Three-color Imaging Research Articles

The actin filament pointed-end depolymerase Srv2/CAP depolymerizes barbed ends, displaces capping protein, and promotes formin processivity

Cellular actin networks exhibit distinct assembly and disassembly dynamics, primarily driven by multicomponent reactions occurring at the two ends of actin filaments. While barbed ends are recognized as the hotspot for polymerization, depolymerization is predominantly associated with pointed ends. Consequently, mechanisms promoting barbed-end depolymerization have received relatively little attention. Here, using microfluidics-assisted three-color single-molecule imaging, we reveal that cyclase-associated protein (CAP), long known for its roles in nucleotide exchange and pointed-end depolymerization, also acts as a processive depolymerase at filament barbed ends. CAP molecules track barbed ends for several minutes, inducing depolymerization rates of up to 60 subunits per second. Importantly, CAP modulates barbed-end dynamics even under cytosol-mimicking assembly promoting conditions. We further show that CAP can colocalize with both formin and capping protein (CP) at barbed ends. CAP enhances formin processivity by 10-fold, allowing CAP-formin complexes to track fast-elongating barbed ends. In contrast, CAP destabilizes CP-bound barbed ends and accelerates dissociation of CP by fourfold. Our findings, combined with CAP's previously reported activities, firmly establish CAP as a key regulator of cellular actin dynamics.

Multiplexed Shortwave Infrared Imaging Highlights Anatomical Structures in Mice.

Multiplexed fluorescence in vivo imaging remains challenging due to the attenuation and scattering of visible and traditional near infrared (NIR-I, 650-950 nm) wavelengths. Fluorescence imaging using shortwave infrared (SWIR, 1000-1700 nm, a.k.a. NIR-II) light enables deeper tissue penetration due to reduced tissue scattering as well as minimal background autofluorescence. SWIR-emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability are powerful contrast agents, yet few imaging demonstrations exclusively use SWIR emission beyond two-color imaging schemes. In this study, we engineered three high quality lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emission peaks ranging from 1100-1550 nm for simultaneous three-color imaging in mice. We first use the exceptional photostability of QDs to non-invasively track lymphatic drainage with longitudinal imaging, highlighting the detailed networks of lymphatic vessels with widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature networks, including clearly distinguishing the liver and spleen. This work establishes optimized SWIR QDs for next generation multiplexed and longitudinal preclinical imaging, unlocking numerous opportunities for preclinical studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

Biocompatible and Water-Soluble Shortwave-Infrared (SWIR)-Emitting Cyanine-Based Fluorescent Probes for In Vivo Multiplexed Molecular Imaging.

Extending molecular imaging into the shortwave-infrared (SWIR, 900-1400 nm) region provides deep tissue visualization of biomolecules in the living system resulting from the low tissue autofluorescence and scattering. Looking at the Food and Drug Administration-approved and clinical trial near-infrared (NIR) probes, only indocyanine green (ICG) and its analogues have been approved for biomedical applications. Excitation wavelength less than 800 nm limits these probes from deep tissue penetration and noninvasive fluorescence imaging. Herein, we present the synthesis of ICG-based π-conjugation-extended cyanine dyes, ICG-C9 and ICG-C11 as biocompatible, and water-soluble SWIR-emitting probes with emission wavelengths of 922 and 1010 nm in water, respectively. Also, ICG-, ICG-C9-, and ICG-C11-based fluorescent labeling agents have been synthesized for the development of SWIR molecular imaging probes. Using the fluorescence of ICG, ICG-C9, and ICG-C11, we demonstrate three-color SWIR fluorescence imaging of breast tumors by visualizing surface receptors (EGFR and HER2) and tumor vasculature in living mice. Furthermore, we demonstrate two-color SWIR fluorescence imaging of breast tumor apoptosis using an ICG-conjugated anticancer drug, Kadcyla and ICG-C9 or ICG-C11-conjugated annexin V. Finally, we show long-term (38 days) SWIR fluorescence imaging of breast tumor shrinkage induced by Kadcyla. This study provides a general strategy for multiplexed fluorescence molecular imaging with biocompatible and water-soluble SWIR-emitting cyanine probes.

Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) can reach sub-50nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Three-color dSTORM Imaging and Analysis of Recombination Foci in Mouse Spread Meiotic Nuclei.

During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience. Key features Preparation of spread nuclei, resulting in a flattened preparation with easy antibody-accessible chromatin-associated proteins on dSTORM-compatible coverslips. dSTORM analysis of immunofluorescent repair foci in meiotic prophase nuclei. Detailed descriptions of data acquisition, (pre-)processing, and nanofoci feature analysis applicable to all proteins that assemble in immunodetection as discrete foci. Graphical overview.

Compact multicolor two-photon fluorescence microscopy enabled by tailorable continuum generation from self-phase modulation and dispersive wave generation.

By precisely managing fiber-optic nonlinearity with anomalous dispersion, we have demonstrated the control of generating plural few-optical-cycle pulses based on a 24-MHz Chromium:forsterite laser, allowing multicolor two-photon tissue imaging by wavelength mixing. The formation of high-order soliton and its efficient coupling to dispersive wave generation leads to phase-matched spectral broadening, and we have obtained a broadband continuum ranging from 830 nm to 1200 nm, delivering 5-nJ pulses with a pulse width of 10.5 fs using a piece of large-mode-area fiber. We locate the spectral enhancement at around 920 nm for the two-photon excitation of green fluorophores, and we can easily compress the resulting pulse close to its limited duration without the need for active pulse shaping. To optimize the wavelength mixing for sum-frequency excitation, we have realized the management of the power ratio and group delay between the soliton and dispersive wave by varying the initial pulse energy without additional delay control. We have thus demonstrated simultaneous three-color two-photon tissue imaging with contrast management between different signals. Our source optimization leads to efficient two-photon excitation reaching a 500-µm imaging depth under a low 14-mW illumination power. We believe our source development leads to an efficient and compact approach for driving multicolor two-photon fluorescence microscopy and other ultrafast investigations, such as strong-field-driven applications.

SplitSMLM, a spectral demixing method for high-precision multi-color localization microscopy applied to nuclear pore complexes

Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. Classical demixing algorithms tend to give suboptimal results in terms of localization precision and correction of chromatic errors. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision and drift correction, compensation of chromatic distortions, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic errors at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.

Minimizing Molecular Misidentification in Imaging Low-Abundance Protein Interactions Using Spectroscopic Single-Molecule Localization Microscopy.

Super-resolution microscopy can capture spatiotemporal organizations of protein interactions with resolution down to 10 nm; however, the analyses of more than two proteins involving low-abundance protein are challenging because spectral crosstalk and heterogeneities of individual fluorescent labels result in molecular misidentification. Here we developed a deep learning-based imaging analysis method for spectroscopic single-molecule localization microscopy to minimize molecular misidentification in three-color super-resolution imaging. We characterized the 3-fold reduction of molecular misidentification in the new imaging method using pure samples of different photoswitchable fluorophores and visualized three distinct subcellular proteins in U2-OS cell lines. We further validated the protein counts and interactions of TOMM20, DRP1, and SUMO1 in a well-studied biological process, Staurosporine-induced apoptosis, by comparing the imaging results with Western-blot analyses of different subcellular portions.

Multi-Color Two-Photon Microscopic Imaging Based on a Single-Wavelength Excitation.

Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues.

Bioorthogonal Ligation-Activated Fluorogenic FRET Dyads.

