Fluorophore labels that transiently and repetitively bind to a target (“exchangeable” or “renewable” labels) lead to a continuous renewal of the fluorescence signal. This dynamic labeling approach minimizes photobleaching and was beneficially exploited in various super-resolution microscopy methods. Here, we report two new developments using exchangeable fluorophores: first, we report fast and long-time live-cell super-resolution microscopy using a weak-affinity protein label and a neural network. Second, we report a novel design for exchangeable DNA labels that show higher brightness and lower background. Together, these two developments further increase the application range for exchangeable fluorophore labels in super-resolution fluorescence microscopy.
Single-molecule localization microscopy (SMLM) in combination with DNA barcoding (DNA-PAINT) enables easy-to-implement multi-target super-resolution imaging. However, image acquisition is slow because of the need to spatio-temporally isolate single emitters and to collect sufficient statistical data to generate a super-resolved image. Here, we bypass this limitation by utilizing a neural network, DeepSTORM, that can predict super-resolved SMLM images from high-emitter density data. This reduces the acquisition time 10- to 20-fold, enabling image acquisition as short as one minute. Integrating weak-affinity DNA labels allows precise control of single-molecule emitter densities, which enables recording of training, ground truth, and testing data from the same sample. Sequential imaging of multiple targets using different DNA barcodes with the same fluorophore enables aberration-free multi-target imaging (Exchange-PAINT). The constant exchange of fluorophore labels at target sites minimizes signal loss for long acquisition times, which allows imaging large samples in a matter of minutes. The concept is transferable to other weak-affinity, non-covalent fluorophore labels.
Single-particle tracking reports on the mobility of biomolecules in living cells with high spatial and temporal resolution. From single-particle trajectories, information such as the diffusion coefficient and diffusion state can be derived. Changes in particle dynamics within single trajectories can be extracted by segmentation, which provides information on transitions between different functional states of a biomolecule. However, such analyses of single-particle tracking data is complex and time-consuming. Here, we present a pipeline that enables a straightforward and rapid analysis of single-particle tracking data. It incorporates mean-squared displacement analysis of trajectories that distinguishes between immobile, confined, and free diffusion states, as well as the analysis of diffusion state transitions within a trajectory with transition counts and hidden Markov modeling. We apply this analysis to single-molecule trajectories of un-activated Fab-bound and internalin B-bound MET receptors in the plasma membrane of live HeLa cells. We found that ligand activated receptors move slower and more confined and exhibit more transitions from free to confined diffusion states than un-activated receptors. This suggests that the confined diffusion state functions as an intermediate between free and immobile, as this state is most likely changing the diffusion type in the following segment. Hidden Markov modeling reported three diffusion states with increased transition probabilities towards the less mobile and immobile states upon ligand activation. The less mobile state operates as an intermediate state, as it has the highest transition probabilities. The analysis pipeline can be readily applied to single-particle tracking data of other membrane proteins and provides rapid access to information that can be associated with functional states.
We present fluorophore labels that transiently and repetitively bind to their targets as probes for various types of super-resolution fluorescence microscopy. Such labels show a weak (~10 µM – 100 nM) affinity to a target and are kept in an imaging buffer that constitutes a reservoir with a high concentration of intact probes, enabling repetitive binding to the same target (we refer to these labels as “exchangeable labels”). This dynamic labeling approach minimizes photobleaching and yields a constant fluorescence signal over time, which has been beneficially exploited in SMLM [1-4], STED [5, 6], and super-resolution optical fluctuation imaging (SOFI) [7]. Multi-color, 3D, and live cell imaging, as well as imaging of large fields of view, is facilitated [4]. We further present the implementation of neural networks for multi-emitter localization to achieve multi-color SMLM with short acquisition times of one minute [8].
We present fluorophore labels that transiently and repetitively bind to their targets as probes for various types of super-resolution fluorescence microscopy. Transient labels typically show a weak affinity to a target, and exchange constantly with the buffer that constitutes a reservoir with a large amount of intact probes, leading to repetitive binding events to the same target (we refer to these labels as “exchangeable labels”). This dynamic labeling approach is insensitive to common photobleaching and yields a constant fluorescence signal over time, which has been successfully exploited in SMLM, STED, single-particle tracking and super-resolution optical fluctuation imaging (SOFI). We discuss properties of suitable exchangeable labels and experimental parameters for optimal performance for the different super-resolution methods. In addition, we show how to combine different classes of exchangeable labels for high-quality multicolor super-resolution imaging.
