This PDF file contains the front matter associated with SPIE Proceedings Volume 11967, including the Title Page, Copyright information, and Table of Contents.

A great interest in high-throughput fluorescence microscopy for biological and medical imaging has led to the flourishing of new imaging methods where the sample is quickly scanned through the optics. Optofluidic microscopes use fluids’ properties as an additional degree of freedom for optical detection and microfluidics to perform simple and low-cost object manipulation. Even though several devices have been optimized for fluorescence-based imaging, these systems can rarely resolve sub-micron details, posing a limit to the structures that can be studied. An exception is represented by systems developed for particle detection, which are capable to quantify protein expression and analyze small molecules even at nanoscale resolution. However, in this case, high resolution requires a low emitter density and it cannot be used to visualize densely packed structures such as membranes and organelles. Hence, we have developed a system for sub-diffractionlimited optofluidic scanning microscopy (OSM) that uses the optofluidics paradigm to extract the inherent super-resolution information of a confocal system. OSM uses the optofluidic flow scanning scheme and a multifocal illumination pattern to obtain resolution doubling with minimal system complexity. In addition, it does not require any mechanical part for the scanning, so that it can be readily adapted to different levels of integration from commercial microscopes to on-chip configurations. This makes our system the most viable configuration for super-resolution optofluidics, being both suitable for continuous flow scanning and compatible with on-chip configurations through the adoption of integrated optics, like custom micro-lenses or Fresnel zone plates. Finally, we demonstrate how the same concept can be adapted to digital slide scanners for super-resolution whole slide imaging.

Photon-HDF5 is an open-source and open file format for storing photon-counting data from single molecule microscopy experiments, introduced to simplify data exchange and increase the reproducibility of data analysis. Part of the Photon-HDF5 ecosystem, is phconvert, an extensible python library that allows converting proprietary formats into Photon-HDF5 files. However, its use requires some proficiency with command line instructions, the python programming language, and the YAML markup format. This creates a significant barrier for potential users without that expertise, but who want to benefit from the advantages of releasing their files in an open format. In this work, we present a GUI that lowers this barrier, thus simplifying the use of Photon-HDF5. This tool uses the phconvert python library to convert data files originally saved in proprietary data formats to Photon-HDF5 files, without users having to write a single line of code. Because reproducible analyses depend on essential experimental information, such as laser power or sample description, the GUI also includes (currently limited) functionality to associate valid metadata with the converted file, without having to write any YAML. Finally, the GUI includes several productivity-enhancing features such as whole-directory batch conversion and the ability to re-run a failed batch, only converting the files that could not be converted in the previous run.

Time-Correlated Single Photon Counting (TCSPC) is a time-resolved and ultrasensitive technique, that provides the analysis of optical pulses to a wide range of different applications both in the biological and chemical domain. Nevertheless, an ultimate constraint to this technique has been historically posed by pile-up distortion, that typically restricts the maximum acquisition speed to few percent of the laser excitation rate. To surpass this fundamental limitation, a novel theoretical solution has been reported in a previous paper: with a perfect matching between detector dead time and laser period, it is possible to achieve a high-speed measurement, still maintaining negligible distortion. In this work, we present the design, characterization and experimental validation of a single-channel TCSPC system that implements the proposed idea. The essential core of the system consists in a compact Detection Head featuring a finely tunable dead time, thanks to a fully-integrated front-end electronics coupled to a custom technology Single-Photon Avalanche Diode (SPAD). This module is providing a picosecond precision timing signal, that is then acquired and digitized by means of a Fast Time to Amplitude Converter (F-TAC) architecture, followed by a high-end Field Programmable Gate Array (FPGA). In order to validate the proposed technique, we carried out on-field fluorescence lifetime measurements employing the newly developed system. The experimental results show good accordance with the previous theoretical framework. It is therefore possible to achieve high acquisition speed (32 Mcps) with an almost null lifetime distortion, thus paving the way to new advanced TCSPC applications.

