A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants

The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa. We rationalize our results in terms of a higher conformational freedom of the chromophore in the protein environment provided by the beta-can. This thorough understanding of the photophysical parameters has allowed us to optimize the acquisition parameters for camera-based sub-diffraction-limit imaging with these photochromic proteins. We show that Dronpa and its mutants are useful for fast photoactivation-localization microscopy (PALM) using common wide-field microscopy equipment, as individual fluorescent proteins can be localized several times. We provide a new approach to achieve fast PALM by introducing simultaneous two-color stroboscopic illumination.     

Second-harmonic generation in GFP-like proteins

The second-order nonlinear optical properties of green fluorescent proteins (GFPs), such as the photoswitchable Dronpa and enhanced GFP (EGFP), have been studied at both the theoretical and experimental levels. In the case of Dronpa, both approaches are consistent in showing the rather counterintuitive result of a larger second-order nonlinear polarizability (or first hyperpolarizability, beta) for the protonated state, which has a higher transition energy, than for the deprotonated, fluorescent state with its absorption at lower energy. Moreover, the value of beta for the protonated form of Dronpa is among the highest reported for proteins. In addition to the pH dependence, we have found a wavelength dependence in the beta values. These properties are essential for the practical use of Dronpa or other GFP-like fluorescent proteins as second-order nonlinear fluorophores for symmetry-sensitive nonlinear microscopy imaging and as nonlinear optical sensors for electrophysiological processes. An accurate value of the first hyperpolarizability is also essential for any qualitative analysis of the nonlinear images.     

Salicylideneaniline is a Photoswitchable Ferroelectric Crystal

Since the discovery of the first ferroelectric Rochelle salt, most ferroelectrics have been investigated showing thermally triggered symmetry-breaking phase transition. Although photochromism arising from geometrical isomerization was reported as early as 1867, such photoswitchable ferroelectric crystals have scarcely been developed to date. Herein, we report that salicylideneaniline is a photochromic ferroelectric crystal. Upon photoirradiation, the dielectric constant shows obvious switching between high and low dielectric states, and more importantly, the ferroelectric polarization demonstrates quick and reversible switching. This work opens the gate to developing photoswitchable ferroelectrics, which holds great potential for applications in optically controlled smart devices.     

Improved resolution in single-molecule localization microscopy using QD-PAINT

Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.Subject terms: Fluorescence imaging, Quantum dots, Oligonucleotide probes, Fluorescent dyes, Super-resolution microscopy     

Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.     

Photoswitchable Fluorescent Nanoparticles: Preparation,Properties and Applications

This minireview highlights recent advances of research dedicated to photoswitchable fluorescent nanoparticles and their applications. Recently, several strategies have been developed to synthesize nanoparticles with optically switchable emission properties: either fluorescence on/off or dual‐alternating‐color fluorescence photoswitching. The underlying mechanisms of fluorescence photoswitching enable many different types of photoswitchable fluorescent nanoparticles to change fluorescence colors, thus validating the basis of the initial photoswitching design. Among all possible applications, the usage of photoswitchable fluorescent nanoparticles to empower super‐resolution fluorescence imaging and to label biological targets was subsequently reviewed. Finally, we summarize the important areas regarding future research and development on photoswitchable fluorescent nanoparticles.     

Single‐Molecule Observation of the Photoregulated Conformational Dynamics of DNA Origami Nanoscissors

We demonstrate direct observation of the dynamic opening and closing behavior of photocontrollable DNA origami nanoscissors using high‐speed atomic force microscopy (HS‐AFM). First the conformational change between the open and closed state controlled by adjustment of surrounding salt concentration could be directly observed during AFM scanning. Then light‐responsive moieties were incorporated into the nanoscissors to control these structural changes by photoirradiation. Using photoswitchable DNA strands, we created a photoresponsive nanoscissors variant and were able to distinguish between the open and closed conformations after respective irradiation with ultraviolet (UV) and visible (Vis) light by gel electrophoresis and AFM imaging. Additionally, these reversible changes in shape during photoirradiation were directly visualized using HS‐AFM. Moreover, four photoswitchable nanoscissors were assembled into a scissor–actuator‐like higher‐order object, the configuration of which could be controlled by the open and closed switching induced by irradiation with UV and Vis light.     

Control of Imine Exchange Kinetics with Photoswitches to Modulate Self‐Healing in Polysiloxane Networks by Light Illumination

Various aldehyde‐containing photoswitches have been developed whose reactivity toward amines can be controlled externally. A thermally stable bifunctional diarylethene, which in its ring‐closed form exhibits imine formation accelerated by one order of magnitude, was used as a photoswitchable crosslinker and mixed with a commercially available amino‐functionalized polysiloxane to yield a rubbery material with viscoelastic and self‐healing properties that can be reversibly tuned by irradiation.     

