Molecular Brightness Research Articles

GPCR-Based Dopamine Sensors-A Detailed Guide to Inform Sensor Choice for In vivo Imaging.

Understanding how dopamine (DA) encodes behavior depends on technologies that can reliably monitor DA release in freely-behaving animals. Recently, red and green genetically encoded sensors for DA (dLight, GRAB-DA) were developed and now provide the ability to track release dynamics at a subsecond resolution, with submicromolar affinity and high molecular specificity. Combined with rapid developments in in vivo imaging, these sensors have the potential to transform the field of DA sensing and DA-based drug discovery. When implementing these tools in the laboratory, it is important to consider there is not a ‘one-size-fits-all’ sensor. Sensor properties, most importantly their affinity and dynamic range, must be carefully chosen to match local DA levels. Molecular specificity, sensor kinetics, spectral properties, brightness, sensor scaffold and pharmacology can further influence sensor choice depending on the experimental question. In this review, we use DA as an example; we briefly summarize old and new techniques to monitor DA release, including DA biosensors. We then outline a map of DA heterogeneity across the brain and provide a guide for optimal sensor choice and implementation based on local DA levels and other experimental parameters. Altogether this review should act as a tool to guide DA sensor choice for end-users.

GAF-CaMP3-sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity.

The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2–superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2–sfGFP indicator, named GAF-CaMP3–sfGFP. The GAF-CaMP3–sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger ΔF/F response to calcium ions. As compared to GAF-CaMP2–sfGFP, in cultured HeLa cells, GAF-CaMP3–sfGFP had similar brightness but a 1.9-fold larger ΔF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3–sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3–sfGFP showed a linear ΔF/F response in the range of 0–20 APs and in this range demonstrated a 1.4-fold larger ΔF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

A general strategy to red-shift green fluorescent protein-based biosensors.

Compared to green fluorescent protein (GFP)-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence, and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization, and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid (ncAA), 3-aminotyrosine (aY), to the chromophores of GFP-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the aY-modified biosensors preserved the molecular brightness, dynamic range, and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.

A rationally enhanced red fluorescent protein expands the utility of FRET biosensors

Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities.

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.

Interplay of Radiative and Non Radiative Rate Constants in the Photophysics of Fluorescent Proteins

Genetically encodable fluorophores like Fluorescent Proteins (FPs) have been engineered to provide better contrast, localization and specificity. Red-FPs (RFPs) are known to have an extended chromophore through an acylimine moiety with respect to their blue shifted and generally brighter counterparts. The molecular brightness is based on the product of the extinction coefficient (EC) and fluorescence quantum yield (QY). Development of brighter FPs has focused on increasing the QY, which is directly proportional to fluorescence lifetime (FL), a time constant corresponding to the inverse sum of the ensemble radiative and non-radiative decay rate constants. We have performed directed evolution of RFPs through random and site-directed mutagenesis on a multi-parametric microfluidic sorting system capable of FL and brightness selections. We also applied site directed mutagenesis on RFPs based on structural analysis to investigate the structural effects on FL and QY. The selected mutants derived from mCherry and FusionRed show that the improvement in FL brings about an expected linear increase in QY (∼3.2 fold for mCherry and ∼2.3 fold for FusionRed). This can be attributed to significant decrease in the non-radiative rate constant, and a modest improvement in radiative rate constant that is supported by hypsochromic shifts and changes in absorption profiles. Both approaches reveal that the improvement in molecular brightness with increased FL is mainly governed by a decrease in the non-radiative rate constant in conjunction with improved absorption (narrower absorption spectrum and higher ECmax). This strategy forms the basis of the best photophysical route to engineer brighter RFPs.