Single-particle tracking reports on the mobility of biomolecules in living cells with high spatial and temporal resolution. From single-particle trajectories, information such as the diffusion coefficient and diffusion state can be derived. Changes in particle dynamics within single trajectories can be extracted by segmentation, which provides information on transitions between different functional states of a biomolecule. However, such analyses of single-particle tracking data is complex and time-consuming. Here, we present a pipeline that enables a straightforward and rapid analysis of single-particle tracking data. It incorporates mean-squared displacement analysis of trajectories that distinguishes between immobile, confined, and free diffusion states, as well as the analysis of diffusion state transitions within a trajectory with transition counts and hidden Markov modeling. We apply this analysis to single-molecule trajectories of un-activated Fab-bound and internalin B-bound MET receptors in the plasma membrane of live HeLa cells. We found that ligand activated receptors move slower and more confined and exhibit more transitions from free to confined diffusion states than un-activated receptors. This suggests that the confined diffusion state functions as an intermediate between free and immobile, as this state is most likely changing the diffusion type in the following segment. Hidden Markov modeling reported three diffusion states with increased transition probabilities towards the less mobile and immobile states upon ligand activation. The less mobile state operates as an intermediate state, as it has the highest transition probabilities. The analysis pipeline can be readily applied to single-particle tracking data of other membrane proteins and provides rapid access to information that can be associated with functional states.

We present the development and application of a novel structured illumination microscope (SIM) in which the grating pattern is generated using two optical beams controlled via two micro-electro-mechanical system (MEMS) 3D scanning micromirrors, each having static angular and piston control. This arrangement enables the generation of a fully controllable spatial interference pattern at the focal plane by adjusting the positions of the beams in the back-aperture of a high numerical aperture (NA) microscope objective. The utilization of MEMS micromirrors to control angular, radial and phase positioning for the structured illumination patterns has advantages of flexible control of the fluorescence excitation illumination, with achromatic beam delivery through the same optical path, reduced spatial footprint and cost-efficient integration.

Optical characterization at the nanoscale currently requires instruments such as NSOM/TERS, or hybrid AFM with specialized far-field optical microscopes that are quite complicated and do not provide any time-resolved data. We have demonstrated a novel class of probes for Scanning Probe Microscopy (SPM) - an Ultrafast Pulsed Atomic Force Microscopy Optical Probe (UFP AAOP) that will enhance characterization capabilities at the nanoscale and provide an exciting opportunity for obtaining both space- and time-resolved chemical information simultaneously. In the UFP AAOP design, a two-section quantum-dot mode-locked laser is monolithically integrated with an SPM probe fabricated from GaAs, with a nanoscale opening at the apex of the tip as the output aperture. With UFP AAOP, the light is supplied through the tip; hence, there is no scattered far-field light and thus significantly reduced background. Furthermore, the difficulties associated with laser alignment onto the tip and with imaging the signal onto a detector are avoided with the UFP AAOP. The UFP AAOP provides pulses with less than 4 ps duration and spatial resolution better than 300 nm at 1240 nm wavelength. It is potentially possible to reduce the pulse width to ~ 0.3 ps and to improve lateral resolution to ~ 1 nm. These unique optical probes will perform the functions of conventional AFM probes and simultaneously provide information about chemical properties of the sample at the nanoscale together with time-resolved spectroscopy. UFP AAOP will facilitate the creation of a new microscopy/spectroscopy instrument with combined single-molecule spatial resolution and ultrafast time-resolved capability.

Understanding cardiomyocyte-extracellular matrix (ECM) interactions at the molecular level is essential for deeper insights into their mechanical signaling function for cardiac development, homeostasis and remodeling. We report a lab-built microscope integrating two-color STED microscopy with second harmonic generation (SHG) microscopy to investigate the detailed architecture of cardiomyocyte-ECM interactions in murine myocardium at a subdiffractive level. SHG microscopy is used to locate possible interaction sites at the cell-ECM interface through the intrinsic SHG signal generated by collagen assemblies and myosin filaments. Two-color STED microscopy is used to obtain a subdiffractive view of proteins at sites of interest registered by SHG microscopy. Because large field-of-view (FOV) STED microscopy is still challenging, with photobleaching often a major concern, imaging only SHG-registered sites is advantageous. Further, using intrinsic contrast in the study reduces the number of biomarkers for fluorescent staining and thereby the number of detection channels for fluorescent imaging, simplifying sample preparation procedures and STED microscopy architectures. For purpose of demonstration, we show images of immunostained type I collagen, type Ⅳ collagen and laminin as ECM structures of interest in rat ventricular sections without counterstaining.