KEYWORDS: Fluorescence resonance energy transfer, Imaging systems, Multiplexing, Super resolution, Signal detection, Microscopy, Super resolution microscopy
Correlating DNA-PAINT (point accumulation for imaging in nanoscale topography) and single-molecule FRET (Förster resonance energy transfer) enables the multiplexed detection with sub-diffraction optical resolution. We designed pairs of short oligonucleotides, labeled with donor and acceptor fluorophores with various distances generating different FRET efficiencies. The strands can transiently bind to a target docking strand, simultaneous binding of both strands results in FRET signals which yield a super-resolved image via DNA-PAINT imaging. We demonstrate FRET-PAINT by designing and imaging DNA origami, which is a useful tool to establish super-resolution methods. The DNA origami structures were equipped with three target binding sites spaced by 55 nm, a sub-diffraction limited distance, however ensuring that no FRET between the target sites occurs. We resolved the individual binding sites in the donor and acceptor channels, and in addition extracted the FRET efficiency for each site in single and mixed populations. The combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging in conjunction with distance-sensitive readout in the 1 to 10 nm range.
Knowledge of how proteins organize into functional complexes is essential to understand their biological function. Optical super-resolution techniques provide the spatial resolution necessary to visualize and to investigate individual protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) builds on the detection of single fluorophore labels, which next to the generation of high-resolution images provides access to quantitative molecular information. We developed various tools for quantitative SMLM (qSMLM), an imaging method that both super-resolves individual protein clusters and reports on molecular numbers by analyzing the kinetics of single emitter blinking. This method is compatible with both fluorescent proteins and organic fluorophores. With qSMLM, we quantify protein copy numbers in single clusters, and we study how changes in the stoichiometry of protein complexes translates into function.
In the brain, the strength of each individual synapse is defined by the complement of proteins present or the “local proteome.” Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.
Knowledge of assembly, subunit architecture and dynamics of membrane proteins in a cellular context is essential to infer their biological function. Optical super-resolution techniques provide the necessary spatial resolution to study these properties of membrane protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) is particularly well suited, as next to high-resolution images, it provides quantitative information on the detection of single emitters. A challenge for current super-resolution methods is to resolve individual protein subunits within a densely packed protein cluster. For this purpose, we developed quantitative SMLM (qSMLM), which reports on molecular numbers by analyzing the kinetics of single emitter blinking. Next to theoretical models for various photophysical schemes, we demonstrate this method for a selection of fluorescent proteins and synthetic dyes and a selection of membrane proteins. We next applied this tool to toll-like receptor 4 (TLR4), and found a ligand-specific formation of monomeric or dimeric receptors. Next to fluorescent proteins, DNA-PAINT offers a novel and flexible approach for quantitative super-resolution microscopy. We demonstrate DNA-PAINT imaging of structurally defined DNA origami structures and robust quantification of target sites, as well as of membrane receptors. Molecular quantification, together with experiments following single receptor mobilities in live cells, will enlighten molecular mechanisms of receptor activation.
We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.
Darius Widera, Christin Klenke, Deepak Nair, Meike Heidbreder, Sebastian Malkusch, Jean-Baptiste Sibarita, Daniel Choquet, Barbara Kaltschmidt, Mike Heilemann, Christian Kaltschmidt
Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12±0.05 to 0.61±0.03 μm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9±1.9 to 15±4.9 μm/s, whereas a velocity increase from 9±1.3 to 14±3 μm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.
The interest in super-resolution microscopy techniques has dramatically increased in the last years due to the
unprecedented insight into cellular structure which has become possible [1]. In all widefield-based techniques, such as
Stochastical Optical Reconstruction Microscopy (STORM) or
Photo-activation localization microscopy (PALM), the
dye-sensor-molecules are switched between a bright and a dark state. Many organic fluorophores exhibit intrinsic dark
states with a lifetime that can be tuned by adjusting the level of oxidants and reductants in the buffer, thereby allowing to
reversibly switch individual fluorophores between an on- and
off-state [2]. This behavior is used in the dSTORM
method.
We exploited this redox-level adjusted photoswitching behaviour based on addition of millimolar amounts of reducing
thiols for high-resolution imaging on a setup based on an inverse microscope coupled with ultrasensitive CCD camera
detection. In order to quickly control the quality of the measurement, we used real-time computation of the
subdiffraction-resolution image [3]. This greatly increases the applicability of the method, as image analysis times are
greatly reduced.
