The over-expression of hyaluronidase has been linked to many types of cancer, and thus we present here a technique for hyaluronidase detection and quantification using Fluorescence Correlation Spectroscopy (FCS). Our probe consists of Hyaluronan macromolecules (HAs) heavily loaded with fluorescein dye to the extent that the dye experiences self-quenching, and these HAs are detected as very bright, slowly moving particles by FCS. Hyaluronidase cleaves HAs into HA fragments, increasing the concentration of independent fluorescent molecules diffusing through the detection volume. The cleavage of HAs releases the self- quenching so that the intensity of emission is drastically increased. Both the concentration of fluorescent particles and intensity are measured simultaneously and correlated to the concentration of hyaluronidase. Also, our time correlated system allows us to assess the heterogeneity of the HA solution. Subpopulations of slowly moving particles with short-lived radiative decay may be separated from fast-moving particles of long-lived radiative decay and studied independently in a technique known as Fluorescence Lifetime Correlation Spectroscopy (FLCS). Further, we assess the use of the AzaDiOxaTriAngulenium (ADOTA) dye for FCS experiments. Its lifetime is significantly longer than that of the autofluorescence that plagues fluorescence experiments involving cells or tissue, and thus the fluorescence decay of the probe can be easily identified and separated from autofluorescence by FLCS. We demonstrate this by labeling HAs with ADOTA and adding free Rhodamine 123 to the solution to simulate the autofluorescence. We show that the combination of ADOTA and FLCS allow construction of an FCS-based hyaluronidase assay despite the presence of severe autofluorescence.

The high sensitivity at single molecule level of fluorescence correlation spectroscopy (FCS) enables measurements of charged polymer molecules at extreme dilution condition, which has been difficulty for conventional techniques as dynamic light scattering. By FCS, the conformation transition of single chain of polyelectrolytes is clearly exposed: the first-order conformation transition by pH of poly 2-vinylpyridine, the scaling analysis of polystyrene sulfonate shows a change from rod-like conformation to random coil conformation with the increase of the molecular weight. Photon count histogram (PCH) helps to determine the electric potential of single charged polymer molecules, explaining the important contribution of the counterion distribution: the breakdown of “counterion condensation.” FCS is a very important method for the study of charged polymers and bio-macromolecules.

We developed an automated approach to Fluorescence Cross-Correlation Spectroscopy (FCCS) to screen for molecular interactions in living cells (HCS-FCS). Similar to manual measurements, we had to address the effects of measurement artifacts. In this paper we used wavelet shrinking to correct for bleaching artifacts. Without correction, bleaching of both dyes used for cross-correlation measurements resulted in a cross-correlation signal indicating molecular interaction even if there was no interaction present in the sample. We used simulations to develop our new method. To evaluate our new approach in vivo we used two control strains in S. cerevisiae. The negative control contained the non-interacting fluorescence proteins eGFP and mCherry. As the positive control we used a tandem of these two proteins. We measured more than 2500 individual cells and demonstrated that we could reduce the erroneous cross-correlation signal of the negative control from 69% to 4% compared to the cross-correlation signal of the positive control in the mCherry channel and from 24% to 5% for the eGFP channel.

F_oF₁-ATP synthase catalyzes the synthesis of adenosine triphosphate (ATP). The F₁ portion can be stripped from the membrane-embedded F_o portion of the enzyme. F₁ acts as an ATP hydrolyzing enzyme, and ATP hydrolysis is associated with stepwise rotation of the γ and ε subunits of F₁. This rotary motion was studied in great detail for the last 15 years using single F₁ parts attached to surfaces. Subunit rotation of γ was monitored by videomicroscopy of bound fluorescent actin filaments, nanobeads or nanorods, or single fluorophores. Alternatively, we applied single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the holoenzyme F_oF₁-ATP synthase which was reconstituted in liposomes. Now we aim to extend the observation times of single FRET-labeled F₁ in solution using a modified version of the anti-Brownian electrokinetic trap (ABELtrap) invented by A. E. Cohen and W. E. Moerner. We used Monte Carlo simulations to reveal that stepwise FRET efficiency changes can be analyzed by Hidden Markov Models even at the limit of a low signal-to-background ratio that was expected due to high background count rates caused by the microfluidics of the ABELtrap.

