When a molecule absorbs a photon of appropriate energy, a chain of photophysical events ensues, such as internal conversion or vibrational relaxation (loss of energy in the absence of light emission), fluorescence, intersystem crossing (from singlet state to a triplet state) and phosphorescence, as shown in the Jablonski diagram for organic molecules (Fig. 1). Each of the processes occurs with a certain probability, characterized by decay rate constants (k). It can be shown that the average length of time τ for the set of molecules to decay from one state to another is reciprocally proportional to the rate of decay: τ = 1/k. This average length of time is called the mean lifetime, or simply lifetime. It can also be shown that the lifetime of a photophysical process is the time required by a population of N electronically excited molecules to be reduced by a factor of e. Correspondingly, the fluorescence lifetime is the time required by a population of excited fluorophores to decrease exponentially to N/e via the loss of energy through fluorescence and other non-radiative processes. The lifetime of photophycal processes vary significantly from tens of femotoseconds for internal conversion1,2 to nanoseconds for fluorescence and microseconds or seconds for phosphorescence.1






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Figure 1


Jablonski diagram and a timescale of photophysical processes for organic molecules.

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Fluorescence Lifetime Measurements and Biological Imaging

Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

/pdf/super-resolution-fluorescence-microscopy-v84c1ojckz.pdf

Super-Resolution Fluorescence Microscopy

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

/pdf/a-guide-to-super-resolution-fluorescence-microscopy-28ekkdaf4n.pdf

A guide to super-resolution fluorescence microscopy

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.

/pdf/evaluation-of-fluorophores-for-optimal-performance-in-2kfvou9h34.pdf

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

/pdf/a-general-method-to-improve-fluorophores-for-live-cell-and-56mvzhhh2n.pdf

A general method to improve fluorophores for live-cell and single-molecule microscopy

Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes†

A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes

We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2–10 mM) of reducing, thiol-containing compounds such as s-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ∼20 nm. The method requires wide-field illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

/pdf/photoswitching-microscopy-with-standard-fluorophores-37b9n78wtu.pdf

Photoswitching microscopy with standard fluorophores

In recent years, fluorescencemicroscopy techniques have been invented that are no longer fundamentally limited by diffraction despite using visible light focused by conventional optical elements. Contrary to earlier attempts to improve the spatial resolution, such as near-field optics and aperture filters, all far-field fluorescence ‘‘nanoscopy’’ methods known to date rely on a judicious exploitation of selected fluorophore properties. In particular, all are based on utilizing a molecular mechanism that renders the fluorophores incapable of responding with fluorescence emission to excitation light. This fluorescence inhibition mechanism is implemented in the image formation in such away that fluorophores that are closer than the diffraction limit emit sequentially in time and hence can be discerned. In stimulated emission depletion (STED) microscopy, fluorescence is inhibited by subjecting the dye molecules to an additional beam of light, thus inducing stimulated emission from the fluorescent state S1 to the ground state S0. The

Single-molecule STED microscopy with photostable organic fluorophores.

Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, ie, chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface In addition, our study shows that the in situ construction of wire molecules, ie, the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of approximately 14 nm Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes

Robert Kasper

Papers

Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes†

A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes

Photoswitching microscopy with standard fluorophores

Single-molecule STED microscopy with photostable organic fluorophores.

Dissecting and Reducing the Heterogeneity of Excited-State Energy Transport in DNA-Based Photonic Wires