Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.
Substances that isomerize upon photoirradiation are useful as molecular devices because their properties can be switched at will with light 1 , 2 , 3 , 4 , 5 , 6. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, exhibit stochastic switching behavior. Despite the great interest in this phenomenon in organic 7 , 8 , coordination 9 , main group 10 , organometallic 11 , 12 , and theoretical chemistry 13 , 14 , only a few applications of fluxional molecules are known 15 , We envision that the juxtaposition of photoswitching and fluxionality could bring about new functional molecules with practical applications.
Specifically, we hypothesize that such a molecule could be useful for single-molecule localization microscopy SMLM in living cells because it could ameliorate both phototoxicity and photobleaching, which are two critical limitations of this technique. Most SMLM experiments rely on photoactivation of dyes or proteins 17 , 18 , 19 , 20 , 21 , 22 , Starting from a dark isomer, photoirradiation transforms a random subset of molecules to their fluorescent state. The emission signals of these sparsely distributed fluorescent molecules can be resolved individually, enabling the localization of single molecules Fig.
This approach has limitations in live-cell imaging because repeated photoactivation with light of high energy induces phototoxicity in the specimen and exacerbates photobleaching of the fluorophores Fig. Whereas some implementations of SMLM achieve switching with a single wavelength 24 , they still rely on relatively high, and therefore toxic, irradiation intensities. Other techniques, such as points accumulation for imaging in nanoscale topography, do not require photoactivation steps 25 , but live-cell imaging of intracellular compartments remains very challenging Mechanisms of single-molecule localization.
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To overcome these difficulties, spontaneously blinking dyes were developed 23 , 27 , These fluorophores exhibit a ground-state equilibrium between a fluorescent and a dark species, providing sparse distribution of fluorescent molecules without photoactivation, which greatly decreases phototoxicity and photobleaching Fig.
Despite the great potential of spontaneously blinking dyes, their performance in terms of resolution achieved, image quality, and apparent photobleaching depends on the fraction of molecules that are fluorescent at equilibrium. This fraction is strongly determined by the pH and polarity of the medium. Although these dyes have been used successfully to image specific molecular targets for short periods of time 28 , 29 , long time-lapse imaging has only been realized in the low-polarity environment of membranes Fig.
In contrast, we argue that a combination of photoactivation and fluxionality would provide a way to control the fraction of fluorescent molecules independently from the properties of the medium. Starting from a dark, nonfluxional isomer, photoactivation would convert a fraction of the total molecules to a fluxional form Fig.
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In this population of fluxional molecules, some would exist in a dark form and others in a fluorescent form. Regardless of what the fraction of fluorescent molecules is in the fluxional equilibrium, the total fraction of fluorescent molecules could always be controlled by photoactivation. Once a population of fluxional molecules is established, their thermal equilibrium between fluorescent and dark species could be used for single-molecule imaging with very low photoxicity. Moreover, even after the whole population of fluxional molecules is photobleached, a new subset of molecules could be photoconverted to the fluxional state Fig.
Here, we report the design, synthesis, validation, and application of fluorescent molecules that become fluxional upon photoactivation. These processes are characterized by single-molecule imaging, demonstrating that the compound indeed becomes fluxional upon photoisomerization. Finally, we apply this fluorophore to study the dynamics and three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.
To create photoregulated fluxional fluorophore PFF- 1 Fig.
As a mechanistic control compound, we also synthesized probe 2 , which could be obtained from intermediate 3 Fig. We hypothesized that prior to photoactivation, PFF- 1 should exist predominantly as the E isomer of the acylhydrazone and a dark, nonfluxional, spirocyclized derivative Fig. This structure was confirmed by X-ray crystallography Supplementary Fig. Synthesis and mechanism of probe PFF- 1. We reasoned that we could use the HPL chromatograms measured at these wavelengths to determine the fraction of molecules in both the E and Z isomers that are present as the open rhodamine form.
This result indicates that formation of the fluorescent rhodamine form is preferred in the Z , but not E isomer. Based on these absorbance measurements Supplementary Fig. Because the population of the Z isomer before irradiation is only 0. These experiments confirm that even after photoactivation, the total fraction of fluorescent molecules is very small, which is a desirable feature for SMLM. These experiments support the mechanism depicted in Fig. This mechanism is further supported by density functional theory DFT modeling Fig. DFT modeling also suggests that the barrier of interconversion between the open fluorescent rhodamine and the dark spirocyclic form is energetically low 6.
To demonstrate that the Z isomer interconverts rapidly between the dark spirocyclic and fluorescent rhodamine forms i. This method has been used before to evaluate the blinking properties of photoactivatable small molecules We validated the method by employing the nonfluxional compounds 2 and rhodamine B as negative controls and a spontaneously blinking molecule HMSiR as a positive control Fig.
