This PDF file contains the front matter associated with SPIE Proceedings Volume 9331, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Post-translational modifications to histone tails nave been shown to play a large role in dictating the conformation of chromatin. Structural changes in chromatin can play a large role in gene expression as compact chromatin can occlude transcriptional machinery. The role of the flexible regions of H4 N terminal tails is investigated using fluorescent correlation spectroscopy and the photon counting histogram. The combination of these techniques allows for the distinction between intra-array and inter-array interactions, as well as reveals structural changes that result from these interactions. The H4 tail was found to partake in attractive intra-array interactions that compact the 6x167 nucleosome arrays but did not partake in inter-array interactions that lead to oligomerization.

Mean-squared displacement (MSD) analysis is one of the most prevalent tools employed in the application of single-particle tracking to biological systems. In camera-based tracking, the effects of “static error” due to photon fluctuations and “dynamic error” due to motion blur on the MSD have been well-characterized for the case of pure Brownian motion, producing a known constant offset to the straight-line MSD. However, particles tracked in cellular environments often do not undergo pure Brownian motion, but instead can for instance exhibit anomalous diffusion wherein the MSD curve obeys a power law with respect to time, MSD=2D*τα, where D* is an effective diffusion coefficient and 0 < α ≤ 1. There are a number of models that can explain anomalous diffusive behavior in different subcellular contexts. Of these models, fractional Brownian motion (FBM) has been shown to accurately describe the motion of labeled particles such as mRNA and chromosomal loci as they traverse the cytoplasm or nucleoplasm (i.e. crowded viscoelastic environments). Despite the importance of FBM in biological tracking, there has yet to be a complete treatment of the MSD in the presence of static and dynamic errors analogous to the special case of pure Brownian motion. We here present a closed-form, analytical expression of the FBM MSD in the presence of both types of error. We have previously demonstrated its value in live-cell data by applying it to the study of chromosomal locus motion in budding yeast cells. Here we focus on validations in simulated data.

3D single-particle tracking (SPT) has been a pivotal tool to furthering our understanding of dynamic cellular processes in complex biological systems, with a molecular localization accuracy (10-100 nm) often better than the diffraction limit of light. However, current SPT techniques utilize either CCDs or a confocal detection scheme which not only suffer from poor temporal resolution but also limit tracking to a depth less than one scattering mean free path in the sample (typically <15μm). In this report we highlight our novel design for a spatiotemporally multiplexed two-photon microscope which is able to reach sub-diffraction-limit tracking accuracy and sub-millisecond temporal resolution, but with a dramatically extended SPT range of up to 200 μm through dense cell samples. We have validated our microscope by tracking (1) fluorescent nanoparticles in a prescribed motion inside gelatin gel (with 1% intralipid) and (2) labeled single EGFR complexes inside skin cancer spheroids (at least 8 layers of cells thick) for ~10 minutes. Furthermore we discuss future capabilities of our multiplexed two-photon microscope design, specifically to the extension of (1) simultaneous multicolor tracking (i.e. spatiotemporal co-localization analysis) and (2) FRET studies (i.e. lifetime analysis). The high resolution, high depth penetration, and multicolor features of this microscope make it well poised to study a variety of molecular scale dynamics in the cell, especially related to cellular trafficking studies with in vitro tumor models and in vivo.

Plants harvest sunlight by converting light energy to electron flow through the primary events in photosynthesis. One important question is how the light harvesting machinery adapts to fluctuating sunlight intensity. As a result of various regulatory processes, efficient light harvesting and photoprotection are balanced. Some of the biological steps in the photoprotective processes have been extensively studied and physiological regulatory factors have been identified. For example, the effect of lumen pH in changing carotenoid composition has been explored. However, the importance of photophysical dynamics in the initial light-harvesting steps and its relation to photoprotection remain poorly understood. Conformational and excited-state dynamics of multi-chromophore pigment-protein complexes are often difficult to study and limited information can be extracted from ensemble-averaged measurements. To address the problem, we use the Anti-Brownian ELectrokinetic (ABEL) trap to investigate the fluorescence from individual copies of light-harvesting complex II (LHCII), the primary antenna protein in higher plants, in a solution-phase environment. Perturbative surface immobilization or encapsulation schemes are avoided, and therefore the intrinsic dynamics and heterogeneity in the fluorescence of individual proteins are revealed. We perform simultaneous measurements of fluorescence intensity (brightness), excited-state lifetime, and emission spectrum of single trapped proteins. By analyzing the correlated changes between these observables, we identify forms of LHCII with different fluorescence intensities and excited-state lifetimes. The distinct forms may be associated with different energy dissipation mechanisms in the energy transfer chain. Changes of relative populations in response to pH and carotenoid composition are observed, which may extend our understanding of the molecular mechanisms of photoprotection.

