Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells.  To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.

This document summarizes recent achievements of the RHIC spin program and their impact on our understanding of the nucleon's spin structure, i.e. the individual parton (quark and gluon) contributions to the helicity structure of the nucleon and to understand the origin of the transverse spin phenomena. Open questions are identified and a suite of future measurements with polarized beams at RHIC to address them is laid out. Machine and detector requirements and upgrades are briefly discussed.

Time and again, spin has been a key element in the exploration of fundamental physics. Spin-dependent observables have often revealed deficits in the assumed theoretical framework and have led to novel developments and concepts. Spin is exploited in many parity-violating experiments searching for physics beyond the Standard Model or studying the nature of nucleon-nucleon forces. The RHIC spin program plays a special role in this grand scheme: it uses spin to study how a complex many-body system such as the proton arises from the dynamics of QCD. Many exciting results from RHIC spin have emerged to date, most of them from RHIC running after the 2007 Long Range Plan. In this document we present highlights from the RHIC program to date and lay out the roadmap for the significant advances that are possible with future RHIC running.

A polarized ep/eA collider (Electron-Ion Collider, or EIC), with polarized proton and light-ion beams and unpolarized heavy-ion beams with a variable center-of-mass energy s∼20 to ∼ 100 GeV (upgradable to ∼ 150 GeV) and a luminosity up to ∼ 10 34 cm-2s-1, would be uniquely suited to address several outstanding questions of Quantum Chromodynamics, and thereby lead to new qualitative and quantitative information on the microscopic structure of hadrons and nuclei. During this meeting at Jefferson Lab we addressed recent theoretical and experimental developments in the spin and the three-dimensional structure of the nucleon (sea quark and gluon spatial distributions, orbital motion, polarization, and their correlations). This mini-review contains a short update on progress in these areas since the EIC White paper (A. Accardi et al., Eur. Phys. J. A 52, 268 (2016)).