In vivo characterization of protein uptake by yeast cell envelope: single cell AFM imaging and μ-tip-enhanced Raman...

Direct detection of biological transformations of single living cells in vivo has been performed by the advanced combination of local topographic imaging by Atomic Force Microscopy (AFM) and label-free sub-surface chemical characterization using new μ-Tip-Enhanced Raman Spectroscopy (μ-TERS). The enhancing mechanism for μ-TERS tips with micrometre range radius differs significantly to that of the conventional tapered structures terminated by a sharp apex and conditioned by the effects of propagating instead of localizing surface plasmon resonance phenomena. Sub-wavelength light confinement in the form of a nonradiative evanescent wave near the tip surface with penetration depth in the sub-micrometre range opens the way for monitoring of subsurface processes near or within the cell wall, inaccessible by other methods. The efficiency of the approach has been demonstrated by the analysis of the cell envelope of genetically modified (by glucose dehydrogenase (GDH) gene bearing Kluyveromyces lactis toxin signal sequence) yeast cells enriched by GDH protein. The presence of trans-membrane fragments in GDH together with the tendency to form active dimers and tetramers causes the accumulation of the proteins within the periplasmic space. These results demonstrate that the advanced combination of AFM imaging and subsurface chemical characterization by the novel μ-TERS technique provides a new analytical tool for the investigation of single living cells in vivo.