Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections

Application of single-molecule switching nanoscopy (SMSN) beyond the coverslip surface poses substantial challenges due to sample-induced aberrations that distort and blur single-molecule emission patterns. We combined active shaping of point spread functions and efficient adaptive optics to enable robust 3D-SMSN imaging within tissues. This development allowed us to image through 30-μm-thick brain sections to visualize and reconstruct the morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer's disease

Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action

Spectral Measurements and Super Resolution Imaging of Single Molecules for Fluoresence Photoactivation Localization Microscopy

Due to the diffraction limit of light, the resolution of fluorescence light microscopy is limited to ~250 nm. Super resolution techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM) can circumvent this limit and improve the final image resolution by roughly a factor of ten. Photoactivatable fluorescent molecules are stochastically converted from a fluorescent dark state to a bright state and imaged until they photobleach. Only a sparse subset of molecules emits light at any given time, and the cycle of emission and bleaching is repeated through time as data are simultaneously acquired. The resultant single molecules are mathematically localized in order to find their spatial positions to a much higher precision than allowed by the diffraction limit. Super resolved images are generated from the localized molecule positions; typically resulting in a final image resolution on the scale of tens of nanometers. While localization microscopy can image nanoscale cellular details, the ability to distinguish multiple fluorescent species simultaneously is invaluable in addressing a number of biological questions. Previously published multispecies schemes have divided the detected fluorescence into two distinct spectral channels, often with the ratio between channels used for species identification. However, such ratiometric methods have been limited in the number of species that can be detected simultaneously, and are unable to obtain the emission spectra of the imaged species, thus limiting their ability to distinguish multiple species. We present a localization microscopy method which detects the emission spectrum of each localized single molecule. In this scheme, a prism in the detection path spatially disperses the fluorescence signal according to the emission spectrum of each single molecule, which is recorded in one of two detection channels. The emission spectrum of each single molecule can be used for fluorescent species identification, theoretically allowing a major increase in the number of different species that can be simultaneously imaged in a sample. This technique enables an exciting new family of super-resolution imaging experiments which can report spectral emission changes due to pH, hydrophobicity, redox state, ion concentrations, temperature, or other factors, while also recording precise nanometer molecular locations

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations.

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample

Spectral-FPALM measurements of spectral wandering and identification of multiple fluorescent species.

<p>Three examples of photoactivatable fluorophores undergoing spectral wandering are shown. Individual molecules spectrally wandered before photobleaching; (a) PAmKate, from mean emission wavelength ~600 nm to ~625 nm and back again; (b) CAGE 590 from ~620 nm to ~645 nm before photobleaching, and (c) Dendra2 from ~590nm to ~620nm before photobleaching. Error bars shown in (a-c) are due to shot noise from the number of detected photons. Single and multi- color images of NIH-3T3 cells were recorded using Spectral-FPALM. Single molecules were localized and identified based on the criteria shown in Fig B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147506#pone.0147506.s001" target="_blank">S1 File</a>. Each panel represents a different sample; Dendra2-HA (d), PAmCherry-cofilin (e), and PAmKate-TfR (f) and all three labels (g). The fraction of molecules identified as each fluorescent species is displayed at the bottom right of the single color cell panels (d-f). Misidentifications of Dendra2 (d) and PAmCherry (e) are less than 5%. Misidentification of PAmKate as PAmCherry is ~12%, largely due to fewer numbers of molecules in the PAmKate sample and large fraction of spectral wanderings of PAmKate (Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147506#pone.0147506.s001" target="_blank">S1 File</a>). Scale bars are 2 μm.</p