Although Single Molecule Localization (SML) techniques have pushed the resolution of fluorescence microscopy beyond the diffraction limit, the accuracy of SML has been limited by the brightness of the fluorophores. The introduction of Quantum Dots (QD) for SML promises to overcome this barrier, and the QD Blueing technique provides a novel approach to SML microscopy. QDs have a higher quantum yield and absorption cross-section, making them brighter, thereby providing a higher accuracy of localization. The blueing technique is also faster and more quantitative than other SML techniques such as dSTORM. The initial bleaching step required by dSTORM is not necessary and each QD is imaged only once as its emission spectrum moves through the blueing window in contrast to dSTORM where the same molecule might be imaged multiple times. Single color QD Blueing has been demonstrated. However in biological imaging, multi-color imaging is essential for understanding the samples under study. Here we introduce two color superresolution microscopy using QD Blueing on biological samples. We demonstrate simultaneous imaging of microtubules and mitochondria in HepG2 cells with a localization accuracy of 40nm. We further show how QD Blueing can be optimized through the control of the sample mounting medium.

F_oF₁-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single F_oF₁-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of F_oF₁-ATP synthase diffusing on the long axis in living E. coli cells.

A new method is described for measuring the absorption of light by single non-emissive nanoparticles. Individual carbon nanofibers are imaged using a photonic transducer to quantify the heat dissipated after the electronic energy is thermalized. Leveraging the high sensitivity of ultrahigh-quality-factor optical microresonators as photothermal transducers provides high sensitivity. Polarization-resolved measurements indicate that the orientation of the absorption dipole of a nanofiber matches the long axis of the fiber. The per-atom absorption cross-section is determined to be (2.9 x 10^-18 cm² /carbon atom), in close agreement with the value for bulk graphite.

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of intracellular structures and multi-protein complexes at the nanometer scale. Using single-molecule 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. One of the key factors in achieving such resolution with single-molecule SRM is the ability to accurately locate each fluorophore while it emits photons. Image quality is also related to appropriate labeling density of the entity of interest within the sample. While ease of detection improves as entities are labeled with more fluorophores and have increased fluorescence signal, there is potential to reduce localization precision, and hence resolution, with an increased number of fluorophores that are on at the same time in the same relative vicinity. In the current work, fixed microtubules were antibody labeled using secondary antibodies prepared with a range of Alexa Fluor 647 conjugation ratios to compare image quality of microtubules to the fluorophore labeling density. It was found that image quality changed with both the fluorophore labeling density and time between completion of labeling and performance of imaging study, with certain fluorophore to protein ratios giving optimal imaging results.

In the field of optical microscopy, it is well known that spatial resolution is limited by diffraction of light. There have been variously efforts to achieve resolution beyond diffraction limit. However, most previous methods rely on nonlinearities of fluorescence, and thus require high-intensity lasers or special labeling strategies. In 2011, a novel superresolution technique based on a dielectric microlens and simple bright field microscope was demonstrated. The scheme is unexpectedly simple, since neither labeling nor intense laser is necessary, while the resolution is significantly higher than diffraction limit. Nevertheless, the contrast of bright field microscope is poor. In this work, we combine a dielectric microlens along with confocal laser scanning microscopy to considerably enhance image contrast. By simply inserting a microsphere onto the sample, the resolution is undoubtedly better than diffraction limit of the objective. Meanwhile, the contrast exhibits almost one order of enhancement. Field of view and magnification of the microlens imaging system are also characterized. Comparing with bright field microscope, laser scanning confocal microscopy provides better contrast under this microslens assisted super-resolution scheme. Our finding will contribute to material science and biomedicine research.

We present a method for Z-super-localization in fluorescence microscopy, based on conical diffraction. By using a thin biaxial crystal, the Point Spread Function (PSF) shape of an objective is made to depend strongly on the z coordinate. This z dependence is then exploited to localize fluorescent emitters axially with a great precision. We study how this method can be used for single molecule imaging with a global assessment by Fisher information analysis. Preliminary experiments demonstrate that this technique can obtain resolutions of tens of nm with the use of high NA objectives.

