Single Fluorophore Research Articles

A modular microscopic smartphone attachment for imaging and quantification of multiple fluorescent probes using machine learning.

Portable smartphone-based fluorescent microscopes are becoming popular owing to their ability to provide major functionalities offered by regular benchtop microscopes at a fraction of the cost. However, smartphone-based microscopes are still limited to a single fluorophore, fixed magnification, the inability to work with a different smartphones, and limited usability to either glass slides or cover slips. To overcome these challenges, here we present a modular smartphone-based microscopic attachment. The modular design allows the user to easily swap between different sets of filters and lenses, thereby enabling utility of multiple fluorophores and magnification levels. Our microscopic smartphone attachment can also be used with different smartphones and was tested with Nokia Lumia 1020, Samsung Galaxy S9+, and an iPhone XS. Further, we showed imaging results of samples on glass slides, cover slips, and microfluidic devices. A 1951 USAF resolution test target was used to quantify the maximum resolution of the microscope which was found to be 3.9 μm. The performance of the smartphone-based microscope was compared with a benchtop microscope and we found an R2 value of 0.99 using polystyrene beads and blood cells isolated from human blood samples collected from Robert Wood Johnson Medical Hospital. Additionally, to count the particles (cells and beads) imaged from the smartphone-based fluorescent microscope, we developed artificial neural networks (ANNs) using multiple training algorithms, and evaluated their performances compared to the control (ImageJ). Finally, we did ANOVA and Tukey's post-hoc analysis and found a p-value of 0.97 which shows that no statistical significant difference exists between the performance of the trained ANN and control (ImageJ).

A Quick Immuno-FISH Protocol for Detecting RNAs, Proteins, and Chromatin Modifications.

Immunofluorescence and fluorescence in situ hybridization (FISH) are widely used cytogenetic techniques for visualization of protein and RNA/DNA molecules. Here, we describe an experimental procedure for quick sequential immunofluorescence and RNA FISH (immuno-FISH), which enables the simultaneous detection of proteins, chromatin modifications, and RNAs on the inactive X-chromosome (Xi) using female mouse embryonic fibroblast (MEF) and tail-tip 3T3 cell lines. Using a pooled array of oligonucleotides labeled with a single fluorophore as an RNA FISH probe, we can reduce the time for RNA FISH from an overnight process to 1-2h without losing its sensitivity. This protocol could be applied to visualization of various protein and RNA molecules, and chromatin modifications.

Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

Design strategy and bioimaging of small organic molecule multicolor fluorescent probes

Multicolor fluorescent probes based on small organic molecules have the advantages of low cost, good biocompatibility, easily modifiable molecular structures and adjustable fluorescence performance. In addition, small molecule multicolor fluorescent probes generally undergo multi-site or multi-step reactions, which means that they can be used for the specific detection of structurally similar substances in complex bio-systems. In this review, we focus on the design and application of multicolor fluorescent probes based on small organic molecules: single fluorophores with multiple reaction sites, multiple fluorophores with single reaction sites, or multiple fluorophores with multiple reaction sites. Moreover, a design strategy for multicolor fluorescent probes and its application in biological imaging are also summarized, providing a systematic plan for future research on fluorescent probes functionalized by small organic molecules. It will also play an important role in the development of additional functions for small organic molecule fluorescent probes.

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the Escherichia coli RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3′ and 5′ ends of the overhang DNA dynamically in vitro for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.

Single molecule light field microscopy

We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views, in combination with a bespoke fitting algorithm, enable the 3D positions of single fluorophores to be determined from parallax. SMLFM achieves up to 20 nm localization precision throughout an extended 6 µ m depth of field. The capabilities of SMLFM are showcased by imaging membranes of fixed eukaryotic cells and DNA nanostructures below the optical diffraction limit.