This experiment revealed that single molecules of PFF- 1 transitioned several times between their dark and emissive forms, confirming the fluxionality of the probe Fig. These parameters are comparable to those of commonly used SMLM dyes 32 , but unlike these previously reported compounds, PFF- 1 does not require continuous photoactivation to switch. Most spots in the color-coded image display multiple colors because single molecules emit photons at various time points.
No switching is observed, only irreversible transformation into a dark state. The chemical structure of each compound is displayed under the indicated panel. PFF- 1 displayed strong fluorescence after photoactivation in the acidic medium of lysosomes Supplementary Fig. Under identical photoactivation conditions, the fraction of fluorescent molecules of PFF- 1 is larger at low pH than in neutral solution because more molecules of PFF- 1 are protonated, which enables fluxionality.
The total fraction of fluorescent molecules can be modulated by photoactivation and, unlike the spontaneously blinking dye HMSiR 28 , PFF- 1 can be employed at either neutral or low pH, simply by tuning the photoactivation step.
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After this initial photoactivation step, cells were either imaged using only the fluxionality of PFF- 1 single photoactivation, Fig. Furthermore, we evaluated the morphology of the cells, disruption of the plasma membrane, and activation of caspase-3 as signs of phototoxicity.
Even after this very long time-lapse acquisition, the irradiated cells displayed only modest signs of phototoxicity Supplementary Fig. The green, dotted square in the bright-field image indicates the area imaged during the SMLM experiment. The green, dotted square indicates the area displayed in panel f.
The magenta, solid-line square indicates the area displayed in panel g. Under these imaging conditions, we were able to distinguish individual vesicles within diffraction-limited areas Supplementary Fig. This excellent spatiotemporal resolution allowed us to track in detail the fast motion of single lysosomes.
The example displayed in Fig. A single lysosome moving primarily along the axial direction could be tracked in this experiment Fig. Besides being useful to image and track lysosomes, probe PFF- 1 could also be used to label other cellular components at higher pH. We synthesized probe MitoPFF- 1 , which is a derivative of PFF- 1 functionalized with a mitochondria-targeting triphenylphosphonium group Although mitochondria display neutral or slightly basic pH, and therefore would decrease the fluorescent fraction of MitoPFF- 1 in its fluxional equilibrium, we were able to image mitochondria with super-resolution using this probe simply by tuning the photoactivation conditions Supplementary Fig.
Additionally, a taxol conjugate of PFF- 1 , termed TaxoPFF- 1 , was prepared to test whether PFF- 1 remains fluxional upon binding to a large macromolecular target such as microtubules. Although further optimization of the labeling strategy and image acquisition could improve this resolution, these experiments already demonstrate that photoregulated fluxional fluorophores could be used to image a variety of targets by optimizing the irradiation pulses, labeling and imaging conditions.
Having shown that probe PFF- 1 enables single-vesicle tracking, in three-dimensions, and with subsecond time resolution, we set out to image synaptic vesicles in human neurons Fig. We first confirmed that PFF- 1 targeted synaptic vesicles by co-localization analysis with FM1—43, a common stain for these vesicles Supplementary Fig. Having established that we could image synaptic vesicles with excellent spatiotemporal resolution, we analyzed their movements. Time-lapse SMLM confirmed that slow vesicles accumulate in hotspots and fast vesicles move along tracks, in agreement with previous reports Our long time-lapse experiments, however, allowed us to discover that vesicles travel along axons by hopping from one hotspot to another within a few seconds, often with long residence times in hotspots.
This behavior is exemplified by vesicle V 3 , which diffuses between hotspots H 1 and H 2 Fig. This hotspot hopping mechanism provides neurons with a constant pool of vesicles localized in defined compartments hotspots , but also with the ability to exchange vesicles rapidly between them through tracks. These experiments revealed that tracks T 1 and T 2 , Fig.
These tracks, however, have connections to these hotspots Fig. The diffusion of a vesicle between H 3 and H 4 confirmed that vesicles are able to jump out of the hotspot, join the track, move rapidly through it, and fall into the next hotspot Fig. More examples of this phenomenon are displayed in Supplementary Fig. Single-vesicle tracking in human neurons.
The green, dotted box indicates the area depicted in panel b. Diffusion coefficients were determined for two vesicles V 1 and V 2. The cyan, dotted rectangle indicates the area in which V 1 moves. Other unlabeled vesicles are also present in the top-right area of the image. The arrows indicate the direction of travel of the fast-moving vesicle. The green, dotted box indicates the area depicted in panel e. A region of interest ROI containing track T 1 and hotspots H 3 and H 4 was selected red, solid-line square to display as a time series and 4D volume in panel g.