Aggregation of misfolded proteins is a characteristic hallmark of many neurodegenerative disorders, such as Parkinson’s, Alzheimer’s and Huntington’s diseases. The ability to observe these aggregation processes and the corresponding structures formed in vitro or in situ is therefore a key requirement to understand the molecular mechanisms of these diseases. We report here on the implementation and application of Stimulated Emission Depletion (STED) microscopy to visualize the formation of amyloid fibrils in vitro.

In a stimulated emission depletion (STED) microscope the region from which a fluorophore can spontaneously emit shrinks with the continued STED beam action after the excitation event. This fact has been recently used to implement a versatile, simple and cheap STED microscope that uses a pulsed excitation beam, a STED beam running in continuous-wave (CW) and a time-gated detection: By collecting only the delayed (with respect to the excitation events) fluorescence, the STED beam intensity needed for obtaining a certain spatial resolution strongly reduces, which is fundamental to increase live cell imaging compatibility. This new STED microscopy implementation, namely gated CW-STED, is in essence limited (only) by the reduction of the signal associated with the time-gated detection. Here we show the recent advances in gated CW-STED microscopy and related methods. We show that the time-gated detection can be substituted by more efficient computational methods when the arrival-times of all fluorescence photons are provided.

Proteins are the most diverse and functionally active biomolecules in the living system. In order to understand their diversity and dynamic functionality, visualization in native form without altering structural and functional properties during the separation from the complex mixtures is very much essential. In the present study, a sensitive methodology for optimal visualization of unstained or untagged proteins in native poly-acrylamide gel electrophoresis (N-PAGE) has been developed where, concentration of the acrylamide and bis-acrylamide mixture, Percentage of the gel, fixing of the N-PAGE by methanol: acetic acid: water and washing of the gel in the mili-Q water has been optimized for highest sensitivity using laser induced autofluorescence. The outcome with bovine serum albumin (BSA) in PAGE was found to be highest at acrylamide and bis-acrylamide concentrations of 29.2 and 0.8 respectively in 12% N-PAGE. After the electrophoresis run, washing of the N-PAGE immediately with miliQ water for 12 times and eliminating the methanol: acetic acid: water, fixing of the N-PAGE yielded better sensitivity of visualization. Using the above methodology 25ng of BSA protein band in PAGE was clearly identified by the technique. The currently used staining techniques for the visualization of proteins are coomassie brilliant blue and silver staining, have the sensitivity of 100ng and 5ng respectively. The current methodology was found to be more sensitive as compared to coomassie staining and less sensitive compared to silver staining respectively. The added advantage of this methodology is the faster visualization of proteins without altering their structure and functional properties.

The lac operon is a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with different DNA constructs. Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences and transient interactions with nonspecific sequences.

Since the behaviour of proteins and biological molecules is tightly related to 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. Since protein dynamics inside a cell involve all three dimensions, we developed an automated routine for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

W. E. Moerner, the Harry S. Mosher Professor of Chemistry and professor, by courtesy, of Applied Physics at Stanford University, conducts research in physical chemistry of single molecules, biophysics, nanoparticle trapping, and nanophotonics. He earned three bachelor's degrees from Washington University in 1975 and master's and doctoral degrees from Cornell University in 1978 and 1982. From 1981 to 1995, he was a research staff member at IBM, receiving two IBM Outstanding Technical Achievement Awards. He was Professor and Distinguished Chair in Physical Chemistry at the University of California, San Diego, from 1995 to 1998, the year he joined the Stanford faculty. Moerner received the 2014 Nobel Prize in Chemistry, along with Eric Betzig and Stefan Hell, for their development of super-resolved fluorescence microscopy.

Stefan W. Hell is a director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, and also leads a research division at the German Cancer Research Center (DKFZ) in Heidelberg. He is credited with having conceived, validated and applied the first viable concept for breaking Abbe's diffraction-limited resolution barrier in a light-focusing microscope. Hell was unable to attend SPIE Photonics West 2015, but participated in the Nobel plenary session by contributing this video, which includes an edited version of his Nobel acceptance speech. Hell received the 2014 Nobel Prize in Chemistry, along with Eric Betzig and W.E. Moerner, for their development of super-resolved fluorescence microscopy.