Single-Molecule Studies of Hetero-FRET Biosensors to Environmental Ionic Strength using Different Modalities of Fluorescence Correlation Spectroscopy

The compartmentalized ionic strength, in the complex environment of the cell, influences a variety of cell behaviors, protein functions, and protein-protein interactions. As a result, there is a need for sensors that can be genetically encoded within living cells, while allowing for quantitative mapping of the ionic strength throughout the intact living cells using noninvasive fluorescence techniques. Here, we investigate a family of genetically encoded, hetero-FRET (mCerulean3-linker-mCitrine) sensors at the single-molecule level using different modalities of fluorescence correlation spectroscopy (FCS) in a controlled environment. These sensors consist of mCerulean3 (the cyan fluorescent protein donor) and mCitrine (the yellow fluorescent protein acceptor) that are tethered together by a flexible linker. We are developing a new single-molecule approach to quantify the energy transfer efficiency and donor-acceptor distance between mCerulean3-mCitrine (as a FRET pair) using the molecular brightness in FCS measurements. Experimentally, the donor is excited, and its emitted fluorescence detected in both intact (i.e., in the presence of the acceptor) and enzymatically cleaved (i.e., in the absence of the acceptor) sensors as a function of the environmental ionic strength. Complementary single-molecule studies on these sensors are currently being conducted using fluorescence cross-correlation spectroscopy, where the donor is excited at 405 nm and the emission of both the donor and acceptor are being cross-correlated as a means for testing existing theoretical models. These single-molecule results will be compared with conventional, ensemble-based techniques such as time-resolved fluorescence of the donor. Our experimental approach should be particularly amenable to live cell studies with the added advantage of requiring very low expression levels of the FRET sensor as compared with conventional, ensemble-based methods.

Expanding Number and Brightness to Determine the Oligomer Size of Membrane Proteins in Live Cells as a Function of Concentration

The oligomer size of many membrane proteins plays a key role in their activity, and changes with increasing concentration and in the presence of ligands. However, many methods struggle to determine the exact oligomer size of complexes larger than dimers. Number and Brightness (N&B) is a fluorescence fluctuation method that is well suited to determine the oligomer size of complexes by comparing the molecular brightness of a monomer control (and ideally dimer, trimer, etc. controls) to the molecular brightness of a protein of interest. By also determining the two-dimensional concentration of the protein in the cell membrane and making measurements over a broad range of receptor concentrations, N&B could be used to create oligomerization curves. Care must be taken to account for cell movement and photobleaching, as small deviations can cause a large error in the measured molecular brightness, and the error is a function of concentration. In order to account for these complications, we have developed an approach which uses a combination of a hydrogel to inhibit cell movement and computational methods to correct for photobleaching and movement. Our preliminary results show differences in the oligomerization behavior of several receptor tyrosine kinases and co-receptors—including EGFR, TrkB, and NRP1—in live cells. Moreover, we directly show how ligand bound to membrane receptors affects their oligomerization state.

Number and molecular brightness analysis reveals Htt25Q protein aggregation upon the uptake of Htt97Q aggregates

Number and molecular Brightness (N&B) analysis is a powerful method used to monitor protein aggregation in living cells. Here, we used the N&B method to characterize the unexpanded HTT protein oligomerization after the internalization of the mutant HTT (mHTT) which contains a CAG repeat extensions encoding for long polyglutamine (polyQ) proteins resulting in misfolding and aggregation. HEK cells expressing Htt25Q-mCherry proteins were infected with Htt97Q-EGFP aggregates, by cell to cell uptake, in cultured conditions resulting in an increasing population of dimers and tetramers compared to our controls. This study shows for the first time the impact of protein aggregation in the unexpanded Htt25Q-mCherry expressing cells that occurs from cell to cell transfer of the expanded Htt97Q-EGFP. These results signify the sporadic behavior of the polyQ inclusion that gives insight into the mechanism of protein dynamics as a consequence of secreted mHTT aggregates.

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

Raster Image Correlation Spectroscopy Performance Evaluation

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three- and fivefold. For D = 1 μm2 s−1, a typical value for intracellular measurements, ∼25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (≪50 × 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E = 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM–μM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical least-squares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient.

