Multicolor Fluorescence Imaging Research Articles

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

All-In-One OsciDrop Digital PCR System for Automated and Highly Multiplexed Molecular Diagnostics.

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all-in-one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation-induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette-based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning-driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high-multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost-effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics.

Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy.

High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20-40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1).

Percentage and Absolute CD4+ Count and Factors Influencing Their Levels in Tuberculosis Patients in a Tetiary Hospital, Kaduna, Nigeria

Background: The role of CD4+ T cells in immunity against tuberculosis have been widely acknowledged. This study determined CD4+ absolute, CD4 percentage and haemoglobin (HB) levels in tuberculosis (TB) patients undergoing TB treatment and compared these levels with presence of some factors which could modify the quantities of CD4+, CD4% and HB in TB patients. Methods: A cross-sectional study of eighty-five persons attending TB DOTS clinic in tertiary hospital between 2020 to 2021. Multicolour fluorescence imaging and absorbance spectrometry microscopy was used to enumerate CD4+ absolute count, CD4+ percentage and HB concentration in whole blood. Questionnaire and interviews were used to determine history, demographic data, BCG vaccination, BCG scar, vitamin A supplementation, awareness of zoonotic diseases and zoonotic TB, maternal BCG vaccination status, maternal BCG scar and undernutrition for each respondent. Results: CD4% significantly differed between forty-seven males (mean age: 33.5) and thirty-eight females (mean age: 32.2) (p <0.05). T-test indicated no significant difference in CD4+ count, CD4% and HB compared with BCG vaccination status, presence of BCG scar, knowledge of zoonotic diseases, knowledge of zoonotic TB and with vitamin A supplementation (p <0.05). There was also significant difference in CD4% compared with undernutrition and in absolute CD4+ counts compared with drug resistance (p <0.05). Conversely, there was significant difference in CD4% and absolute CD4+ count when compared with maternal BCG vaccination, presence of maternal BCG Scar. Pearson correlation coefficient indicated weak negative relationship between CD4%, absolute CD4+ count and age. Also, weak positive relationship between HB and age. Both relationships were not significant (p <0.05). Conclusion: Undernutrition, gender, maternal BCG vaccination (with or without scaring) and age influence levels of CD4+ in TB patients.

Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy.

Understanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30nm spatial resolution. We developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30nm lateral resolution. We used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM). We demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites. Our optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.

Super-resolution multicolor fluorescence microscopy enabled by an apochromatic super-oscillatory lens with extended depth-of-focus

Planar super-oscillatory lens (SOL), a far-field subwavelength-focusing diffractive device, holds great potential for achieving sub-diffraction-limit imaging at multiple wavelengths. However, conventional SOL devices suffer from a numerical-aperture-related intrinsic tradeoff among the depth of focus (DoF), chromatic dispersion and focusing spot size. Here, we apply a multi-objective genetic algorithm (GA) optimization approach to design an apochromatic binary-phase SOL having a prolonged DoF, customized working distance (WD), minimized main-lobe size, and suppressed side-lobe intensity. Experimental implementation demonstrates simultaneous focusing of blue, green and red light beams into an optical needle of ~0.5λ in diameter and DOF > 10λ at WD = 428 μm. By integrating this SOL device with a commercial fluorescence microscope, we perform, for the first time, three-dimensional super-resolution multicolor fluorescence imaging of the “unseen” fine structures of neurons. The present study provides not only a practical route to far-field multicolor super-resolution imaging but also a viable approach for constructing imaging systems avoiding complex sample positioning and unfavorable photobleaching.

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.

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.

A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and with acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color invivo imaging. Its simple design, using readily available optical components, and its easy integration provide a low-cost solution for multi-color fluorescence imaging with increased efficiency and speed.

Miniaturized multicolor fluorescence imaging system integrated with a PDMS light-guide plate for biomedical investigation

Miniaturized fluorescence imaging systems are promising platforms that overcome the limited use of conventional microscopes in the biomedical field. However, there are physical limitations for multicolor fluorescence imaging in existing miniaturized imaging systems because multiple filters have to be integrated into a small structure. Here, we present a miniaturized multicolor fluorescence imaging system integrated with single polydimethylsiloxane (PDMS) light-guide plate (LGP) for multicolor fluorescence imaging. The PDMS LGP allows guiding the transmitted light from the light source only to the fluorescent samples regardless of the wavelength of the light source. Thus, our system is capable of multicolor fluorescence imaging without multiple filters that block the excitation light. We demonstrated the usability of our system in the biomedical field by observing green- and red-labeled cells in the incubator. Our proposed system can be used in a wide range of applications for studies that require multicolor fluorescence imaging in the biomedical field.

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.

Critical Steps in DAPI and FISH Imaging of Chromosome Spread Preparations.

The final step in a long period of chromosome slide experiments is the publication of DAPI and multicolor fluorescence images. Quite often the result of published artwork is disappointing due to insufficient knowledge of image processing and presentation. In this chapter we describe some errors of fluorescence photomicrographs and how to avoid them. We include suggestions of processing chromosome images with simple examples of image processing in Photoshop® or the like, without the need of complex knowledge of the software programs.

