Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2.

Single-molecule localization microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass-transition temperature hampers efficient cryo-photoswitching. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at ∼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of two off-states in cis conformation with blue-shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these off-states can be switched back to the fluorescent on-state by 405 nm light, while both of them are sensitive to UV light at 355 nm. Superior recovery to the fluorescent on-state by 355 nm light was confirmed at the single-molecule level. This suggests, as also shown by simulations, that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly other FPs could improve the effective labeling efficiency achievable with this technique. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins.

Structural myelin defects are associated with low axonal ATP levels but rapid recovery from energy deprivation in a mouse model of spastic paraplegia.

In several neurodegenerative disorders, axonal pathology may originate from impaired oligodendrocyte-to-axon support of energy substrates. We previously established transgenic mice that allow measuring axonal ATP levels in electrically active optic nerves. Here, we utilize this technique to explore axonal ATP dynamics in the Plpnull/y mouse model of spastic paraplegia. Optic nerves from Plpnull/y mice exhibited lower and more variable basal axonal ATP levels and reduced compound action potential (CAP) amplitudes, providing a missing link between axonal pathology and a role of oligodendrocytes in brain energy metabolism. Surprisingly, when Plpnull/y optic nerves are challenged with transient glucose deprivation, both ATP levels and CAP decline slower, but recover faster upon reperfusion of glucose. Structurally, myelin sheaths display an increased frequency of cytosolic channels comprising glucose and monocarboxylate transporters, possibly facilitating accessibility of energy substrates to the axon. These data imply that complex metabolic alterations of the axon-myelin unit contribute to the phenotype of Plpnull/y mice.

Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos.

The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.

Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis.

A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.

Modeling charge separation in charged nanochannels for single-molecule electrometry.

We model the transport of electrically charged solute molecules by a laminar flow within a nanoslit microfluidic channel with electrostatic surface potential. We derive the governing convection-diffusion equation, solve it numerically, and compare it with a Taylor-Aris-like approximation, which gives excellent results for small Péclet numbers. We discuss our results in light of designing an assay that can measure simultaneously the hydrodynamic size and electric charge of single molecules by tracking their motion in such nanoslit channels with electrostatic surface potential.

Rapid nonlinear image scanning microscopy.

Image scanning microscopy (ISM) doubles the resolution of a conventional confocal microscope for super-resolution imaging. Here, we describe an all-optical ISM design based on rescanning microscopy for two-photon-excited fluorescence and second-harmonic generation that allows straightforward implementation into existing microscopes. The design offers improved sensitivity and high frame rates relative to those of existing systems. We demonstrate its utility using fixed and living specimens as well as collagen hydrogels.

Wide-Field Fluorescence Lifetime Imaging of Single Molecules.

Fluorescence lifetime imaging (FLIM) has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Förster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy (CLSM) and time-correlated single-photon counting (TCSPC) and the other based on wide-field microscopy and phase fluorometry. Although the first approach (CLSM-TCSPC) assures high sensitivity and allows one to detect single molecules, it is slow and has a small photon yield. The second allows, in principal, high frame rates (by 2-3 orders of magnitude faster than CLSM), but it suffers from low sensitivity, which precludes its application for single-molecule imaging. Here, we demonstrate that a novel wide-field TCSPC camera (LINCam25, Photonscore GmbH) can be successfully used for single-molecule FLIM, although its quantum yield of detection in the red spectral region is only ∼5%. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. We performed single-molecule FLIM of different red fluorophores, and we use the lifetime information for successfully distinguishing between different molecular species. Finally, we demonstrate single-molecule metal-induced energy transfer (MIET) imaging which is a first step for three-dimensional single-molecule localization microscopy (SMLM) with nanometer resolution.

Excitation and Emission Transition Dipoles of Type-II Semiconductor Nanorods.

The mechanisms of exciton generation and recombination in semiconductor nanocrystals are crucial to the understanding of their photophysics and for their application in nearly all fields. While many studies have been focused on type-I heterojunction nanocrystals, the photophysics of type-II nanorods, where the hole is located in the core and the electron is located in the shell of the nanorod, remain largely unexplored. In this work, by scanning single nanorods through the focal spot of radially and azimuthally polarized laser beams and by comparing the measured excitation patterns with a theoretical model, we determine the dimensionality of the excitation transition dipole of single type-II nanorods. Additionally, by recording defocused patterns of the emission of the same particles, we measure their emission transition dipoles. The combination of these techniques allows us to unambiguously deduce the dimensionality and orientation of both excitation and emission transition dipoles of single type-II semiconductor nanorods. The results show that in contrast to previously studied quantum emitters, the particles possess a 3D degenerate excitation and a fixed linear emission transition dipole.