During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.

Photoswitchable and photoactivable proteins Dronpa and PhotoActivable mCherry (PA-mCherry) respectively, were used in order to perform FRET (Förster Resonance Energy Transfer) imaging at the single molecule level, using a FRET standard construct consisting of Dronpa and PA-mCherry separated by seven amino acids expressed in cells. Given Dronpa’s complex photophysical properties and the existence of a preswitched emissive state, irradiation conditions at 491 and 405 nm were optimised. We discuss strategies for observing FRET at the single molecule level with photoactivatable proteins by monitoring modifications in the donor and acceptors emissive states.

Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful tool used to investigate the properties of individual molecules. Single-Photon Avalanche Diodes (SPADs) are the detectors of choice for these applications. Recently a new type of SPAD detector was introduced, dubbed red-enhanced SPAD (RE-SPAD), with good sensitivity throughout the visible spectrum and with excellent timing performance. We report a characterization of this new detector for single-molecule fluorescence resonant energy transfer (smFRET) studies on freely diffusing molecules in a confocal geometry and alternating laser excitation (ALEX) scheme. We use a series of doubly-labeled DNA molecules with donor-to-acceptor distances covering the whole range of useful FRET values. Both intensity-based (μs-ALEX) and lifetime-based (ns-ALEX) measurements are presented and compared to identical measurements performed with standard thick SPADs. Our results demonstrate the great potential of this new detector for smFRET measurements and beyond.

Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to address outstanding problems in biology and biophysics. In order to study freely diffusing molecules, current approaches consist in exciting a low concentration (<100 pM) sample with a single confocal spot using one or more lasers and detecting the induced single-molecule fluorescence in one or more spectrally- and/or polarization-distinct channels using single-pixel Single-Photon Avalanche Diodes (SPADs). A large enough number of single-molecule bursts must be accumulated in order to compute FRET efficiencies with sufficient statistics. As a result, the minimum timescale of observable phenomena is set by the minimum acquisition time needed for accurate measurements, typically a few minutes or more, limiting this approach mostly to equilibrium studies. Increasing smFRET analysis throughput would allow studying dynamics with shorter timescales. We recently demonstrated a new multi-spot excitation approach, employing a novel multi-pixel SPAD array, using a simplified dual-view setup in which a single 8-pixel SPAD array was used to collect FRET data from 4 independent spots. In this work we extend our results to 8 spots and use two 8-SPAD arrays to collect donor and acceptor photons and demonstrate the capabilities of this system by studying a series of doubly labeled dsDNA samples with different donor-acceptor distances ranging from low to high FRET efficiencies. Our results show that it is possible to enhance the throughput of smFRET measurements in solution by almost one order of magnitude, opening the way for studies of single-molecule dynamics with fast timescale once larger SPAD arrays become available.

The most prevalent fungal pathogen in humans Candida albicans expresses on its surface Als (Agglutinin-like sequence) proteins which mediate both yeast-to-host tissue adherence and yeast aggregation. Although adhesins often show weak binding to specific ligands, Als mediate remarkably strong adherence. By combining single-molecule atomic force microscopy (AFM) with the tools of genetics and cell biology, we unraveled the various Als protein domains to understand how they synergize to strengthen the cell adhesion and aggregation. The complementary role of the ligandbinding domains, amyloid-forming regions and the less specific hydrophobic tandem repeat domains give raise to a fascinating new mechanism of cell-activation resulting in force-activated clustering of hundreds of adhesion molecules. The amyloid heptapeptide sequence plays a crucial role in this mechanism providing a cohesive strength to the Als protein by forming molecular zipper that mediates protein interactions between cells.

