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Single-molecule photobleaching: Instrumentation and applications

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Abstract

Single-molecule photobleaching (smPB) technique is a powerful tool for characterizing molecular assemblies. It can provide a direct measure of the number of monomers constituting a given oligomeric particle and generate the oligomer size distribution in a specimen. A major current application of this technique is in understanding protein aggregation, which is linked to many incurable diseases. Quantitative measurement of the size distribution of an aggregating protein in a physiological solution remains a difficult task, since techniques such as dynamic light scattering or fluorescence correlation spectroscopy (FCS) can provide an average size, but cannot accurately resolve the underlying size distribution. Here we describe the smPB method as implemented on a home-built total internal reflection fluorescence microscope (TIRF). We first describe the construction of a TIRF microscope, and then demonstrate the power of smPB by characterizing a solution of Amylin (hIAPP) oligomers, a 37-residue peptide whose aggregation is associated with Type II diabetes. We compare our results with FCS data obtained from the same specimen, and discuss the advantages and disadvantages of the two techniques.

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Acknowledgements

We thank Dr. Anoop Rawat for preparing the IAPP peptide and Anustup Chakraborty for his help with the Chung–Kennedy algorithm for fitting the step photobleaching data. This work was supported by intramural grants from the Tata Institute of Fundamental Research.

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  1. Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai, 400 005, India

    Simli Dey & Sudipta Maiti

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  1. Simli Dey
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  2. Sudipta Maiti
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Correspondence to Sudipta Maiti.

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Dey, S., Maiti, S. Single-molecule photobleaching: Instrumentation and applications. J Biosci 43, 447–454 (2018). https://doi.org/10.1007/s12038-018-9770-5

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  • DOI: https://doi.org/10.1007/s12038-018-9770-5

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