30 research outputs found
Multi-Color Ultra-High Resolution Imaging
Fluorescence microscopy, which allows multiple-color imaging, plays an important role in observing structures inside cells with high specificity. The advent of super-resolution fluorescence microscopy, or nanoscopy techniques such as single-molecule switching nanoscopy (SMSN), has extended the application range of fluorescence microscopy beyond the diffraction limit, achieving up to 10-fold improvement in spatial resolution. At the same time, the recent development of expansion microscopy (ExM) allows samples to be physically expanded by 4-fold in the lateral dimensions providing another independent method to resolve structures beyond the diffraction limit. When combined, ExM-SMSN makes it possible to achieve another significant leap in resolution of light microscopy. However, the need for specialized protein labels prevents the efficient combination of these two techniques, especially for multiple-color imaging. Here, we demonstrate our work in progress to effectively combine these two super-resolution techniques to provide another 2-4-fold improvement from the current achievable resolution for multi-color imaging. The developed technique will further push the boundary of achievable resolution of fluorescence microscopy and pave the way towards resolving protein-specific ultra-structures in the cellular context
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
Quantitative Shape Analysis of Giant Unilamellar Vesicles as a Function of Cholesterol Content
Viral infection, bacterial infection, and certain diseases such as Alzheimer\u27s and Parkinson\u27s are known to involve specific cell membrane domains referred to as rafts. Rafts, which also play a role in cellular signaling, endocytosis, and membrane trafficking, are sub-micrometer sized domains of the cell membrane enriched in saturated lipids and cholesterol. In this experiment, unsaturated and saturated lipids, cholesterol, and minute amounts (total \u3c 0.5 mole%) of two different fluorescent dyes are used to create model cell membranes called Giant Unilamellar Vesicles (GUVs). These GUVs separate into two distinct phases, a liquid disordered (La) domain enriched in unsaturated lipids and a liquid ordered (L0) domain, analogous to the raft phase, enriched in saturated lipids and cholesterol. Equatorial cross sections of GUVs are imaged with confocal fluorescence microscopy and analyzed with custom programs. Three different GUV compositions were examined at various cholesterol concentrations, with equi-molar unsaturated lipid, dioleoylphosphatidylcholine (DOPC), and saturated lipid, egg sphingomyelin (ESM). Computer programs analyze the distribution of fluorescent dye intensity, the area fraction, and distribution of meridional curvature in each phase of the GUVs and line tension values at the phase boundary between the Ld and L0 domains. The results indicate that GUVs with higher concentrations of cholesterol feature significant negative curvature due to similar bending rigidities between the two phases, a large area fraction of L0 phase, and a wide distribution of line tension values in the range of 10 13N. The GUVs with the lowest cholesterol concentration contain very little negative curvature especially in the L0 phase, significantly less overall L0 area, and much lower fluorescent probe partitioning in the L0 phase. These results give some insight into the structure and shape of interior cell organelles such as the Golgi and the possible effects on rafts in live cells at various cholesterol concentrations
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
Probing Spatial Organization of mRNA in Bacterial Cells using 3D Super-Resolution Microscopy
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