Single-Molecule Optical Imaging with Improved Spatial Resolution and Chiral Fingerprinting for Disease Biophysics

Abstract

Optical light microscopy has a limitation in resolving finer details below the diffraction limit. This diffraction limit is explained by Abbe and Rayleigh, and it depends on the Numerical Aperture and wavelength of collected light, which is close to (λ/2NA). This diffraction limit can be surpassed by super-resolution microscopy techniques such as STED, SIM,(f)PALM, STORM and dnaPAINT. The STED and SIM surpass the diffraction limit by modifying the illumination light. Whereas the techniques (f)PALM, STORM and dnaPAINT use the temporal emission of fluorescence signals from a single molecule to surpass the diffraction limit, collectively they are called Single Molecule Localization Microscopy. The first aspect of the work presents a technique to enhance localization precision by identifying molecules that emit photons over an extended temporal window(fortunate molecules). Two SMLM techniques leSMLM and corrSMLM are developed to enhance localization precision and spatial resolution. The leSMLM technique involves the detection of fortunate molecules using a long exposure time to generate enough statistics for high-quality reconstruction of the target protein distribution in a cellular system. Studies show a significant PArticle Resolution Shift (PAR-shift) of about 6 nm and 11 nm towards Single-molecule-limit (away from diffraction-limit) for an exposure time window of 60 ms and 90 ms, respectively. In addition, a significant decrease in the fraction of fortunate molecules (single molecules with small localization precision) is observed. Specifically, 8.33% and 3.43% molecules are found to emit in 30−60 ms and 60−90 ms, respectively, when compared to SMLM. The long exposure has enabled better visualization of the Dendra2HA molecular cluster, resolving sub-clusters within a large cluster. The corrSMLM technique identifies fortunate molecules using a single-molecule PSF that appears in consecutive frames via PSF correlation, followed by data integration to determine their position and effective localization precision. The technique addresses two significant problems that plague existing SMLM : (1) false detection due to random noise that contributes to a strong background and (2) poor localization leading to overall low resolution. To demonstrate, corrSMLM is used for imaging fixed NIH3T3 cells (transfected with Dendra2-Actin, Dendra2-Tubulin, and mEos-Tom20 plasmid DNA). Quantitatively, the technique achieved more than 1.5-fold improvement in signal-to-background ratio and nearly 2-fold enhancement in localization precision. Intensity analysis based on the number of molecules suggests that corrSMLM better corroborates the raw data and preserves finer features (e.g., edges), which are wiped out in standard SMLM. Overall, an improvement in the localization precision and spatial resolution is noted. The method does not require major hardware modifications and can be implemented computationally on standard SMLM systems. The second work presents a new SMLM technique (chiralSMLM) to explore singlemolecule chirality. This involves the detection of right and left circularly polarized fluorescence from single molecules. The detection of left and right ircularly polarized light is done with the help of a chiral detection module. The chiral detection module consists of a Quarter Wave Plate (QWP) and a Polarization Beam Splitter (PBS). The QWP turns the circularly polarized light into linearly polarized light, which is further split by PBS. The chiral dissymmetry factor for each molecule is calculated using the intensity information from the polarized light. The system is calibrated with fluorescent beads and a known polarized light source. Further, the system is tested with real specimens. We examined three samples, such as Dendra2-Actin, Dendra2-HA and Dendra2-NS3 protein, in NIH3T3 cells. The results show that chiral dissymmetry factor histogram spreads are relatively higher for the samples Dendra2-Actin, Dendra2-HA and Dendra2-NS3 compared with Dendra2 alone. This is possibly because of the conjugated protein along with Dendra2. Also, the chirality-based cluster analysis shows LCP active molecules participate more in clustering compared with RCP active molecules. The final and third part elaborates on the use of SMLM to understand the role of viral proteins in infected/transfected cells. We studied the distribution of NS2B and NS3 viral proteins from Dengue virus type 2. In this study, the photoactivatable probes Dendra2NS2B and Dendra2-NS3 are prepared using conventional cloning methods. Furthermore, the plasmid is tested using PCR amplification and restriction digestion. Additionally, confocal images of transfected cells confirm protein expression in the cellular system. The confocal study reveals the formation of NS2B clusters on the Endoplasmic Reticulum. The NS2B cluster properties, such as the number of molecules in a cluster, cluster area and cluster density, are measured using a lab-made SMLM microscope. where a total of ∼ 41 NS2B aggregates are noted with an area-spread of 0.055±0.008μm2, the molecular density of 43581170 mol/μm2, and an average of 174±38 NS2B molecules per cluster. Moreover, the super-resolved volume image revealed NS2B clusters spanning several planes with a few extending up to 5 planes (∼ 2.5 μm from the coverslip). In addition, the collective dynamics of NS2B proteins leading to the formation of clusters are evident from timelapse super-resolved data, which provides conclusive evidence of NS2B accumulation 24 hours post-transfection. In the case of the NS3 protein, the confocal study reveals the formation of clusters on the mitochondrial network. The cluster parameters are estimated using a DBSCAN-based clustering method, which reveals that, on average, NS3 forms 785±105 molecules per cluster with an area of 0.2864±0.11μm2, and the average density is 2520±815 mol/μm2. Further, the multicolour SMLM shows the super-resolved images of NS3 clusters along with the mitochondrial network. In the future, organelle-specific drug targeting may help reduce the rate of dengue infection by disrupting the accumulation of NS2B and NS3.