An energy transfer‐based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue‐excitable coumarins to yellow/red emitting cyanine frames is presented. Such relay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes‐shifts allowing lower background fluorescence even in no‐wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells. These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three‐color imaging of intracellular targets using one single excitation source with separate emission windows. Sub‐diffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopy is also presented.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Comparison and progress review of various super-resolution fluorescence imaging techniques

所见即所得是生命科学研究的中心哲学,贯穿在不断认识单个分子、分子复合体、分子动态行为和整个分子网络的历程中。活的动态的分子才是有功能的,这决定了荧光显微成像在生命科学研究中成为不可替代的工具。但是当荧光成像聚焦到分子水平的时候,所见并不能给出想要得到的。这个障碍是由于受光学衍射极限的限制,荧光显微镜无法在衍射受限的空间内分辨出目标物。超分辨荧光成像技术突破衍射极限的限制,在纳米尺度至单分子水平可视化生物分子,以前所未有的时空分辨率研究活细胞结构和动态过程,已成为生命科学研究的有力工具,并逐渐应用到材料科学、催化反应过程和光刻等领域。超分辨成像技术原理不同,其具有的技术性能各异,限制了各自特定的技术特色和应用范围。目前主流的超分辨成像技术包括3种:结构光照明显微镜技术(structured illumination microscopy, SIM)、受激发射损耗显微技术(stimulated emission depletion, STED)和单分子定位成像技术(single molecule localization microscopy, SMLM)。这些显微镜采用不同的复杂技术,但是策略却是相同和简单的,即通过牺牲时间分辨率来提升衍射受限的空间内相邻两个发光点的空间分辨。该文通过对这3种技术的原理比较和在生物研究中的应用进展介绍,明确了不同超分辨成像技术的技术优势和适用的应用方向,以方便研究者在未来研究中做合理的选择。

Structural and compositional diversity in the kainate receptor family.

SUMMARYThe kainate receptors (KARs) are members of the ionotropic glutamate receptor family and assemble into tetramers from a pool of five subunit types (GluK1–5). Each subunit confers distinct functional properties to a receptor, but the compositional and stoichiometric diversity of KAR tetramers is not well understood. To address this, we first solve the structure of the GluK1 homomer, which enables a systematic assessment of structural compatibility among KAR subunits. Next, we analyze single-cell RNA sequencing data, which reveal extreme diversity in the combinations of two or more KAR subunits co-expressed within the same cell. We then investigate the composition of individual receptor complexes using single-molecule fluorescence techniques and find that di-heteromers assembled from GluK1, GluK2, or GluK3 can form with all possible stoichiometries, while GluK1/K5, GluK2/K5, and GluK3/K5 can form 3:1 or 2:2 complexes. Finally, using three-color single-molecule imaging, we discover that KARs can form tri- and tetra-heteromers.

Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

General Considerations for Acquiring a Three-Color Image by Laser Scanning Confocal Microscopy.

Laser scanning confocal microscopy is the workhorse epifluorescence imaging technique used in laboratories worldwide to acquire three-dimensional images of both fixed and live specimens with fine, high-contrast optical sections to discern details that cannot be afforded by standard widefield microscopy. This basic protocol steps the user through a typical three-color imaging experiment using a Zeiss LSM 880 confocal microscope for the example. The extensive Notes section attempts to generalize the method so that concepts and considerations can be applied to other laser scanning confocal systems.

Instant multicolor super-resolution microscopy with deep convolutional neural network.

Multicolor super-resolution (SR) microscopy plays a critical role in cell biology research and can visualize the interactions between different organelles and the cytoskeleton within a single cell. However, more color channels bring about a heavier budget for imaging and sample preparation, and the use of fluorescent dyes of higher emission wavelengths leads to a worse spatial resolution. Recently, deep convolutional neural networks (CNNs) have shown a compelling capability in cell segmentation, super-resolution reconstruction, image restoration, and many other aspects. Taking advantage of CNN's strong representational ability, we devised a deep CNN-based instant multicolor super-resolution imaging method termed IMC-SR and demonstrated that it could be used to separate different biological components labeled with the same fluorophore, and generate multicolor images from a single super-resolution image in silico. By IMC-SR, we achieved fast three-color live-cell super-resolution imaging with ~100 nm resolution over a long temporal duration, revealing the complicated interactions between multiple organelles and the cytoskeleton in a single COS-7 cell.

