This PDF file contains the front matter associated with SPIE Proceedings Volume 8950, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Here we present an automated microscope capable of 3D multi-color single molecule localization of individual messenger RNA molecules in a wide range of cell types. We have implemented astigmatic imaging with a cylindrical lens to improve z-localization, and a maximum likelihood estimator on a graphics processing unit to improve localization precision and speed. This microscope will aid in gene expression analysis by its capability to perform high throughput imaging of thick cells and tissues while still maintaining sufficient z resolution to resolve single RNA transcripts in three dimensions. Enhanced z-localization allows for resolving membrane localized and co-localized transcripts.

In aqueous solution, diffusion generally limits the observation window of a nano-meter sized single molecule to milliseconds and prevents quantitative determination of spectroscopic and transport properties molecule-by-molecule. The anti-Brownian electrokinetic (ABEL) trap is a feedback-based microfluidic device that enables prolonged (multiseconds) observation of single molecules in solution. The amount of information that can be extracted from each molecule in solution is thus boosted by three orders of magnitude. We describe recent advances in extending the ABEL trap to conduct both spectroscopic and transport measurements of single trapped molecules. First, by combining the trap with multi-parameter fluorescence detection, synchronized dynamics in different observables can be visualized in solution. We use single molecules of Atto 633 as an example and show that this popular label switches between different emissive states under common imaging conditions. Next, we show how transport properties of trapped single molecules can be extracted in addition to spectroscopic readouts. Due to their direct sensitivity to molecular size and charge, measured transport coefficients can be used to distinguish different molecular species and trace biomolecular interactions in solution. We demonstrate this new paradigm by monitoring DNA hybridization/melting in real-time.

Typically the signal-to-background ratio is the limiting aspect of fluorescence-based detecting and imaging. The background signal can be composed of a variety of sources-excitation scattering, contaminants, and autofluorescence from cellular constituents. Most of these sources have a short-lived lifetime (ps to ns range). In order to increase the signal-to-background ratio, fluorophores with high brightness or in large concentrations are typically used along with time-gated detection. This unfortunately sacrifices the probe’s signal unless it has a very long lifetime. Herein we are presenting a simple method to enhance the detection of widely available and well-known mid-range lifetime (~20 ns) fluorophores’ signal against short-lived backgrounds. This requires a repetition rate of ~300 MHz to pump a 20 ns probe sufficiently. Typical laser sources today are not equipped with repetition rates above 80 MHz. However, this multipulse method allows these rates to be attainable for nearly any pulsed laser source. Multiple pulses of excitation are separated by a variable temporal length, which is short compared to the lifetime of the long-lived fluorophore, to increase the excited state population of a long-lived fluorophore, while the short-lived background decays almost completely between pulses. This is accomplished by simply redirecting the pulsed excitation beam through glass and then a delay length any number of times and lengths as desired to control the number of pulses and separation times.

Micro-fabrication and surface functionalization imply to know the equilibrium surface concentration of various kinds of molecules. Paradoxically, this crucial parameter is often poorly controlled and even less quantified. We have used a technique belonging to the family of fluorescence fluctuation microscopy, namely Image Correlation Spectroscopy (ICS), to measure the absolute surface concentration of fibrinogen molecules adsorbed on glass substrates. As these molecules are immobile, the width of the autocorrelation of the confocal image obtained by scanning the sample only reflects that of the confocal Point Spread Function. Conversely, the amplitude of the autocorrelation is directly related to the average number of proteins simultaneously illuminated by the laser beam and therefore to their surface concentration. We have studied the surface concentration of fibrinogen proteins versus the initial concentration of these molecules, solubilized in the solution which has been deposited on the surface. The estimation of this relation can be biased for several reasons: the concentration of fibrinogen molecules in solution is difficult to control; the measurement of the surface concentration of adsorbed molecules can be strongly underestimated if the surface coverage or the molecular brightness is not uniform. We suggest methods to detect these artifacts and estimate the actual surface concentration, together with control parameters. Globally, fluorescence fluctuation microscopy is a powerful set of techniques when one wants to quantify the surface concentration of molecules at the micrometer scale.

One major limitation of single-molecule fluorescence spectroscopy is the finite residence time of diffusing molecules in the confocal detection volume. The time ranges in the order of milliseconds. This has typical size of femtoliter. Here, we present a concept of extending the residence time using nanochannels of ca. 60 nm x 60 nm cross-section to restrict the molecular motion. We use solid-state nanochannels of silicon and silicon dioxide. This work is aimed to use for dual-focus fluorescence detection combining Anti-Brownian ELectrophoretic or ABEL trap to actively trap single molecule.

