The review covers the knowledge on photoremovable protecting
groups and includes all relevant chromophores studied in the
time period of 2000–2012; the most relevant earlier works are
also discussed.

/pdf/photoremovable-protecting-groups-in-chemistry-and-biology-2n33qs6p7b.pdf

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

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

Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.

Fluorescence nanoscopy in cell biology

We report broad bandwidth, 0.1–10 THz time-domain spectroscopy of linear and electro-optic polymers. The common THz optical component materials high-density polyethylene, polytetrafluoroethylene, polyimide (Kapton), and polyethylene cyclic olefin copolymer (Topas) were evaluated for broadband THz applications. Host polymers polymethyl methacrylate, polystyrene, and two types of amorphous polycarbonate were also examined for suitability as host for several important chromophores in guest-host electro-optic polymer composites for use as broadband THz emitters and sensors.

/pdf/broadband-terahertz-characterization-of-the-refractive-index-4m1y1fojz0.pdf

Broadband terahertz characterization of the refractive index and absorption of some important polymeric and organic electro-optic materials

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push−pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.

/pdf/superresolution-imaging-of-targeted-proteins-in-fixed-and-2opcl034dq.pdf

Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores

We report an induction of the blue phase III (BPIII) at a relatively low and wide (over 20 °C) temperature range in nematogenic achiral bent-core liquid crystals doped with a high twisting power chiral material. The pitch decreases with increasing chiral dopant ratio, and easily reaches the ultraviolet wavelength, so that completely dark texture is obtained under crossed polarizers. Electrooptical switching was achieved in a time range of a few to a few tens of milliseconds. We propose for the stabilization of BPIII that broad-temperature range smectic nano-clusters inhibit the long-range order of the double twisted helical structures, and also inhibit possible separation of chiral dopants from the mixture.

/pdf/stable-amorphous-blue-phase-of-bent-core-nematic-liquid-1e6qxh1gvy.pdf

Stable Amorphous Blue Phase of Bent-Core Nematic Liquid Crystals Doped with a Chiral Material

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precis...

http://pubs.acs.org/doi/pdf/10.1021/ja508028h

Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

We report the first synthesis of step-growth aromatic polyamide (PA) aerogels made using amine end-capped polyamide oligomers cross-linked with 1,3,5-benzenetricarbonyl trichloride (BTC). Isophthaloyl chloride (IPC) or terephthaloyl chloride (TPC) were combined with m-phenylenediamine (mPDA) in N-methylpyrrolidinone (NMP) to give amine-capped polyamide oligomers formulated with up to 40 repeat units. Addition of the cross-linker, BTC, typically induces gelation in under 5 min. Solvent exchange of the resulting gels into ethanol followed by supercritical CO2 drying gives colorless aerogels with densities ranging from 0.06 to 0.33 g/cm3, compressive moduli between 5 and 312 MPa, and surface areas as high as 385 m2/g. Dielectric properties were also measured in the X-band frequency range. It was found that relative dielectric constant decreased with density as seen with other aerogels with the lowest relative dielectric constant being 1.15 for aerogels with densities of 0.06 g/cm3. Because of their superior ...

Jarrod Williams

Papers

Broadband terahertz characterization of the refractive index and absorption of some important polymeric and organic electro-optic materials

Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores

Stable Amorphous Blue Phase of Bent-Core Nematic Liquid Crystals Doped with a Chiral Material

Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

Synthesis and Properties of Step-Growth Polyamide Aerogels Cross-linked with Triacid Chlorides