Journal of Fluorescence

, Volume 28, Issue 3, pp 855–862 | Cite as

Two Photon Spectroscopy Can Serve as a Marker of Protein Denaturation Pathway

  • Dipak Kumar DasEmail author
  • Sk Imadul Islam
  • Nirnay Samanta
  • Yogendra Yadav
  • Debabrata GoswamiEmail author
  • Rajib Kumar MitraEmail author


Rhodamine group of molecules are widely used dyes for imaging of biological molecules. Application of these dyes however includes a limitation that these molecules absorb in the visible range of the spectrum, which does not fall in the ‘biologically transparent window’ (BTW). Two photon absorption (TPA) process could come up with an alternate solution to this as these dyes could be excited in the near infrared (NIR) window to extract similar information. To validate this we have investigated TPA cross section (TPACS, σ2) of two rhodamine dyes, namely Rhodamine 6G (R6G), Rhodamine B (RhB), site selectively bound with a model protein, bovine serum albumin (BSA), by exciting at 800 nm. Two photon spectroscopy and imaging confirms the binding of the dye to the protein. The decreases in TPACS with increasing temperature at a fixed BSA concentration excellently follows the temperature induced structural transition of BSA as the protein transforms from a molten globule to unfolded conformation beyond 60 °C, which has previously been established through circular dichroism (CD) measurements. The thus established resemblance in TPACS and CD measurement trends thus strongly affirms the suitability of TPA process in protein imaging and as an alternative marker to tracking its conformational transformations using NIR radiation.


Two photon Rhodamine Human serum albumin Circular dichroism 



RKM acknowledge the funding from 12th Five Year Plan project at SNBNCBS (SNB/AB/12-13/96). SII wants to thank UGC, India for graduate fellowship. DG acknowledges ISRO funds for imaging.

Supplementary material

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ESM 1 (DOCX 1426 kb)


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Authors and Affiliations

  1. 1.Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic SciencesKolkataIndia
  2. 2.Department of ChemistryIndian Institute of TechnologyKanpurIndia

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