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Practical Aspects of PALM Imaging

Although single-molecule superresolution imaging techniques such as PALM and STORM are simple in concept, successful implementation requires a considerable amount of skill and attention to detail. In particular, the registration of the data and correct image assembly require caution in order to avoid two molecules appearing in the same focal area. Furthermore, the choice of photoactivatable probe dictates the maximum achievable resolution, which is a function of the number of photons emitted by each molecule. A number of other factors are important, including eliminating aberrations, mechanical stage drift, reducing autofluorescence, and general aspects of specimen preparation.

Shroff, H., White, H. and Betzig, E.

Photoactivated Localization Microscopy (PALM) of adhesion complexes.  Current Protocols in Cell Biology 4: 4.21.1-4.21.27 (2008).  An excellent review on the instrument configuration details and practical aspects of PALM imaging from the inventors of the technique. Also discussed are sample preparation, camera details, coverslip cleaning, dual-color imaging, and troubleshooting.

Gould, T. J., Verkhusha, V. V. and Hess, S. T.

Imaging biological structures with fluorescence photoactivation localization microscopy.  Nature Protocols 4: 291-308 (2009).  The authors describe experimental design for FPALM imaging, fluorescent probes, instrument parameters, camera calibration, illumination sources, filters, and the basic technique of single-molecule imaging. Included are a detailed reagent list and step-by-step instructions.

Dempsey, G. T., Wang, W. and Zhuang, X.

Fluorescence imaging at sub-diffraction-limit resolution with stochastic optical reconstruction microscopy.  Handbook of Single-Molecule Biophysics 4: 95-127 (2009).  Several aspects of implementing multicolor and three-dimensional STORM experiments are addressed in this excellent review. Also examined are instrumentation and methods for performing STORM experiments.

Lippincott-Schwartz, J. and Manley, S.

Putting super-resolution fluorescence microscopy to work.  Nature Methods 6: 21-23 (2009).  A nice overview of the potential benefits and pitfalls of superresolution imaging with emphasis on PALM and related single-molecule techniques. The authors suggest a set of guidelines for presenting images and point out inconsistencies in the current literature.

Small, A. R.

Theoretical limits on errors and acquisition rates in localizing switchable fluorophores.  Biophysical Journal 96: L16-L18 (2009).  The tradeoff between speed and error rates in single-molecule imaging is addressed, and a formalism for defining these error rates is presented. Algorithms that can infer molecular positions from images having overlapping blurs are also assessed.

Shroff, H., Galbraith, C. G., Galbraith, J. A. and Betzig, E.

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.  Nature Methods 5: 417-423 (2008).  A nice overview of live-cell imaging with PALM that discusses a number of factors influencing spatial resolution in terms of the density of localized molecules and feature motion. The supplement outlines the dependence of signal-to-noise on resolution and molecular density.

Gould, T. J. and Hess, S. T.

Nanoscale biological fluorescence imaging: Breaking the diffraction barrier.  Methods in Cell Biology 89: 329-358 (2008).  The authors present a general review of FPALM imaging with particular attention given to methodology. Discussed are probe choices, instrument alignment, background rejection, position stability, and a number of other factors.

Shroff, H., Galbraith, C. G., Galbraith, J. A., White, H., Gillette, J., Olenych, S., Davidson, M. W. and Betzig, E.

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes.  Proceedings of the National Academy of Sciences (USA) 104: 20308-20313 (2007).  In the first demonstration of dual-color PALM, the authors review the effects of contrast ratio on molecular localization precision, as well as other factors that affect the signal-to-noise in single-molecule superresolution imaging.

Baddeley, D., Cannell, M. B. and Soeller, C.

Visualization of localization microscopy data.  Microscopy and Microanalysis 16: 64-72 (2010).  An excellent review of how molecular localization data is presented in single-molecule superresolution imaging. Several techniques are discussed with regards to pitfalls and accuracy, and several algorithms are examined.

Ram, S., Ward, E. S. and Ober, R. J.

A stochastic analysis of performance limits for optical microscopes.  Multidimensional Systems and Signal Processing 17: 27-57 (2006).  The authors derive formulations of the Fisher information matrix for models that allow both stationary and moving objects. They also discuss background, detector size, pixelation, and noise sources in single-molecule fluorescence imaging.