Optics Within Life Sciences 2016

Aggregation of α-Synuclein (α-Syn) into amyloid fibrils is known to be associated with the pathogenesis of Parkinson's disease (PD). Several missense mutations of the α-Syn gene have been associated with rare, early onset familial forms of PD. Despite several studies done so far, the local/residue-level structure and dynamics of α-Syn in its soluble and aggregated fibril form and how these are affected by the familial PD associated mutations are still not clearly understood. Here, we review studies performed by our group as well as other research groups, where time-resolved fluorescence spectroscopy has been used to understand the site-specific structure and dynamics of α-Syn under physiological conditions as well as under conditions that alter the aggregation properties of the protein such as low pH, high temperature, presence of membrane mimics and familial PD associated mutations. These studies have provided important insights into the critical structural properties of α-Syn that may govern its aggregation. The review also highlights time-resolved fluorescence as a promising tool to study the critical conformational transitions associated with early oligomerization of α-Syn, which are otherwise not accessible using other commonly used techniques such as thioflavin T (ThT) binding assay.

Understanding protein folding and the functional properties of intrinsically disordered proteins (IDPs) requires detailed knowledge of the forces that act in polypeptide chains. These forces determine the dimensions and dynamics of unfolded and disordered proteins and have been suggested to impact processes such as the coupled binding and folding of IDPs, or the rate of protein folding reactions. Much of the progress in understanding the physical and chemical properties of unfolded and intrinsically disordered polypeptide chains has been made possible by the recent developments in single-molecule fluorescence techniques. However, the interpretation of the experimental results requires concepts from polymer physics in order to be understood. Here, I review some of the theories used to describe the dimensions of unfolded polypeptide chains under varying solvent conditions together with their more recent application to experimental data.

Subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measurement of their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, review the relevant types of probes and labeling techniques, and highlight the advantages and disadvantages of these technologies for addressing specific inquiries.

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSD_ACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSD_ACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1  ×  10⁵ M⁻¹. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.

We report investigations, using time-resolved and polarised evanescent wave-induced fluorescence methods, into the location, orientation and mobility of a fluorescently labelled form of the antimicrobial peptide, melittin, when it interacts with vesicles and supported lipid bilayers (SLBs). This melittin analogue, termed MK14-A430, was found to penetrate the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water region when cholesterol was included. MK14-A430 formed lytic pores in SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the Cholesterol-containing SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. This study indicates that the lytic activity of MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label, where MK14-A430 displayed pore information, indicated that pore formation was best described by the toroidal pore model.

The nucleobase 5-hydroxymethylcytosine (5-hmC), a modified form of cytosine, is an important epigenetic mark related to regulation of gene expression. 5-hmC levels are highly dynamic during early development and are modulated during the progression of neurodegenerative disease and cancer. We describe a spectroscopic method for the global quantification of 5-hmC in genomic DNA. This method relies on the enzymatic glucosylation of 5-hmC, followed by a glucose oxidation step that results in the formation of aldehyde moieties that are covalently linked to a fluorescent reporter by oxime ligation. The fluorescence intensity of the labeled sample is directly proportional to its 5-hmC content. We show that this simple and cost-effective technique is suitable for quantification of 5-hmC content in different mouse tissues.

Multiphoton absorbing dye-coated trapped spherical bead at the focal plane of femtosecond optical tweezers shows nonlinear optical (NLO) phenomena. One such NLO process of two-photon fluorescence (TPF) has been used for the background-free imaging of a femtosecond laser-trapping event. Due to the high peak powers of femtosecond laser pulses with low average powers, it is possible to not only trap single nanospheres, but encourage optically directed self-assembly. The TPF signatures of trapped particles show evidence of such a directed self-assembly process which, in turn, can provide information about the structural dynamics during the process of cluster formation. We are able to trap and characterize structure and dynamics in 3D until pentamer formation from the decay characteristics of trapping at the focal plane.