Real-time 3D single molecule tracking

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm2/s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

Ratiometric fluorescence detection of bleomycin based on proximity-dependent fluorescence conversion of DNA-templated silver nanoclusters

We design a ratiometric fluorescent sensing platform for bleomycin (BLM) by using proximity-dependent DNA-templated silver nanoclusters (DNA-AgNCs) probe. This ratiometric sensing system is constructed with DNA-AgNCs as single fluorophore. The proposed strategy is based on the two following facts: (1) a covert DNA can approach and transform the DNA-AgNCs with green emission (G-DNA-AgNCs) into red emission through hybridization reaction. (2) The specific cleavage of the convert DNA by BLM in the presence of Fe(II) inhibits the discoloration of G-DNA-AgNCs. Thus, benefiting from the specific recognition of BLM and unique properties of G-DNA-AgNCs, a highly-sensitive ratiometric sensor for BLM has been successfully developed. The detection limit is as low as 30 pmol/L. This label-free fluorescence probe possesses advantages of convenient synthetic process and low cost. Moreover, this ratiometric method has been applied to the detection of BLM in human serum samples, illustrating a promising tool for analysis of BLM in cancer therapy.

Accurate 4Pi single-molecule localization using an experimental PSF model.

Interferometric single-molecule localization microscopy (iPALM, 4Pi-SMS) uses multiphase interferometry to localize single fluorophores and achieves nanometer isotropic resolution in 3D. The current data analysis workflow, however, fails to reach the theoretical resolution limit due to the suboptimal localization algorithm. Here, we develop a method to fit an experimentally derived point spread function (PSF) model to the interference 4Pi-PSF. As the interference phase is not fixed with respect to the shape of the PSF, we decoupled the phase term in the model from the 3D position of the PSF. The fitter can reliably infer the interference period even without introducing astigmatism, reducing the complexity of the microscope. Using a spline-interpolated experimental PSF model and by fitting all phase images globally, we show on simulated data that we can achieve the theoretical limit of 3D resolution, the Cramér-Rao lower bound (CRLB), also for the 4Pi microscope.

Ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays.

The detection and quantification of low-abundance molecular biomarkers in biological samples is challenging. Here, we show that a plasmonic nanoscale construct serving as an ‘add-on’ label for a broad range of bioassays improves their signal-to-noise ratio and dynamic range without altering their workflow and read-out devices. The plasmonic construct consists of a bovine-serum-albumin scaffold with approximately 210 IRDye 800CW fluorophores (with fluorescence intensity approximately 6700-fold that of a single 800CW fluorophore), a polymer-coated gold nanorod acting as a plasmonic antenna, and biotin as a high-affinity biorecognition element. Its emission wavelength can be tuned over the visible and near-infrared spectral regions by modifying its size, shape and composition. It is compatible with multiplexed bead-based immunoassays (it improves the limit of detection by up to 4,750-fold in fluorescence-linked immunosorbent assays), immuno-microarrays, flow-cytometry and immunocytochemistry methods, and it shortens overall assay times and lowers sample volumes, as shown for the detection of a pro-inflammatory cytokine in mouse interstitial fluid and of urinary biomarkers in patient samples.

Front-Face Fluorescence of Honey of Different Botanic Origin: A Case Study from Tuscany (Italy)

Honey is a natural pure food produced by honeybees from the nectar of various plants, and its chemical composition includes carbohydrates, water, and some minor compounds, which are very important for honey quality and authentication. Most of honey’s minor components are related to the botanic origin, climate, and geographic diversity. In this work, we report an original case study on monofloral honey samples of twelve different botanic origins produced in Tuscany (Italy) based on the ‘semi-quantitative’ analysis of emission, excitation, and synchronous front-face fluorescence spectra. This is the first front-face fluorescence study of Italian honey samples and, to our knowledge, the first fluorescence investigation of honey from inula (Inula viscosa (L.) Aiton), marruca (Paliurus spina-christi Mill.), lavender (Lavandula L. 1753), sulla (Hedysarum coronarium L.), arbutus (or strawberry tree) (Arbutus unedo L., 1753), and alfalfa (Medicago sativa L.) plants. Results obtained from fluorescence spectroscopy are discussed in terms of characteristic spectral emission profiles typical of honey of different botanic origins. Moreover, the spectral analysis based on the decomposition of the front-face fluorescence (FFF) spectra in terms of single main fluorophores’ components is here proposed to identify several minor compounds, such as amino acids, phenolic acids, vitamins, and other fluorescent bioactive molecules.

DNA-Templated Timer Probes for Multiplexed Sensing.