Spatial Cumulant Analysis to Study D2-Like Dopamine Receptor Dynamics on Plasma Membrane

Fluorescence fluctuation spectroscopy (FFS) measures physical properties of fluorescent samples by analyzing the statistical nature of signals measured by photodetectors. In biological contexts, the most frequently measured parameters include concentration, molecular brightness, and motion characteristics (i.e. diffusion coefficients). Molecular brightness holds salience because it can be used to determine the stoichiometry of homomeric and heteromeric interactions between fluorescent species. We use spatial cumulant analysis (SpCA), an adaptation of cumulant analysis of point measurements, to determine molecular brightness from images recorded using two-photon laser scanning microscopy. Spatial cumulant analysis combines the advantages of imaging and single point cumulant analysis. Imaging provides decreased photobleaching, the ability to measure fluctuations from a larger set of molecules, and spatial resolution of the measured parameters. Cumulants are favorable in FFS because they are additive for multiple molecular species and provide computational ease. Accurate determination of stoichiometry from molecular brightness can be difficult for several reasons including non-ideal photodetection, incomplete chromophore maturation, photobleaching, and resonance energy transfer. Our approach accounts for these complications. D2-like dopamine receptors (D2Rs) are cell surface receptors that couple to trimeric G-protein complexes. Activation of D2Rs catalyzes the dissociation of the G-protein complex into Gα and Gβγ subunits. Gα and Gβγ subunits mediate the action of D2Rs by interaction with downstream effectors including adenylyl cyclase, inwardly rectifying K+ channels, and voltage-gated Ca2+ channels. Physiologically, D2R activation inhibits cAMP production and Ca2+ influx while activating hyperpolarizing K+ currents. While it is known that D2Rs, G-proteins, and their effectors interact to produce these physiological effects, the details of these interactions are difficult to probe experimentally in live cells. We show how multicolor spatial cumulant analysis of labeled proteins of interest can accurately measure the stoichiometry of receptors, G-proteins, and downstream signaling targets.

FRET at the Single Molecule Level using Molecular Brightness and Fluorescence Correlation Spectroscopy

Förster resonance energy transfer (FRET) is a noninvasive, quantitative method to study intermolecular interactions and conformational changes. The energy transfer efficiency of FRET pairs are routinely quantified using steady-state fluorescence spectroscopy or time-resolved fluorescence for which the main challenge of these approaches is the averaging over a larger ensemble of molecules. Here, we present a new approach for determining the energy transfer efficiency of hetero-FRET probes at the single-molecule level, using time-resolved fluorescence correlation spectroscopy (FCS) and molecular brightness (i.e., the number of fluorescence photons per molecule emitted while diffusing in the open observation volume). In this approach, the molecular brightness of the donor, in the presence and absence of the acceptor, is related to the energy transfer efficiency. We used the engineered hetero-FRET probe, RD, which consists of a mCerulean-linker-mCitrine construct, as the model system. Laser intensity-dependent FCS experiments were carried out on both the intact and cleaved RD indicating an energy transfer efficiency of (50 ± 10)% for RD in buffer, corresponding to a distance of 7 ± 2 nm between the donor and acceptor. These results demonstrate that our FCS approach is applicable to hetero-FRET constructs in well-controlled environments. Further it should be useful in living cells because it has the added advantage of requiring low expression levels of the FRET sensor, in contrast to more conventional FRET methods.