Saturation spectroscopy is a powerful method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). As we have presented in a previous paper [1], we interpret the evolution of the brightness with the laser power by cascade absorption of two and three photons within a five-level molecular system. This multi-photon consecutive absorption leads us to reconsider the common expression of the saturation curve of fluorescent molecule. Furthermore, this multi-photon absorption process also affects the observation volume of microscope. So, in this paper we propose to interpret the size increase of the confocal observation volume according to simulations based upon two often used expressions of the Point Spread Functions (PSF) in fluorescence microscopy.

F₁-ATPase is the soluble portion of the membrane-embedded enzyme F_oF₁-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F₁ part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F₁ (or F_oF₁) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F₁. We investigate this proposed mechanism by labeling F₁ specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F₁ molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F₁ in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F₁ and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

Localization microscopy methods allow the acquisition of fluorescence microscopy images with a resolution of tens of nanometres. However, these techniques are generally limited in speed because of the requirement that in each frame fluorophores must be well separated. This condition can be lifted by developing improved analysis techniques which allow data to be extracted from images in which the point spread functions of the fluorophores overlap (at the expense of an increased computational load). These improved analysis techniques build more information into the model for the system, either about the probable appearance of overlapping fluorophores in the spatial domain, or about blinking behaviour in the temporal domain. Here we discuss a method which incorporates both types of information, where we create a Hidden Markov Model including the photophysical processes occuring when fluorophores blink and bleach. This Bayesian analysis of blinking and bleaching (3B) technique allows super-resolution information to be obtained from live cells labelled with standard fluorescent proteins, with a temporal resolution of a few seconds and a spatial resolution of 50 nm.

We present a means of measuring the dipole orientation of a fluorescent, rotationally fixed single molecule (SM), using a specially designed phase mask, termed a “quadrated pupil,” conjugate to the back focal plane of a conventional widefield microscope. In comparison to image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies, defocus, and optical aberrations. Precision on the order of 1°-5° is achieved in proofof- concept experiments for both azimuthal (φ) and polar (θ) angles. Since the phase mask is implemented on a liquid-crystal spatial light modulator (SLM) that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into conventional imaging studies.

Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. However, most available technologies for mRNA detection rely on cell extraction that inherently destroys the tissue context and provide only average expression levels from cell populations or whole tissues. In this paper, we proposed a novel super resolution concept, second harmonic generation (SHG) super-resolution microscopy (SHaSM), and apply that to detect single short mRNA transcript, Her2 mRNA, beyond the diffraction limit. Nano-sized SHG crystals, barium titanium oxide BaTiO₃ (BTO), were functionalized with two complimentary strands of Her2 mRNA after the chemical surface-modification. Dimer schematic was used to improve the specificity of detection and quantification, where two BTO monomers bind to the Her2 mRNA to form a dimer and being visualized via the SHaSM. SHaSM is able to detect single BTO nanocrystal with ~20 nm spatial resolution, and differentiate BTO dimers (Her2 mRNA) from BTO monomers (non-specific bounded BTO nanocrystal) with high specificity.

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of cellular structures and multi-protein complexes at the nanometer scale. Using singlemolecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ~10-20 nm, an order of magnitude improvement over conventional microscopy. However, the spectral resolution of current SRM techniques are limited by existing fluorophores with only up to four colors imaged simultaneously, limiting the number of intracellular components that can be studied in a single sample. In the current work, a library of novel BODIPY-based fluorophores was synthesized using a solid phase synthetic platform with the goal of creating a set of photoswitchable fluorophores that can be excited by 5 distinct laser lines but emit throughout the spectral range (450-850 nm) enabling multispectral super resolution microscopy (MSSRM). The photoswitching properties of all new fluorophores were quantified for the following key photoswitching characteristics: (1) the number of photons per on cycle (2) the number of on cycles (switching events), (3) the percentage of time the fluorophore spends in the fluorescent on and off states, and (4) the susceptibility of the fluorophore to photobleaching (time of last event). To ensure the accuracy of our photoswitching measurements, our methodology to detect and quantitate the photoswitching properties of individual fluorophore molecules was validated by comparing measured photoswitching properties of three commercial dyes to published results.¹ We also identified two efficient methods to positionally isolate fluorophores on coverglass for screening of the BODIPY-based library.

