, and exclusively metasurfaces-based optics are exposed. Further routes in the development of low-cost multicolor and polarization-sensitive THz imaging are discussed.

The phenotype of cryopreserved platelets influences the formation of platelet-leukocyte aggregates in an in vitro model

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.