Molecular Counting by Photon Statistics in Confocal Fluorescence Imaging

Quantifying molecules in subdiffraction-sized structures is crucial for understanding various fundamental biological processes. Ideally, a non-destructive measurement would result in an image of a cellular structure along with the number of fluorescent molecules in each region of interest. Recently, a considerable amount of work has gone into developing such methods. Counting by Photon Statistics (CoPS) is a promising new technique based on photon antibunching, which exploits the fact that a single molecule can only generate one photon at a time [1]. The number of independent fluorescent emitters can be determined by measuring photon coincidences on a time-resolved confocal microscope with four single- photon detectors (e.g., MicroTime 200, PicoQuant) [2]. Both a molecular brightness and the spatial density of fluorophores per image pixel are estimated. By summing over all corresponding pixels, the number of molecules in a structure can be calculated. The only calibration required is to determine the instrument's Point Spread Function (PSF) size. We investigated imaging with CoPS under a wide range of experimental conditions. The applicability of the method both to artificial samples such as immobilized DNA origami, as well as to biological samples is explored. We also discuss sample preparation requirements and practical guidelines for performing CoPS measurements. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching. Laser Phys. 20:119 (2010) [2] Ta, H.et al., Mapping molecules in scanning far-field fluorescence nanoscopy.Nat. Commun. 6:7977 (2015)

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes

We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single-molecule measurements on samples inside regular cuvettes. The cuvette FCS uses a horizontally mounted extra-long working distance, 0.7 NA, air objective with a working distance of >1.8 mm instead of a high NA water or oil immersion objective. The performance of the cuvette FCS is found to be highly sensitive to the quality and alignment of the cuvette. The radial resolution and effective observation volume obtained using the optimized setup are ∼340 nm and 1.8 fL, respectively. The highest molecular brightness and the signal/noise ratio in the autocorrelation data achieved using an aqueous solution of rhodamine B are greater than 44 kHz and 110, respectively. Here, we demonstrate two major advantages of cuvette FCS. For example, the cuvette FCS can be used for measurements over a wide range of temperatures that is beyond the range permitted in the microscope-based FCS. Furthermore, cuvette FCS can be coupled to automatic titrators to study urea-dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette FCS in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS.

Optimal fluorescent protein tags for quantifying protein oligomerization in living cells

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization.

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells.

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors

Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural change triggered by alterations in the bio-parameter is monitored as a change in either fluorescent intensity, or lifetime. Potentially, other photo-physical properties of fluorophores, such as fluorescence anisotropy, molecular brightness, concentration, and lateral and/or rotational diffusion could also be used. Furthermore, while it is likely that multiple photo-physical attributes of a biosensor might be altered as a function of the bio-parameter, standard measurements monitor only a single photo-physical trait. This limits how biosensors are designed, as well as the accuracy and interpretation of biosensor measurements. Here we describe the design and construction of an automated multimodal-microscope. This system can autonomously analyze 96 samples in a micro-titer dish and for each sample simultaneously measure intensity (photon count), fluorescence lifetime, time-resolved anisotropy, molecular brightness, lateral diffusion time, and concentration. We characterize the accuracy and precision of this instrument, and then demonstrate its utility by characterizing three types of genetically encoded calcium sensors as well as a negative control.

Photophysical Behavior of mNeonGreen, an Evolutionary Distant Green Fluorescent Protein

Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion-proteins in model systems and live cells. In particular, molecular brightness depends on the dynamics of short-lived dark states leading to flickering of the fluorescence signal. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from Branchiostoma lanceolatum, in comparison to EGFP and AcGFP from Aequorea species. Fluorescence correlation spectroscopy (FCS) is used to determine flickering and molecular brightness of purified proteins in solutions of different pH. The molecular brightness of mNG is consistently higher as compared to the traditional GFPs, but decreases slightly under acidic and basic conditions. Consistently, an advanced analysis of steady state spectroscopy data resolves two additional chromophore states with reduced quantum yield that build up at low and high pH. Thus, mNG is distinguished from traditional GFPs by a two-step protonation response that reflects a chemically distinct chromophore environment. Despite the more complex pH dependence, mNG represents a superior GFP under a broad range of conditions.

Oligomerization and Nuclear Shuttling Dynamics of Viral Proteins Studied by Quantitative Molecular Brightness Analysis using Fluorescence Correlation Spectroscopy

Oligomerization and Nuclear Shuttling Dynamics of Viral Proteins Studied by Quantitative Molecular Brightness Analysis using Fluorescence Correlation Spectroscopy