Multi-target spectrally resolved fluorescence lifetime imaging microscopy.

We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

Fluorescence lifetime correlation spectroscopy: Basics and applications.

This chapter presents a concise introduction into the method of Fluorescence Lifetime Correlation Spectroscopy (FLCS). This is an extension of Fluorescence Correlation Spectroscopy (FCS) that analyses fluorescence intensity fluctuations from small detection volumes in samples of ultra-low concentration. FCS has been widely used for investigating diffusion, conformational changes, molecular binding/unbinding equilibria, or chemical reaction kinetics, at single molecule sensitivity. In FCS, this is done by calculating intensity correlation curves for the measured intensity fluctuations. FLCS extends this idea by calculating fluorescence-lifetime specific intensity correlation curves. Thus, FLCS is the method of choice for all studies where a parameter of interest (conformational state, spatial position, molecular environmental condition) is connected with a change in the fluorescence lifetime. After presenting the theoretical and experimental basis of FLCS, the chapter gives an overview of its various applications.

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

Photon Yield Enhancement of Red Fluorophores at Cryogenic Temperatures.

Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 ⋅ 106 to 11 ⋅ 106 photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

Three-dimensional single-molecule localization with nanometer accuracy using Metal-Induced Energy Transfer (MIET) imaging.

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films.

Cell-Substrate Dynamics of the Epithelial-to-Mesenchymal Transition.

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.

Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution.

The cell is metabolically highly compartmentalized. Especially, mitochondria host many vital reactions in their different microcompartments. However, due to their small size, these microcompartments are not accessible by conventional microscopy. Here, we demonstrate that time-correlated single-photon counting (TCSPC) fluorescence lifetime-imaging microscopy (FLIM) classifies not only mitochondria, but different microcompartments inside mitochondria. Sensor proteins in the matrix had a different lifetime than probes at membrane proteins. Localization in the outer and inner mitochondrial membrane could be distinguished by significant differences in the lifetime. The method was sensitive enough to monitor shifts in protein location within mitochondrial microcompartments. Macromolecular crowding induced by changes in the protein content significantly affected the lifetime, while oxidizing conditions or physiological pH changes had only marginal effects. We suggest that FLIM is a versatile and completive method to monitor spatiotemporal events in mitochondria. The sensitivity in the time domain allows for gaining substantial information about sub-mitochondrial localization overcoming diffraction limitation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Photoactivation of Luminescent Centers in Single SiO2 Nanoparticles.

Photobleaching of fluorophores is one of the key problems in fluorescence microscopy. Overcoming the limitation of the maximum number of photons, which can be detected from a single emitter, would allow one to enhance the signal-to-noise ratio and thus the temporal and spatial resolution in fluorescence imaging. It would be a breakthrough for many applications of fluorescence spectroscopy, which are unachievable up to now. So far, the only approach for diminishing the effect of photobleaching has been to enhance the photostability of an emitter. Here, we present a fundamentally new solution for increasing the number of photons emitted by a fluorophore. We show that, by exposing a single SiO2 nanoparticle to UV illumination, one can create new luminescent centers within this particle. By analogy with nanodiamonds, SiO2 nanoparticles can possess luminescent defects in their regular SiO2 structure. However, due to the much weaker chemical bonds, it is possible to generate new defects in SiO2 nanostructures using UV light. This allows for the reactivation of the nanoparticle's fluorescence after its photobleaching.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Dead-time correction of fluorescence lifetime measurements and fluorescence lifetime imaging.

We present a comprehensive theory of dead-time effects on Time-Correlated Single Photon Counting (TCSPC) as used for fluorescence lifetime measurements, and develop a correction algorithm to remove these artifacts. We apply this algorithm to fluorescence lifetime measurements as well as to Fluorescence Lifetime Imaging Microscopy (FLIM), where rapid data acquisition is necessarily connected with high count rates. There, dead-time effects cannot be neglected, and lead to distortions in the observed lifetime image. The algorithm is quite general and completely independent of the particular nature of the measured signal. It can also be applied to any other single-event counting measurement with detector and/or electronics dead-time.

MD simulations and FRET reveal an environment-sensitive conformational plasticity of importin-ß.

The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-ß in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Förster resonance energy transfer experiments using dual-fluorophore-labeled Importin-ß. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.