Subwavelength metal apertures significantly enhance single molecule fluorescence signaling systems, but require efficient illumination and collection optics. On-chip micromirror structures offer a way to markedly improve the coupling efficiency between such subwavelength metal apertures and the external fluorescence illumination and collection optics, which in turn greatly simplifies several aspects of instrument design including optics, optomechanics, and thermal control. Modeling and experimental verification of the gains in illumination and collection efficiency for subwavelength metal apertures leads to a micromirror design that is both highly efficient yet also manufacturable. A combination of ray-based and finite-difference-time-domain models is used to optimize conical micromirrors colocated with subwavelength metal apertures for the case where the illumination light interacts strongly with the micromirror and the collection optics have modest numerical aperture (NA~0.5). Experimental methods employing either freely diffusing or immobilized dye molecules are used to measure the illumination and collection efficiencies of fabricated micromirror prototypes. An overall fluorescence gain of ~100x, comprising a 20x improvement with flood illumination efficiency together with a 5x improvement in collection efficiency, are both predicted and experimentally verified.

Raman spectra are most commonly analyzed using the ordinary least squares (LS) method. However, LS is sensitive to variability in the spectra of the analyte and background materials. We previously addressed this problem by successfully proposing a novel hybrid least squares and principal components (HLP) algorithm. HLP extended LS by allowing the reference spectra to vary in accordance with the principal components observed in calibration sets. Previously, HLP assumed zero-mean Gaussian measurement noise. In this work, we show that the noise in fact follows a Poisson distribution, and update the mathematical framework of our algorithm accordingly. Since the name ’least squares’ referred to the Gaussian noise model, we also generalize the name of our algorithm to the Hybrid reference Spectrum and Principal component analysis (HSP) algorithm. The performance of the Gaussian and Poisson noise models is compared using both simulated and measured spectra. The simulated spectra were computed by adding various concentrations of Raman-enhanced gold-silica nanoparticles to three different backgrounds (paraffin, glass and quartz). The measured spectra were acquired from a serial dilution of gold-silica nanoparticles placed on an excised pig colon. For the simulated spectra, the Poisson model consistently outperformed the Gaussian model, on average reducing the mean absolute concentration error as well as its standard deviation by ~15-20%. For the measured data, the Gaussian and Poisson noise models yielded similar concentration estimates. Both HSP algorithms also outperformed the LS algorithm, indicating that the incorporation of the principal components yields a larger improvement in accuracy than the optimization of the noise model. Further comparison between the two HSP algorithms (with Gaussian and Poisson noise models) was precluded by a lack of precise ground truth knowledge of the nanoparticle concentrations on the colon tissue. However, the simulation results already demonstrated that the optimization of noise models can improve the detection accuracy of Raman spectroscopy, and that it may therefore be an important consideration in future high-sensitivity Raman imaging studies.

Single-molecule fluorescence (SMF) permits imaging with nanometer-scale resolution. This technique is particularly useful for cellular imaging as it provides a non-invasive, minimally perturbative means to examine macromolecular localization and dynamics, even in live cells. Here, we demonstrate that nanometer-scale SMF imaging can be extended to a new category of experiments: intracellular imaging of live, obligate anaerobic cells on the benchtop. We investigate the starch-utilization system (Sus) proteins in the gut symbiont Bacteroides thetaiotaomicron and discuss three different labels that we implemented to detect these proteins: fluorescent proteins, the tetracysteine-based FlAsH tag, and the enzymatic HaloTag.

Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or 2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the molecule’s transition dipole moment, as well as its z-position. Failure to account for this fact can result in localization errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF) microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).

Double-helix point spread functions (DH-PSFs) have been used to extend localization and super-resolution microscopy to three dimensions. Current DH-PSF design provides a long depth-of-field for 3D imaging. However, it is not optimal for imaging under high background conditions. We present a method to design unconventional DH-PSFs with control on characteristics such as efficiency, transverse spread, and Fisher information. This allows tailoring the PSFs for specific applications. In particular, we introduce a design suitable for 3D localization-based super-resolution imaging under typical background conditions and demonstrate imaging with resolution one order of magnitude beyond the diffraction limit over a depth-of-field of 1.2 μm.