Cells adapt their actin cytoskeletons architecture to structural cues of the environment in all three dimensions. Nevertheless, how manipulating cell shape influences the actin cytoskeletons z-dimension is unstudied, but crucial for an understanding of the mutual influence of cell shape, cell tension and actin architecture. To study the effect of shape on the z-dimension of the actin cytoskeleton we combine metal-induced energy transfer as a super-resolution technique with micropatterning. This allows us not only to precisely manipulate the shape of the cell but also to regulate forces by changing the shape while studying specific actin structures with super-resolution.

Focal adhesions function are cellular anchoring points to the extracellular matrix and enable cells to sense and exert forces on their environment . They are complex structures consisting of a multitude of different proteins. Despite the important role of the focal adhesion complex in cellular adhesion, its structure and mechanoresponse remain difficult to resolve. Knowing the exact position of the proteins in the focal adhesion complex under strain is necessary to understand their working principle. For a detailed analysis of the focal adhesion architecture coupled with force response, we require a method to measure small distances with super resolution precision while manipulating force acting on the cell. To meet this challenge, we couple life-cell atomic force spectroscopy with Metal Induced Energy Transfer (MIET) to resolve forces with pN and with nanometer accuracy. Here, we show an initial analysis of how forces are transduced from the extracellular space to the actin cytoskeleton.

Cells actively probe the mechanical properties of their environment and adapt their physiological state accordingly. For this fundamental task, they exert forces on their environment using their actomyosin machinery. This machinery consists of protein fibers consisting of dynamically assembling and dissembling actin units, so called actin stress fibers, on which motor proteins act to generate forces. This machinery is also used to react to external forces applied to cells. Transduction of forces from the actomyosin complexes to the extracellular space occurs via adaptive multi-protein complexes, so called focal adhesions. Focal adhesions re-structure depending on biochemical and mechanical signals. The distance of the extracellular matrix to the actomyosin fibers is determined by the structure of these complexes. Monitoring this distance reveals cellular adaptions to force. Here, we use metal induced energy transfer (MIET) to monitor the distance of the extracellular matrix to focal adhesions for understanding force transduction through focal adhesions. We manipulate the cells either with drugs influencing cellular force generation or using an atomic force microscope. We find that high forces lead to a multistep-restructuring of focal adhesions, rendering force transduction more efficient.

This presentation will introduce a modified blaze condition of a digital micromirror device for structured illumination microscopy. This condition aims to remove a special mask at the Fourier plane, which blocks unwanted orders to make two or three beams interference. The proposed alignment is intrinsically free from the zeroth order of diffraction light. So, we can generate structured light by uploading proper patterns onto DMD with an iris diaphragm instead of the special mask. By adjusting the size of the iris diaphragm, structured light with various periods can be illuminated to the sample. The basic concept, and experiment results with a modified blaze condition will be presented.

Small oligomers are widely accepted to be the major toxic agents in amyloid diseases. However, it is difficult to decipher their structure or conformation. Small oligomers of different sizes are in dynamic equilibrium with each other, and are therefore not amenable to separation techniques. Single molecule approaches can study individual members of an ensemble without separating them. Here we apply a single molecule photobleaching technique, called Q-SLIP (quencher induced step length increase in photobleaching), which allows us to probe the arrangement of monomers in human Islet Amyloid Poly Peptide (hIAPP, or simply IAPP) oligomers. IAPP oligomers are the toxic species associated with Type II diabetes, and understanding the monomer-wise arrangement of membraneattached IAPP oligomers is crucial for understanding the toxic mechanism. QSLIP probes the accessibility of fluorophore labels in each monomer of individual membrane-attached IAPP oligomers. We show that the arrangement is far from uniform. When two monomers form a dimer, the Nterminus of the second monomer gets buried near the middle of the bilayer (which has a doxyl quencher at the 16’ position of the lipid chain). We study oligomers until the tetramer, and our results show that the sequential growth of the oligomers produces a structure very different from a tetrameric subunit of a mature fibril. This may explain the difference in toxicity between the oligomeric species and the fibrils.