Alcohol directly modulates the BK potassium channel to alter behaviors in species ranging from invertebrates to humans. In the nematode Caenorhabditis elegans, mutations that eliminate the BK channel, SLO-1, convey dramatic resistance to intoxication by ethanol. We hypothesized that certain conserved amino acids are critical for ethanol modulation, but not for basal channel function. To identify such residues, we screened C. elegans strains with different missense mutations in the SLO-1 channel. A strain with the SLO-1 missense mutation T381I in the RCK1 domain was highly resistant to intoxication. This mutation did not interfere with other BK channeldependent behaviors, suggesting that the mutant channel retained normal in vivo function. Knock-in of wild-type versions of the worm or human BK channel rescued intoxication and other BK channel-dependent behaviors in a slo-1-null mutant background. In contrast, knock-in of the worm T381I or equivalent human T352I mutant BK channel selectively rescued BK channel-dependent behaviors while conveying resistance to intoxication. Single-channel patch-clamp recordings confirmed that the human BK channel engineered with the T352I missense mutation was insensitive to activation by ethanol, but otherwise had normal conductance, potassium selectivity, and only subtle differences in voltage dependence. Together, our behavioral and electrophysiological results demonstrate that the T352I mutation selectively disrupts ethanol modulation of the BK channel. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes that are critical for behavioral responses to ethanol.

New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multicolor, live-cell, single-molecule localization microscopy (SMLM). Here we report the design, synthesis, and biological evaluation of Yale 676sb , a photostable, near-IR-emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale 676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives.

This protocol describes how to assemble a 4Pi single-molecule switching (SMS or SMLM) super-resolution microscope. Detailed instructions for beam-path alignment, testing, application to cellular samples, and troubleshooting are provided.TWEET New Protocol for setting up a 4Pi super-res microscope from @MicronOxford @GurdonInstitute @JonasRies and @bewersdorflab COVER TEASER Implementing a 4Pi super-resolution microscope ABSTRACTThe development of single-molecule switching (SMS) fluorescence microscopy (also called singlemolecule localisation microscopy (SMLM)), over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection, has demonstrated three-dimensional (3D) resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and how to analyse the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a trouble-shooting guide and present examples of anticipated results. An experienced optical instrument builder will require approximately 12 months from the start of ordering hardware components to acquiring high-quality biological images.Optical overview. The microscope presented here is centred around two opposing 100X/1.35 NA silicone oil immersion objective lenses (Olympus, UPLSAPO 100XS), OBJ0 and OBJ1 in Figure 1. Critically, these objectives must have closely matched magnification (within 0.3%) to allow for alignment of the two imaged fields of view for interference. Each objective lens is immediately followed by a quarterwave plate (Figure 1a, QWP0-1) set at 45 degrees with respect to the plane of the setup 2,4 . The quarterwave plates ensure that the emitted light is split in both beam paths into s-and p-polarisation components of equal intensity, independent of the initial polarisation orientation. Each objective's back pupil plane is imaged onto a deformable mirror (Figure 1a, lenses L1 and L3 for Objective OBJ0 and lenses L2 and L4 for OBJ1). The deformable mirrors (DMs) serve three functions: 1) introduce astigmatism to assist in emitter z-position determination, 2) correct for system aberrations

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a 3-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate and photobleaching rate of mEos3.2 sensitive to the buffer conditions by permeabilizing the yeast cell membrane.Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that some red-state mEos3.2 molecules entered an intermediate dark state that is converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to compare quantitatively conditions for imaging and counting of mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells. STATEMENT OF SIGNIFICANCEMaking quantitative measurements with single-molecule localization microscopy (SMLM) has been impeded by limited understanding of the photophysics of the fluorophores, which is very sensitive to the sample preparation and imaging conditions. We characterized the photophysics of the green-to-red photoconvertible fluorescent protein mEos3.2, which is widely used in SMLM. We combined quantitative fluorescence microscopy and mathematical modeling to measure the fluorescence signal and rate constants for photoconversion and photobleaching of mEos3.2 in live and fixed cells under a wide range of illumination intensities. Our findings

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a 3-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate and photobleaching rate of mEos3.2 sensitive to the buffer conditions by permeabilizing the cell membrane. Under some imaging conditions we tested, the time-integrated mEos3.2 signal per cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT as a reducing agent, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405nm irradiation converts some mEos3.2 molecules from the green state to an intermediate state that requires 561-nm illumination for conversion to the red fluorescent state. Our findings provide a guide to compare quantitatively and optimize conditions for imaging and counting of mEos3.2-tagged molecules. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescence proteins in cells.3