The synaptic efficacy of neurons depends on the number of neurotransmitter receptors in the plasma membrane. The availability of these receptors is controlled by their specific intracellular trafficking routes. γ-Aminobutyric acid type B receptors (GABABRs) are heteromeric proteins consisting of GABA_BR1 and GABA_BR2 subunits. These receptors are found at the plasma membrane of somatodendritic postsynaptic sites and in axons. It is unknown whether the assembly of the subunits occurs directly in the somatic endoplasmic reticulum (ER) followed by vesicular transport, or whether the assembly occurs after the separate transport of the subunits to the dendritic ER compartment. To address this question we have studied the assembly of the GABA_BRs in hippocampal neurons with dual-color, 3D super-resolution optical fluctuation imaging (SOFI). SOFI is a fluorescence imaging modality which yields superresolved spatial resolution, 3D-sectioning and high image contrast. We will use the SOFI images to quantify the distribution of the GABA_BR subunits in the plasma membrane and in the dendritic intracellular compartments. Finally, we want to apply quantitative co-localization analysis to determine the compartments in which the assembly of the GABA_BR subunits occurs.

Single-molecule fluorescence is a powerful tool for imaging structures below the standard diffraction limit of light. The resolution gain comes from fitting the emission of isolated fluorophores, and localization accuracy improves with number of photons detected. Here, we control single-molecule emission by coupling fluorescence to gold nanoparticle local plasmon resonances. These strongly localized electromagnetic modes are generated by the interaction of light with particles smaller than the incident wavelength. We discuss the effects of this plasmonic mode coupling on the fluorescence intensity and lifetime of dye molecules and quantum dots.

In this paper, we analyze the Cramer-Rao lower bound on the accuracy of 3-D emitter locations estimated from a STORM movie with a cylindrical lens. Numerical evaluation for randomly and uniformly distributed emitters indicates that as the emitter density in the image plane increases, the Cramer-Rao lower bounds on the average full-width half-maximum (FWHM) of location estimates in the lateral plane and axial direction increase exponentially fast; and the exponents can be numerically accurately estimated. The Cramer-Rao lower bounds are inversely proportional to the square root of mean number of photons of an emitter and monotonically decrease as the signal to noise ratio increases. The result reveals the insightful property of 3-D STORM movies and provides a benchmark for the achievable accuracy of location estimation algorithms. The developed algorithm with multiemitter colocalization can improve the temporal resolution by five folds compared with the single-emitter location estimator.

The determination of 3-dimensional arrangement of subcellular assemblies has become a necessary requirement in cellular biology. Unfortunately, the size of most assemblies lies beyond the diffraction limit and therefore they cannot be visualized using conventional fluorescence microscopy techniques. Photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) enable the localization of fluorescent molecules with nanometric resolution. We used these microscopy methods together with MicAO 3D-SR – the first adaptive optics device which introduces the three-dimensional imaging capability for PALM/STORM. MicAO also corrects various types of aberrations induced by optical elements inside the microscope and by the biological sample. The correction of these aberrations almost doubles the number of detected photons, increasing the localization precision of PALM/STORM by 40%. At 1000 detected photons the localization precision of our setup is 8 nm in lateral and 16 nm in axial directions. The separate optimization performed for two different colors delivers superb imaging quality, as demonstrated by dual-color 3-dimentional imaging of two centrosomal proteins in HeLa cells.

Superresolution fluorescence microscopy techniques such as PALM, STORM, STED, and Structured Illumination Microscopy (SIM) enable imaging of live cells at nanometer resolution. The common theme in all of these techniques is that the diffraction limit is circumvented by controlling the states of fluorescent molecules. Although the samples are labeled very densely (i.e. with spacing much smaller than the Airy distance), not all of the molecules are emitting at the same time. Consequently, one does not encounter overlapping blurs. In the deterministic techniques (STED, SIM) the achievable resolution scales as the wavelength of light divided by the square root of the intensity of a beam used to control the fluorescent state. In the stochastic techniques (PALM, STORM), the achievable resolution scales as the wavelength of light divided by the square root of the number of photons collected. Although these limits arise from very different mechanisms (parabolic beam profiles for STED and SIM, statistics for PALM and STORM), in all cases the resolution scales inversely with the square root of a measure of the number of photons used in the experiment. We have developed a proof that this relationship between resolution and photon count is universal to techniques that control the states of fluorophores using classical light. Our proof encompasses linear and nonlinear optics, as well as computational post-processing techniques for extracting information beyond the diffraction limit. If there are techniques that can achieve a more efficient relationship between resolution and photon count, those techniques will require light exhibiting non-classical correlations.