Simultaneous analysis based on encoded fluorophores suffers from potential crosstalk between fluorophores and the limited number of colors that can be practically resolved. Inspired by nontrivial temporal patterns in living organisms, we developed a DNA-templated probe by utilizing DNA polymerase (DNAP) for multiplexed detection of nucleic acids. These probes use differential delay times of signaling by a DNAP-mediated extension to distinguish different targets, which serve as the primers. Taking advantage of the high processivity and the controllable kinetics of DNAP, we find that multiplexed detection can be achieved in homogeneous solution using a single fluorophore. As a proof of concept, we developed assays for genomic DNA from four different bacteria. In addition, we designed and implemented probes to undergo a single oscillation in signal as an alternative way for multiplexing. We anticipate this approach will find broad applications not only in sensing but also in synthetic DNA nanosystems.

Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions.

Intrinsic molecular brightness (MB) is a number of emitted photons per second per molecule. When a substrate labeled by a fluorophore and a second unlabeled substrate form a complex in solution, the MB of the fluorophore changes. Here we use this change to determine the equilibrium constant (K) for the formation of the complex at pM concentrations. To illustrate this method, we used a reaction of DNA hybridization, where only one of the strands was fluorescently labeled. We determined K at the substrate concentrations from 80 pM to 30 nM. We validated this method against Förster resonance energy transfer (FRET). This method is much simpler than FRET as it requires only one fluorophore in the complex with a very small (a f̃ew percent) change in MB.

Coating that self-reports cracking and healing using microcapsules loaded with a single AIE fluorophore

A crack and crack healing were both detectable with a microcapsule-based dual-reporting self-healing coating system developed using a single aggregation-induced emission (AIE) fluorophore, (2Z,2′Z)-3,3′-(2,5-bis(diphenylamino)-1,4-phenylene)bis(2-(4-fluorophenyl)acrylonitrile) (DPA-PPV-F). This selected and synthesized single AIE dye was tested for changes in its fluorescence color and fluorescence intensity following a solution/solid-state phase change. These unique AIE-fluorophore-embedded microcapsules were successfully synthesized, and a crack/healing dual-reporting self-healing system with a top coating and an epoxy matrix resin was prepared. The prepared self-healing coating system was indicated by optical microscopy and scanning electron microscopy (SEM) after a scratch was created with a razor. The self-healing coatings showed red fluorescence with weak intensity immediately after being scratched, and the fluorescence color changed from red to orange as healing occurred via photocuring. The fluorescence intensity also dramatically increased at the healing region soon after the scratch was inflicted. The fluorescence color changed from red to orange according to the crack/healing process, and the fluorescence intensity also dramatically increased.

Concentration Enrichment, Separation, and Cation Exchange in Nanoliter-Scale Water-in-Oil Droplets.

Droplet-based techniques have had a profound impact in chemistry, owing to their ability to perform rapid and massively parallel reactions in minute fluid volumes. In many applications, concentration enrichment is required to increase the speed of reactions or the sensitivity of assays; but in-droplet concentration enrichment remains challenging. Here, we interface electrokinetic concentration polarization with droplet microfluidics to accomplish in-droplet demixing. This result is significant because the concentration of any charged species in the droplet can be enriched and the approach can be readily integrated into existing droplet workflows. Further, we show that such electrokinetic enrichment is rapid, on the order of seconds, and is robust, occurring over a wide parametric space. We further demonstrate electrokinetic separation of two anionic fluorophores within the droplet. Such a capability potentiates the droplet-templated synthesis of particles with gradient composition and the development of mobility-shift assays, which rely on discrimination of multiple species tagged with a single color fluorophore. Finally, by using a calcium-binding dye as an indicator, we demonstrate in-droplet cation exchange. This demonstration of cation exchange in droplets is significant because of its broad applicability to strategies for synthesis and bioassays. These results lay the foundation for new advanced droplet techniques with transformative applications.