We performed super-resolution imaging of isolated olfactory sensory neurons (OSNs) using a custom-built Stimulated Emission Depletion (STED) microscope. The design for the STED microscope is based on the system developed in the laboratory of Dr. Stefan Hell¹. Our system is capable of imaging with sub-diffraction limited resolution simultaneously in two color channels (at Atto 590/Atto 647N wavelengths). A single, pulsed laser source (ALP; Fianium, Inc.) generates all four laser beams, two excitation and two STED. The two STED beams are coupled into one polarization maintaining (PM) fiber and the two excitation beams into another. They are then collimated and both STED beams pass through a vortex phase plate (RPC Photonics) to allow shaping into a donut at the focus of the objective lens. The beams are then combined and sent into an inverted research microscope (IX-71; Olympus Inc.) allowing widefield epifluorescence, brightfield and DIC imaging on the same field of view as STED imaging. A fast piezo stage scans the sample during STED and confocal imaging. The fluorescent signals from the two color channels are detected with two avalanche photodiodes (APD) after appropriate spectral filtering. The resolution of the system was characterized by imaging 40 nm fluorescent beads as ~60 nm (Atto 590) and ~50 nm (Atto 647N). We performed STED imaging on immunolabeled isolated OSNs tagged at the CNGA2 and ANO2 proteins. The STED microscope allows us to resolve ciliary CNGA2 microdomains of ~54 nm that were blurred in confocal.

IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with other techniques will allow the molecular architecture of

We report a method to fabricate SERS fiber probe by femtosecond laser micromachining on a microfiber tip. Multimode optical fibers are tapered to outer diameter of 4 to 20 μm. Femtosecond laser pulses are used to form nanostructures on the cleaved endface of the microfiber tip. This endface is then activated by silver sputter coating. To confine the excitation and reflection signal within the tapered core of the microfiber tip, the probe is side-coated with silver plating. High quality SERS signal of Rhodamine 6G molecules is detected through back excitation and collection from a lead in fiber of up to several meters long. The small size of the SERS micro-probe is promising for intra-cellular or even single cell detection, while the back excitation and collection setup make it suitable for remote sensing applications.

We present a generic sub-diffraction-limited imaging method – photobleaching imprinting microscopy (PIM) – for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a two-fold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community.

Recently, we discovered, for the first time, reverse saturable scattering in a single gold nanoparticle. When incident intensity increases, the scattering intensity dependence of 80-nm gold nanoparticles evolves from linear, to saturation, and to reverse saturation sequentially. The intensity dependence in reverse saturable scattering region is significantly steeper than that in the linear region. With the aid of a confocal microscope, the full width half maximum of the single-particle point spread function can be reduced down to 80 nm, which is beyond the diffraction limit. Our finding shows great potential for superresolution imaging application without bleaching.

Since the behaviour of proteins and biological molecules is tightly related to the cell’s environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.

Three dimensional single-molecule tracking (3D-SMT) has revolutionized the way we study fundamental cellular processes. By analyzing the spatial trajectories of individual molecules (e.g. a receptor or a signaling molecule) in 3D space, one can discern the internalization or transport dynamics of these molecules, study the heterogeneity of subcellular structures, and elucidate the complex spatiotemporal regulation mechanisms. Sub-diffraction localization precision, sub-millisecond temporal resolution and tens-of-seconds observation period are the benchmarks of current 3D-SMT techniques. We have recently built two molecular tracking systems in our labs. The first system is a previously reported confocal tracking system, which we denote as the 1P-1E-4D (one-photon excitation, one excitation beam, and four fiber-coupled detectors) system. The second system is a whole new design that is based on two-photon excitation, which we denote as the 2P-4E-1D (two-photon excitation, four excitation beams, and only one detector) system. Here we compare these two systems based on Monte Carlo simulation of tracking a diffusing fluorescent molecule. Through our simulation, we have characterized the limitation of individual systems and optimized the system parameters such as magnification, z-plane separation, and feedback gains.

Engineering of quantum emissions is regarded as the heart of nano-optics and photonics; local density of optical states (LDOS) around the quantum emitters are critical to engineer quantum emissions, thus detection of the LDOS will impact areas related to illumination, communication, energy, and even quantum-informatics. In this report, we demonstrated a far-field approach to detect and quantify the near-field LDOS of a nanorod via using CdTe quantum dots (QDs) tethered to the surface of nanorods as beacons for optical read-outs. The spontaneous decay of QD emission in the proximity of nanorod was used as a ruler for elucidating the LDOS. Our analysis indicates that the LDOS of the nanorod at its ends is 2.35 times greater than that at the waist. Our approach can be applied for further evaluation and elucidation of the optical states of other programmed nanostructures.

Fluorescent nanodiamonds (NDs) are new and emerging nanomaterials that have potential to be used as fluorescence imaging agents and also as a highly versatile platform for the controlled functionalization and delivery of a wide spectrum of therapeutic agents. We will utilize two experimental methods, TIRF, a relatively simple method based on total internal reflection fluorescence and SPRF, fluorescence enhanced by resonance coupling with surface plasmons. We estimate that the SPRF method will be 100 times sensitive than currently available similar detectors based on detectors. The ultimate goal of this research is to develop microarray platforms that could be used for sensitive, fast and inexpensive gene sequencing and protein detection.