Multimodal algorithm for differential diagnosis of tumors and tumor-like diseases of the ocular fundus

Diagnosing pathologies of the ocular fundus and performing differential diagnosis of intraocular tumors along with conventional ophthalmoscopy can involve additional visualization methods such as ultrasonography, fluorescein angiography and optical coherence tomography (OCT). Many researchers note the importance of employing a multimodal approach in differential diagnosis of intraocular tumors, but there is no universally established algorithm for a rational choice of both the combination of visualizing methods, and the sequence of their application with consideration of the ophthalmoscopy findings and the results of first-line diagnostic methods. The article presents author's own multimodal algorithm developed for differential diagnosis of tumors and tumor-like diseases of the ocular fundus. This approach involves the use of such methods as OCT and Multicolor fluorescence imaging, with exact sequence and combination determined on the basis of ophthalmoscopy and ultrasonography findings.

Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.

The ciliary membrane is continuous with the plasma membrane but has distinct lipid and protein composition, which is key to defining the function of the primary cilium. Ciliary membranes dynamically assemble and disassemble in association with the cell cycle and directly transmit signals and molecules through budding membranes. Various imaging approaches have greatly advanced the understanding of the ciliary membrane function. In particular, fluorescence live-cell imaging has revealed important insights into the dynamics of ciliary membrane assembly by monitoring the changes of fluorescent-tagged ciliary proteins. Protein dynamics can be tracked simultaneously using multi-color live cell imaging by coupling ciliary-associated factors with different colored fluorescent tags. Ciliary membrane and membrane associated-proteins such as Smoothened, 5-HTr6, SSTR3, Rab8a, and Arl13b have been used to track ciliary membranes and centriole proteins like Centrin1/2, CEP164, and CEP83 are often used to mark the ciliary basal body. Here, we describe a method for studying ciliogenesis membrane dynamics using spinning disk confocal live-cell imaging.

Noninvasive Detection of Salt Stress in Cotton Seedlings by Combining Multicolor Fluorescence–Multispectral Reflectance Imaging with EfficientNet-OB2

Salt stress is considered one of the primary threats to cotton production. Although cotton is found to have reasonable salt tolerance, it is sensitive to salt stress during the seedling stage. This research aimed to propose an effective method for rapidly detecting salt stress of cotton seedlings using multicolor fluorescence–multispectral reflectance imaging coupled with deep learning. A prototyping platform that can obtain multicolor fluorescence and multispectral reflectance images synchronously was developed to get different characteristics of each cotton seedling. The experiments revealed that salt stress harmed cotton seedlings with an increase in malondialdehyde and a decrease in chlorophyll content, superoxide dismutase, and catalase after 17 days of salt stress. The Relief algorithm and principal component analysis were introduced to reduce data dimension with the first 9 principal component images (PC1 to PC9) accounting for 95.2% of the original variations. An optimized EfficientNet-B2 (EfficientNet-OB2), purposely used for a fixed resource budget, was established to detect salt stress by optimizing a proportional number of convolution kernels assigned to the first convolution according to the corresponding contributions of PC1 to PC9 images. EfficientNet-OB2 achieved an accuracy of 84.80%, 91.18%, and 95.10% for 5, 10, and 17 days of salt stress, respectively, which outperformed EfficientNet-B2 and EfficientNet-OB4 with higher training speed and fewer parameters. The results demonstrate the potential of combining multicolor fluorescence–multispectral reflectance imaging with the deep learning model EfficientNet-OB2 for salt stress detection of cotton at the seedling stage, which can be further deployed in mobile platforms for high-throughput screening in the field.

Simultaneous labeling and multicolor fluorescence imaging of multiple immune cells on liver frozen section by polychromatic quantum dots below freezing points

A method for simultaneous labeling and multicolor fluorescence imaging of different hepatic immune cells below freezing point is established based on quantum dots. In the experiment, carbon quantum dots with emission wavelength of 435 nm, CdTe@CdS quantum dots at 542 nm and CdSe@ZnS quantum dots at 604 nm are synthesized respectively, it is found that when the mass fractions of KCl (as antifreeze) are 12 %, 14 %, and 12 %, respectively, the three quantum dot dispersion systems remain liquid state at −20 °C. After they are conjugated with the corresponding secondary antibodies, agarose gel electrophoresis, circular dichroism and capillary electrophoresis confirm the effectiveness of conjugation. By indirect immunofluorescence method, the above three quantum dot fluorescent probes are used to simultaneously and specifically target a variety of liver immune cells, and the multi-color simultaneous imaging of different liver immune cells is realized under the same excitation wavelength, it is found that hepatic macrophages are arranged radially in the liver, hepatic stellate cells present punctate distribution, and hepatic sinusoidal endothelial cells present circular distribution, which is consistent with the results of H&E staining and ultrathin section TEM. This study provides an important technical means for elucidating the structure and function of the liver.

Werner syndrome protein works as a dimer for unwinding and replication fork regression.

The determination of the oligomeric state of functional enzymes is essential for the mechanistic understanding of their catalytic activities. RecQ helicases have diverse biochemical activities, but it is still unclear how their activities are related to their oligomeric states. We use single-molecule multi-color fluorescence imaging to determine the oligomeric states of Werner syndrome protein (WRN) during its unwinding and replication fork regression activities. We reveal that WRN binds to a forked DNA as a dimer, and unwinds it without any change of its oligomeric state. In contrast, WRN binds to a replication fork as a tetramer, and is dimerized during activation of replication fork regression. By selectively inhibiting the helicase activity of WRN on specific strands, we reveal how the active dimers of WRN distinctly use the energy of ATP hydrolysis for repetitive unwinding and replication fork regression.