The upconversion (UC) process in lanthanide-doped nanophosphors has attracted great research interest for its extensive application potential in biological in vitro and in vivo imaging due to the high tissue penetration depth of near-infrared excitation and low autofluorescence background. In this article, the authors report the synthesis of oxide nanophosphor of size  ⩽50 nm, which forms stable aqueous dispersion. The photoluminescence study is made on the nanophosphor upon 808 and 980 nm diode laser excitations. The 808 nm excitation resulted in strong emission at 795 nm due to the ⁴K_13/2  →  ⁴I_13/2 transition along with other emissions from the Nd³⁺ ion. The 980 nm excitation has resulted in it turning green in the 525–560 nm range and is assigned to the ⁴S_3/2  →  ⁴I_15/2 transition of the Er³⁺ ion, which is supposed to be present in a trace amount in the sample. The observation of strong UC emission indicates that the sample can be used for UC-based bio-imaging applications.

The advancement of high-resolution bioimaging has always been dependent on the discovery of bright and easily available fluorescent probes. Fluorescent carbon nanodots, an interesting class of relatively new nanomaterials, have emerged as a versatile alternative due to their superior optical properties, non-toxicity, cell penetrability and easy routes to synthesis. Although a plethora of reports is available on bioimaging using carbon dots, single-molecule-based super-resolution imaging is rare in the literature. In this study, we have systematically characterized the single-molecule fluorescence of three carbon dots and compared them with a standard fluorescent probe. Each of these carbon dots showed a long-lived dark state in the presence of an electron acceptor. The electron transfer mechanism was investigated in single-molecule as well as in ensemble experiments. The average on–off rate between the fluorescent bright and dark states, which is one of the important parameters for single-molecule localization-based super-resolution microscopy, was measured by changing the laser power. We report that the photon budget and on–off rate of these carbon dots were good enough to achieve single-molecule localization with a precision of ~35 nm.

Mechanical coupling between the nucleus and the cytoskeleton is indispensable for direct force transduction from the extra cellular matrix (ECM) to the chromatin. Although this physical coupling has been shown to be crucial for nuclear positioning and its function, the quantification of nuclear-cytoskeleton interaction has been lacking. In this paper, using various quantitative fluorescence spectroscopy techniques, we investigate the nature of this connection. High-resolution 3D imaging shows that nesprin2G forms short linear structures along actin stress fibers (ASFs) in the apical region of the nucleus. Fluorescence recovery after photobleaching (FRAP) revealed that the alignment of nesprin2G becomes heterogeneous when cell shape is engineered from elongated rectangular shape to square using micropatterned substrates. Further, fluorescence cross-correlation spectroscopy (FCCS) revealed that actin interacts transiently with outer nuclear membrane protein nesprin2G with a time scale of 12 ms. In addition, fluorescence resonance energy transfer (FRET) experiments show that the apical ASFs and nesprin2G are in close physical proximity. This interaction is spatially heterogeneous with high FRET along the ASFs. Lastly, we show that the disruption of actin to nuclear connection by over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) leads to an increase in nuclear height. These results not only reveal the characteristics of actin-nesprin2G interaction and its significance in regulating nuclear morphology, but also validate the utility of quantitative fluorescence techniques in deciphering physical connections that are essential for mechanotransduction.

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels.

Lumichrome (Lc), a molecule consisting of a trinuclear alloxazine moiety is our present subject of interest. This molecule is subjected to tautomerization in the presence of pyridine, acetic acid, etc, through the formation of an eight-membered ring. In our present contribution, we have attempted to analyze the influence of the presence of an aliphatic amine, triethylamine (TEA) and an aromatic amine, N,N-dimethylaniline (DMA) in the double proton transfer step of the tautomerization as well as the photo-induced electron transfer (PET) from those amines to Lc. We have studied these phenomena within micelles, anionic and neutral, to observe the effect of confinement. Through our experiments, it could be stated that along with tautomerization and proton transfer, there is also evidence of PET in triplet excited state.