The volumes were colored according to the bar in panel f. The unit cell is 1. The combination of photoactivation and fluxionality in the same fluorophore allowed us to control its emission in a manner that alleviates photobleaching and phototoxicity in live-cell SMLM. To the best of our knowledge, our time-lapse SMLM experiments are the longest ever reported, and even after such long acquisition times, no apparent photobleaching was observed. The performance of these dyes was not affected by the medium, allowing for super-resolved imaging of both neutral and acidic intracellular organelles, and thereby providing a generalizable mechanism for the development of small-molecule dyes for long-term SMLM.
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Despite its many advantages, PFF- 1 also has a few potential shortcomings. For example, the fraction of Z isomers must be kept low to avoid overlap of multiple emitters. Because of this low density of emitting molecules, it would be more difficult to resolve complex cellular structures that require a large number of localizations with high-temporal resolution. The development of new photoregulated fluxional fluorophores with increased brightness and blinking speed might ameliorate this situation.
Employing this dye and imaging methodology, a series of time-lapse 2D and 3D SMLM experiments in neurons revealed that synaptic vesicles move along well-defined tracks and accumulate within hotspots. Although tracks and hotspots have been identified before 37 , our long time-lapse acquisitions revealed that vesicles hop between hotspots using tracks. We also determined that vesicles in tracks move more than three orders of magnitude faster than vesicles at hotspots. Moreover, time-lapse 3D SMLM demonstrated that tracks and hotspots occupy distinct locations along the axial direction of the cell, but they fuse to one another at defined points to enable the exchange of vesicles.
These investigations confirm that the probes reported in this work are robust, generally applicable, and enable the discovery of biological mechanisms that would have been overlooked with other probes or imaging techniques. All reagents were purchased from commercial sources and used without further purification. Solvents were obtained from Sigma-Aldrich. Coupling constants are reported in Hz.
Peak assignments are based on calculated chemical shifts, multiplicity and 2D experiments. UV-visible spectra were obtained employing a Cary Scan spectrometer. Fluorescence spectra were acquired using a Micromax plate reader coupled to a Fluorolog 3 fluorimeter Horiba Jobin-Yvon. All measurements were performed in triplicate. The transmitted light and the total effective absorbed power were monitored during each acquisition. All experiments were measured in triplicate.
The time range of the peaks of interest were selected, a minimum value of each curve was subtracted from all the values within the peak serving as a baseline correction. The area under the curve and linear fit of the areas were calculated using Prism 7 GraphPad.
For small areas with irregular baseline Supplementary Fig. The slope and y -intercept obtained from this linear regression were used to estimate the concentration of photoproducts for both E and Z isomers. The total number of molecules in the ON state was calculated from the ratio of the sum of ON Z and E molecules and the total concentration 0.
A total of 29, reflections were measured 6. Final 0. The two minima were characterized by zero imaginary vibrational frequencies. The transition state was located by performing a relaxed scan of elongation of the C—N bond that forms the lactam ring steps of 0. The structure of highest energy in this relaxed potential energy surface was used as input for optimization of the transition state, which was characterized by one imaginary vibrational frequency involving the elongation of the C—N bond of interest.
Energies of stationary states were corrected by their zero-point energy. A well with DMSO only 0. Absolute IC 50 values were determined with Prism 7 GraphPad and error bars represent standard deviations. Laser powers and exposure times were kept constant within specific experiments. HeLa cells were transiently transfected with plasmids encoding for the cyan fluorescent protein mTurquoise2 fused to targeting sequences for mitochondria Addgene , ER Addgene , and Golgi Addgene The assembled product was transformed into competent E.
A polyvinylalcohol solution 0. Both preparation methods gave comparable results. HeLa cells were cultured and plated in phenol red-free growth medium Gibco. HeLa cells were plated in phenol red-free growth medium Gibco. Cells were washed with PBS 0. An in-built focus-lock system PFS was used to prevent axial drift of the sample during data acquisition.
As a second step, a controllable and aberration-free astigmatism was introduced to the point spread function PSF of single molecules, which allowed us to perform 3D SMLM based on astigmatism by determining the z positions of the fluorescent molecules based on the ellipticity of their PSF Signals were detected searching the local intensity maxima in each frame, which were fit using an integrated Gaussian PSF.
Thresholding for 2D was done as follows: a radius of 12 pixels was selected with an absolute intensity value of 0. Polyhedral Isomerizations: Microscopic Models. Polyhedral Isomerizations: Macroscopic Models. Discovery Activation Parameters of [1,3]-B Shifts.
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