BioFiles Volume 6, Number 3 — Superior Fluorescent Products

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BioFiles Volume 6 Number 3 - Superior Fluorescent ProductsTable of Contents

 


Introduction

Monika Bäumle
Monika Bäumle

Fluorescent techniques are widespread and fast-growing analytical methods used in life science. They allow sensitive and selective investigation of biological processes, diagnostic screening, kinetics, and conformational studies. Research is evolving from identification of a large number of target molecules to isolation and investigation at the level of a single molecule. Key innovations for microscopy and nanoscopy in the last decade have been:

  • Confocal laser scanning microscopy (CLSM)
  • Fluorescence correlation spectroscopy (FCS)
  • Total internal reflection of fluorescence (TIRF)
  • Stimulated emission depletion (STED) microscopy
  • Spectral precision distance microscopy (SPDM)
  • Scanning nearfield optical microscopy (SNOM)
  • Fluorescence photoactivation localization microscopy (FPALM)
  • Stochastic optical reconstitution microscopy (STORM).1

These super-resolution microscopic and spectroscopic techniques allow a tremendous increase in lateral resolution and enable scientists to have a new perspective of cellular studies. For example, STED microscopy, the revolutionary technique invented by Professor Stefan Hell, enables a microscopic resolution limit below the theoretical limit of resolution and permits more detailed studies in cellular processes.2,3

These techniques require suitable fluorescent dyes that fulfill the demands of the applications, including a high cross section for stimulated emission, an absence of excitation at the depletion wavelength, dye photostability at depletion and excitation wavelengths, low triplet-state formation, and low non-linear photobleaching. Atto dyes are a series of fluorophores that can be used in the most sensitive applications including STED microscopy, and are especially suitable for target-specific detection. In a study performed by Donnert, et al., Atto 532 and Atto 647N were used for the study of synaptic vesicle proteins and demonstrated superior properties in two-color STED microscopy.4 Due to its super resolution property, the two-color STED microscopy technique was able to reveal ring-shaped synaptophysin domains and differentiate colocalized proteins with greater resolution than confocal microscopy.

With increasing instrument application limits, there is a growing demand for more sensitive, photostable and selective probes. Based on our extensive experience, expertise in organic synthesis and life sciences, and stringent quality assurance, Sigma offers a comprehensive selection of reagents for superior application results including the fluorescent Atto dyes series, MegaStokes dyes, and Chromeo™ Py-dyes. We are continuously increasing our portfolio of detection products and optimized protocols to meet the needs of fluorescence applications in life science research.

In this issue, scientists from the Swiss Institute of technology ETH, Zurich, Switzerland, show extraordinary application results using the red-emitting dyes Atto 647N, Atto 633, and Atto 655 in microarray experiments. Atto dyes, kits for protein labeling with Atto dyes, and Atto dye conjugates used to detect recombinant polyhistidine-tagged proteins and carbohydrates are also reviewed.

Additional sections in this issue review cellular processes and signaling by oxygencontaining molecules, and a new series of pI markers for isoelectric focusing (IEF) that are detectable by ultraviolet light for greater sensitivity.

We hope you find our articles and products helpful and interesting. For more information on detection products available from Sigma, please visit sigma.com/fluorescence.

References

  1. A guide to super-resolution fluorescence microscopy. Schermelleh, L., Heintzmann, R., Leonhardt, H., J. Cell. Biol., 190, 166–75 (2010).
  2. Confocal Application Letter No. 32, Leica Microsystems (2009).
  3. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Hell, S.W. and Wichmann, J., Opt. Lett., 19, 780–2 (1994).
  4. Two-color far-field fluorescence nanoscopy, Donnert, G., Keller, J., Wurm, C.A., Rizzoli, S.O., Westphal, V., Schönle, A., Jah, R., Jakobs, S., Eggeling, C., Hell, S.W., Biophysical Journal, 92, 67–9 (2007).

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