Large Stokes-shift bioorthogonal probes for STED, 2P-STED and multi-color STED nanoscopy

Synthesis and multiple STED imaging applications of four, red-emitting (610–670 nm), tetrazine-functionalized fluorescent probes (CBRD = Chemical Biology Research group Dye 1–4) with large Stokes-shift is presented. Present studies revealed the super-resolution microscopy applicability of the probes as demonstrated through bioorthogonal labeling scheme of cytoskeletal proteins actin and keratin-19, and mitochondrial protein TOMM20. Furthermore, super-resolved images of insulin receptors in live-cell bioorthogonal labeling schemes through a genetically encoded cyclooctynylated non-canonical amino acid are also presented. The large Stokes-shifts and the wide spectral bands of the probes enabled the use of two common depletion lasers (660 nm and 775 nm). The probes were also found suitable for super-resolution microscopy in combination with two-photon excitation (2P-STED) resulting in improved spatial resolution. One of the dyes was also used together with two commercial dyes in the three-color STED imaging of intracellular structures.

Three-Color Imaging Enables Simultaneous Screening of Multiple RNA Targets on Small Molecule Microarrays.

Small molecule microarray (SMM) technology has become a powerful tool used in high-throughput screening for target-based drug discovery. One area in which SMMs have found use is the identification of small molecule ligands for RNA. RNAs with unique secondary or tertiary three-dimensional structures are considered to be attractive targets for small molecules. Complex RNA structures can form hydrophobic pockets suitable for small molecule binding, representing an opportunity for developing novel therapeutics. Our lab has previously taken a target-based approach, screening a single target against many small molecules on an SMM platform. Here, we report a screening protocol for SMMs to investigate multiple RNAs simultaneously using multi-color imaging. By introducing a mixture containing different fluorophore-labeled RNAs, the fluorescence signal of each binding event can be observed simultaneously. Thus, the specificity of a hit compound binding to one RNA target over other highly abundant RNAs (such as tRNA or rRNA) can be easily evaluated. © 2020 Wiley Periodicals LLC. Basic Protocol: RNA screening on SMMs by multi-color imaging Support Protocol 1: Preparation of SMM slides Support Protocol 2: Fluorophore labeling of RNA through maleimide chemistry.

Fluorescent Probe for Simultaneous Discrimination of GSH, Cys, and SO2 Derivatives.

Biothiols and SO2 derivatives, as essential reactive sulfur species (RSS), play vital roles in various physiological processes and have a close network of generation and metabolic pathways among them. To clarify their complex correlations, fluorescent probes to simultaneously detect GSH, Cys, and SO2 derivatives are highly desirable. Herein, we develop the first fluorescent probe (BO-HEM) to simultaneously discriminate GSH, Cys, and SO2 derivatives. The fluorescent probe is designed by integration of hemicyanine and BODIPY fluorophores through an ether bond. The ether bond of the probe is rapidly replaced by thiolates through nucleophilic aromatic substitution (SNAr) to generate hemicyanine with NIR fluorescence and sulfur-BODIPY. The amino groups of Cys but not GSH then further replace the thiolate to form amino-BODIPY. As for SO32-, nucleophilic addition to the double bond of BO-HEM generates adduct O-BODIPY with green fluorescence. To further improve the sensing performance, the nanoprobe with increased reactivity and biocompatibility is constructed by encapsulation of BO-HEM into the polymeric micelle. More importantly, by taking advantage of the hydrophilicity of the reaction products, the spectral discrimination was further enhanced to avoid signal interference. The nanoprobe is applied to discriminate biothiols and SO2 derivatives in living cells through three-color fluorescence imaging.