Nonlinear structured illumination microscopy (SIM) allows full-field imaging at resolutions <100 nm. Two nonlinear effects, excitation saturation (SSIM) and the photo-switching of protein had been applied to nonlinear SIM. We report a new SIM technique which utilizes the nonlinearity of STED effect. Resolution and signal noise ratio simulation shows that STED-SIM may serve as a better alternative to SSIM and SIM with photo-switchable protein. SIM requires a strong nonlinear effect in a large area. We use Surface Plasmon Resonant to enhance of evanescence field near a dielectric-metal-dielectric interface. An 8 times STED effect enhancement is achieved on an optimized glass-silver-glass-water planar structure. We further use the interference of two SPR-enhanced STED fields propagating at opposite direction to generate a 1D structured STED field. Combined with a uniform excitation field, the structure STED field allows full field total internal reflection imaging with an enhanced resolution along the structured dimension. Less than 50 nm resolution is demonstrated. A STED-SIM microscope with 2D structured STED field is under development. Future research will apply the microscope to superresolution imaging of membrane resident or near membrane structure at super-resolution in live cells.

Superresolution localization microscopy requires accurate and precise localization algorithms. We have developed a plugin for ImageJ, called M2LE, which can localize molecules quickly and distinguish between single-molecule and multiple-molecule images using a shape test that requires only a single iteration. Localization is accomplished via a fast maximum-likelihood algorithm that uses the separable property of the Gaussian to independently fit two 1-D Gaussians along the x- and y-directions. To assess the performance of M2LE, we tested the plugin with realistic simulated images of single and multiple molecule images. We first found the optimal shape test parameters that accept most single-molecule images, and then the optimal signal-to-noise cutoff parameter for identifying potential molecules from noise. These two parameters have the greatest impact on what parts of the image go on to be analyzed. Using these optimal parameters, we then assessed (1) the tendency of the algorithm to find molecules from the tail of a point-spread function in high signal-to-noise cases, (2) the effects of regions-of-interest size and overlap tolerances, (3) the ability of shape tests to identify multi-molecule images as a function of molecular separation and ratio of photon counts from two molecules, and (4) the performance of the entire process{the number of molecules identified and their corresponding localization precision and accuracy. These methods and results can be used to identify the optimal M2LE parameters to use for experiments, as well as to compare the performance with other localization microscopy software.

Förster resonance energy transfer (FRET) is a powerful tool to investigate biochemical and biophysical processes in vitro and in vivo. We present the computational study of a novel FRET system, namely, a fuse protein that is composed of two far-red fluorescent proteins FusionRed and eqFP670 joined with a linker (FusionRed-Linker1-eqFP670). The latter contains a tetrapeptide DEVD motif which can be specifically cleaved by caspase-3. FusionRed acts as an energy donor and eqFP670 as an energy acceptor in this pair. We carried out the comprehensive study of the factors that influence FRET efficiency, including, in particular, distances between donor and acceptor, as well as relative orientations of transition dipole moments from donor to acceptor and the structure of linker between donor and acceptor. We started from the primary structure of the proteins and also used available data on the similar β-barrels to construct the full-atom 3D structure of FusionRed-Linker1-eqFP670 sensor. We used a protein-protein docking procedure to construct the tetramer structure of the fuse protein. We compared this sensor with another FLIM/FRET-based sensor on caspase-3, TagRFP-23-KFP and explained the differences in their properties.

Fluorescent microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile even the most advanced confocal microscopy can only yield optical resolution approaching Abbe diffraction limit of ~200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade.

In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction.

In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.

Circumventing the limit imposed by diffraction is a major issue in the instrumental development to realize finer resolutions in biological samples. With STED microscopy, we exploit the molecular transitions of the fluorescent marker to image well below the Rayleigh criterion. Also in combination with STED, we propose to use an alternative technique for optically sectioning fluorescent emitters close to the water-glass interface by selectively filtering the supercritical emission at the pupil plane. We discuss the instrumental development of such a system and its combination with other imaging techniques.

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