Color-tunable single-fluorophore supramolecular system with assembly-encoded emission

Regulating the fluorescent properties of organic small molecules in a controlled and dynamic manner has been a fundamental research goal. Although several strategies have been exploited, realizing multi-color molecular emission from a single fluorophore remains challenging. Herein, we demonstrate an emissive system by combining pyrene fluorophore and acylhydrazone units, which can generate multi-color switchable fluorescent emissions at different assembled states. Two kinds of supramolecular tools, amphiphilic self-assembly and γ-cyclodextrin mediated host-guest recognition, are used to manipulate the intermolecular aromatic stacking distances, resulting in the tunable fluorescent emission ranging from blue to yellow, including a pure white-light emission. Moreover, an external chemical signal, amylase, is introduced to control the assembly states of the system on a time scale, generating a distinct dynamic emission system. The dynamic properties of this multi-color fluorescent system can be also enabled in a hydrogel network, exhibiting a promising potential for intelligent fluorescent materials.

Quantifying Heme-Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion.

Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccp1-GFP with GFP fused C-terminally to Ccp1 by the linker GRRIPGLIN. Time-correlated single-photon counting reveals a single fluorescence lifetime for heme-free apoCcp1-GFP, τ0 = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially quenches GFP fluorescence since holoCcp1-GFP exhibits two lifetimes, τ1 = 0.95 ± 0.02 and τ2 = 2.46 ± 0.03 ns with fractional amplitudes a1 = 38 ± 1.5% and a2 = 62 ± 1.5%. Also, τ and a are independent of Ccp1-GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of a1 vs % holoCcp1-GFP in mixtures with apoCcp1-GFP is linear, establishing that the fraction of Ccp1-GFP with heme bound can be determined from a1. Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1-GFP fusion revealed 30% holoCcp1-GFP (i.e., mature Ccp1) and 70% apoCcp1-GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme-protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements.

Investigating ultrafast two-pulse experiments on single DNQDI fluorophores: a stochastic quantum approach.

Ultrafast two-pulse experiments on single molecules are invaluable tools to investigate the microscopic dynamics of a fluorophore. The first pulse generates electronic or vibronic coherence and the second pulse probes the time-evolution of the coherence. A protocol that is able to simulate ultrafast experiments on single molecules is applied in this study. It is based on a coupled quantum-mechanical description of the fluorophore and real-time dynamics of the system vibronic wave packet interacting with an electric field, described by means of the stochastic Schrödinger equation within the Markovian limit. This approach is applied to the DNQDI fluorophore, previously investigated experimentally [D. Brinks et al., Nature, 2010, 465, 905-908]. We find this to be in good agreement with the experimental outcomes and provide microscopic and atomistic interpretation.

Single-molecule detection on a portable 3D-printed microscope

Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson’s disease, with an improved sensitivity of >100,000-fold over bulk measurements.

Solvent vapour induced rare single-crystal-to-single-crystal transformation of stimuli-responsive fluorophore: Solid state fluorescence tuning, switching and role of molecular conformation and substituents

Supramolecular interactions and molecular conformations play significant role on producing fluorescent polymorphs with tunable emission in the solid state from single fluorophore. External stimuli-induced structural transformation from one polymorph to another polymorph without losing single crystalline character (single-crystal-to-single-crystal transition (SC-SC)) provides perfect model to understand the structure-property of molecules in the solid state and designing new functional fluorescent materials. Herein we have tailored the substituent groups in the aggregation-induced-emissive (AIE) tetraphenylethylene (TPE) based donor (D)-acceptor (A) fluorophores (TPPA-1-6) and explored fluorescent polymorphs formation and SC-SC transition in the solid state. TPPA-1 showed conformational fluorescent polymorphs via twisted and co-planar conformation between TPE phenyl group and cyanophenyl acceptor phenyl ring. However, single crystal analysis of other derivatives revealed that substituent groups and supramolecular interactions control the molecular conformation and SC-SC transition. Fluorophore with same substituent but positional change also show drastic effect on the fluorescent polymorphs formation and SC-SC transition. Substituents that can form strong intermolecular interactions stabilize the less stable co-planar structure whereas bulky substituents with weak intermolecular interactions produced twisted or both conformers. Photophysical, powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimteric (TG) analysis were performed to substantiate and get insight on the structural transformation. The formation of fluorescent polymorphs and SC-SC transition exhibited tunable and switchable solid state fluorescence. The non-planar TPE core has been made use to demonstrate high contrast stimuli induced reversible fluorescence switching. Overall, the present studies attempted to gain information on the structural design for developing SC-SC transforming functional organic fluorescent materials that could be of potential interests for optoelectronic and display devices.