KEYWORDS: Luminescence, Microscopy, Image processing, Data processing, Real time imaging, Super resolution, Image resolution, Diffraction, Optical resolution, Stochastic processes
In the recent past, a variety of methods have been developed to circumvent the diffraction barrier of light which restricts
optical resolution to about 200 nm in the image plane. Single-molecule based photoswitching microscopy such as direct
stochastic optical reconstruction microscopy (dSTORM) has been successfully implemented for subdiffraction-resolution
fluorescence imaging. The major drawback of this technique has been that the reconstruction of subdiffraction-resolution
images requires substantially more time than the actual experiment and prevented real-time imaging. Here we present a
new computational algorithm enabling subdiffraction-resolution fast imaging of cellular structures with ~20 nm optical
resolution in less than 10 seconds.
High-resolution fluorescence imaging has a vast impact on our understanding of intracellular organization. The
key elements for high-resolution microscopy are reversibly photo-switchable fluorophores that can be cycled
between a fluorescent and a non-fluorescent (dark) state and can be localized with nanometer accuracy. For
example, it has been demonstrated that conventional cyanine dyes (Cy5, Alexa647) can serve as efficient photoswitchable
fluorescent probes. We extended this principle for carbocyanines without the need of an activator
fluorophore nearby, and named our approach direct stochastic optical reconstruction microscopy (dSTORM).
Recently, we introduced a general approach for superresolution microscopy that uses commercial fluorescent
probes as molecular photoswitches by generating long lived dark states such as triplet states or radical states.
Importantly, this concept can be extended to a variety of conventional fluorophores, such as ATTO520, ATTO565,
or ATTO655. The generation of non-fluorescent dark states as the underlying principle of superresolution
microscopy is generalized under the term photoswitching microscopy, and unlocks a broad spectrum of organic
fluorophores for multicolor application. Hereby, this method supplies subdiffraction-resolution of subcellular
compartments and can serve as a tool for molecular quantification.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Spectroscopy, Fluorescence spectroscopy, Proteins, Single molecule spectroscopy, Data hiding, Distance measurement, Data acquisition, Polymers
We present single-molecule fluorescence studies of σ54-dependent gene-transcription complexes using singlemolecule
fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX)
spectroscopy. The ability to study one biomolecule at the time allowed us to resolve and analyze sample
heterogeneities and extract structural information on subpopulations and transient intermediates of
transcription; such information is hidden in bulk experiments.
Using site-specifically labeled σ54 derivatives and site-specifically labeled promoter-DNA fragments, we
demonstrate that we can observe single diffusing σ54-DNA and transcription-initiation RNA polymerase-σ54-
DNA complexes, and that we can measure distances within such complexes; the identity of the complexes has
been confirmed using electrophoretic-mobility-shift assays. Our studies pave the way for understanding the
mechanism of abortive initiation and promoter escape in σ54-dependent transcription.
KEYWORDS: Luminescence, Chromophores, Energy transfer, Molecular photonics, Molecules, Fluorescence resonance energy transfer, Absorption, Energy efficiency, Glasses, Single molecule spectroscopy
We report a new approach to an unidirectional photonic wire based on fluorescent dyes as chromophores and DNA as a rigid scaffold. The physical functioning of the wire is realized by dipole-dipole intreraction, i.e. resonant energy transfer, between chromophores. The use of four dyes (Alexa 430, TAMRA, Cy3.5, and Cy5) with different excited state energies creates an energy cascade constituting the driving force of the energy current and providing the unidirectionality of the device. The unique molecular properties of DNA, its scaffold-like structure, combined with straightforward synthesis methods allowed the engineering of a 30 base pair double-stranded DNA with inter-dye distances of 10 base pairs (3.4 nm), respectively, a range where electronic interactions between the chromophores can be neglected but dipole-dipole induced fluorescence resonance energy transfer (FRET) is expected to be still highly efficient. Steady-state and time-resolved ensemble spectroscopic measurements show an overall energy transfer efficiency of approximately 0.60. That is, the unidirectional transport of photonic energy over a distance of approximately 10 nm and a spectral separation of approximately 250 nm. Furthermore, pulsed diode laser excitation at 440 nm in combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) was applied to characterize the effectiveness of individual photonic wires dispersed on glass coverslips.
We present a new technique for high-resolution colocalization of fluorescent dyes. The technique is based on polarization modulated excitation and spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as on coincidence analysis of the detected photon counts following pulsed laser excitation. The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To reveal the number of absorbing and emitting molecules polarization modulated excitation and coincidence analysis of the detected photon counts is used. We demonstrate that by the use of appropriately selected dyes, the presented technique permits (1) the counting of the number of molecules present in the observation volume, and (2) the determination of the distance between two single molecules down to approximately 30 nm with a precision of approximately 10 nm without any chromatic aberrations. The developed techniques are promising for applications in molecular biology, e.g. to determine the number of polymerase molecules active within a transcription factory and/or to measure their distances to nanscent transcripts.
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