Hydrogen Bond Fluctuations Control Photochromism in a Reversibly Photo‐Switchable Fluorescent Protein

Reversibly switchable fluorescent proteins (RSFPs) are essential for high‐resolution microscopy of biological samples, but the reason why these proteins are photochromic is still poorly understood. To address this problem, we performed molecular dynamics simulations of the fast switching Met159Thr mutant of the RSFP Dronpa. Our simulations revealed a ground state structural heterogeneity in the chromophore pocket that consists of three populations with one, two, or three hydrogen bonds to the phenolate moiety of the chromophore. By means of non‐adiabatic quantum mechanics/molecular dynamics simulations, we demonstrated that the subpopulation with a single hydrogen bond is responsible for off‐switching through photo‐isomerization of the chromophore, whereas two or more hydrogen bonds inhibit the isomerization and promote fluorescence instead. While rational design of new RSFPs has so far focused on structure alone, our results suggest that structural heterogeneity must be considered as well.     

Photoswitchable nanoparticles enable high-resolution cell imaging: PULSAR microscopy

Beyond-diffraction-limit optical imaging of cells will reveal biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties of spiropyran fluorophores, we achieved nanoresolution fluorescence imaging using photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy. The PULSAR microscope successfully resolved nanostructures and subcellular organelles when the photoswitchable nanoparticles containing spiropyran dyes were used as fluorescent probes.     

Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

The in situ imaging of soft matter is of paramount importance for a detailed understanding of functionality on the nanoscopic scale. Although super‐resolution fluorescence microscopy methods with their unprecedented imaging capabilities have revolutionized research in the life sciences, this potential has been far less exploited in materials science. One of the main obstacles for a more universal application of super‐resolved fluorescence microscopy methods is the limitation of readily available suitable dyes to overcome the diffraction limit. Here, we report a novel diarylethene‐based photoswitch with a highly fluorescent closed and a nonfluorescent open form. Its photophysical properties, switching behavior, and high photostability make the dye an ideal candidate for photoactivation localization microscopy (PALM). It is capable of resolving apolar structures with an accuracy far beyond the diffraction limit of optical light in cylindrical micelles formed by amphiphilic block copolymers.     

Fast,very fast,and ultra-fast gas chromatography-mass spectrometry of thermally labile steroids,carbamates, and drugs in supersonic molecular beams

Gas chromatography-mass spectrometry (GC-MS) analyses of thermally labile compounds have been studied by using a short column fast gas chromatograph, coupled with fly-through electron ionization in supersonic molecular beams. Thirty-two compounds, which include steroids, carbamate pesticides, antibiotic drugs, and other pharmaceutical compounds, have been analyzed and the details of their GC-MS analysis are provided. The ability to analyze thermally labile compounds is discussed in relation to the speed of analysis. A new term, “speed enhancement factor” (SEF), is defined as the product of column length reduction and the carrier gas linear velocity increase, as compared with normal GC-MS conditions. Fast, very fast, and ultra-fast GC-MS are defined with a SEF in the ranges of 5–30, 30–400, and 400–4000, respectively. Trade-offs in the degree of dissociation, speed, gas chromatograph resolution, and sensitivity were studied and examined with thermally labile molecules. The experimental factors that affect the dissociation are described with emphasis on its reduction. We claim that the use of supersonic molecular beams for sampling and ionization provides the ultimate capability in the GC-MS of thermally labile compounds. The obtained 70-eV electron ionization mass spectra are shown, and an enhanced relative abundance of the molecular ion is demonstrated together with library search capability of these mass spectra, which is better than that reported with particle beam liquid chromatography-mass spectrometry. The performance of fast GC-MS in supersonic molecular beams is compared with other methods of fast GC-MS and with particle beam liquid chromatography-mass spectrometry.     

亲水性二芳基乙烯荧光分子开关的研究进展

二芳基乙烯荧光分子开关因具有优良的抗疲劳性和双稳态特征而被广泛地研究与应用,亲水化成为其作为荧光开关探针走向应用的关键点之一。本文综述了亲水性二芳基乙烯荧光分子开关当前的研究进展,归纳了实现亲水性的几种重要途径和结构,分析了各种亲水化方法的优缺点,并着重介绍了亲水性二芳基乙烯荧光分子开关作为荧光开关探针在化学传感、生物传感、生物成像以及超分辨成像等领域的应用现状,并指出当前应用研究中存在的一些问题,同时也对其未来的应用前景进行了展望。     

Conjugated polymer nanoparticles as fluorescence switch for selective cell imaging

Fluorescence switch plays a vital role in bioelectronics and bioimaging.Herein,we presented a new kind of facile electrostatic complex nanoparticles(ECNs)for fluorescence switching in cells and marking of individual cell.The ECNs were prepared by mixing positively charged poly(6-(2-(thiophen-3-yl)ethoxy)hexyl trimethylammonium bromide)(PT)and negatively charged diarylethene sodium salt(DAECOONa).DAE-COONa is a photoswitchable molecule which can be transformed between the ring-closed fo rm and ring-open form under the irradiation of UV or visible light.The closed-form of DAE-COONa can efficie ntly quench the fluorescence of PT through intermolecular energy transfer,while the open form of DAE-COONa does not influence the emission of PT.Thus,the fluorescence of ECNs can be modulated by light irradiation,and the ECNs with good fluorescence switching performance have been employed for fluorescence imaging and individual cell lighting up process successfully.We demonstrate that the electrostatic complex strategy provides a facile method to construct fluorescence switch fo r selective cell marking and imaging applications.     

Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

Subdiffraction‐Resolution Fluorescence Microscopy of Myosin–Actin Motility

Subdiffraction‐resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of ～20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video‐like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore‐labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of ～100 Hz, only 100 consecutive frames are sufficient to generate a single high‐resolution image of moving actin filaments with a lateral resolution of ～30 nm. A video‐like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to ～0.18 μm s^?1, observed strong bending and disruption of filaments as well as locally immobile fragments.     

Color-Change Photoswitching of an Alkynylpyrene Excimer Dye

We describe a photoswitchable DNA-based dimeric dye that visibly changes fluorescence from green to blue upon UV irradiation. A novel bis-alkyne-dependent [2+2+2] cycloaddition is proposed as a mechanism for the color change in air. The photoinduced structural switching results in spatial separation of stacked pyrene units, thereby causing selective loss of the excimer emission. We demonstrate and suggest several applications for this novel photoswitch.     

A new SIMS paradigm for 2D and 3D molecular imaging of bio-systems

With the implementation of focused primary ion beams, secondary ion mass spectrometry (SIMS) has become a significant technique in the rapidly emerging field of mass spectral imaging in the biological sciences. Liquid metal ion guns (LMIG) offered the prospect of sub-100 nm spatial resolution, however this aspiration has yet to be reached for molecular imaging. This brief review shows that using LMIG the limitations of the static limit and low ionization probability will restrict useful imaging to around 2 μm spatial resolution with high-yield molecules. The only prospect of going beyond this in the absence of factors of 100 increase in ionization probability is to use polyatomic ion beams such as C₆₀⁺, for which bombardment induced damage is low. In these cases sub-micron imaging becomes possible, using voxels together with molecular depth profiling and 3D imaging. The discussion shows that conventional ToF-SIMS instrumentation then becomes a limitation in that the pulsed ion beam has a very low duty cycle which results in inordinately long analysis times, and pulsing the beam means that high-mass resolution and high spatial resolution are mutually incompatible. New instrumental configurations are described that allow the use of a dc ion beam and separate the mass spectrometry for the ion formation process. Early results from these instruments suggest that sub-micron analysis and imaging with high mass resolution and good ion yields are now realizable, although the low ion yield issue still needs to be solved.     

Photoswitchable fluorescent dyads incorporating BODIPY and [1,3]oxazine components

We designed and synthesized three compounds incorporating a BODIPY fluorophore and an oxazine photochrome within the same molecular skeleton and differing in the nature of the linker bridging the two functional components. The [1,3]oxazine ring of the photochrome opens in less than 6 ns upon laser excitation in two of the three fluorophore-photochrome dyads. This process generates a 3H-indolium cation with a quantum yield of 0.02-0.05. The photogenerated isomer has a lifetime of 1-3 μs and reverts to the original species with first-order kinetics. Both photochromic systems tolerate hundreds of switching cycles with no sign of degradation. The visible excitation of the dyads is accompanied by the characteristic fluorescence of the BODIPY component. However, the cationic fragment of their photogenerated isomers can accept an electron or energy from the excited fluorophore. As a result, the photoinduced transformation of the photochromic component within each dyad results in the effective quenching of the BODIPY emission. Indeed, the fluorescence of these photoswitchable compounds can be modulated on a microsecond time scale with excellent fatigue resistance under optical control. Thus, our operating principles and choice of functional components can ultimately lead to the development of valuable photoswitchable fluorescent probes for the super-resolution imaging of biological samples.     

Circular dichroism probed by two-photon fluorescence microscopy in enantiopure chiral polyfluorene thin films

Two-photon fluorescence scanning confocal microscopy sensitive to circular dichroism with a diffraction-limited resolution well below 500 nm is demonstrated. With this method, the spatial variation of the circular dichroism of thermally annealed chiral polyfluorene thin films has been imaged. We observed circular dichroism associated with submicrometer-sized domains showing helicoidally twisted macromolecular organization. Domains with opposite chiroptical properties, corresponding to left- or right-handed molecular organization, coexist in the film. Our results are consistent with those obtained by one-photon imaging and illustrate the potential of two-photon imaging for use in studying helical macromolecular organization.