Time-lapse Chemical Microscopy using Intracellular Chemical Nanosensors

K. Rector
Los Alamos National Lab, US

Keywords: nanosensor, microscopy

Summary:

A vision for 21st century biophysical measurement is to employ whole live cell and tissue systems as miniature laboratories that can be analyzed while being perturbed with drug-based agents. To achieve this vision, next generation nano-sized chemical sensors must enable the reporting of chemical signatures over background and also use the natural cellular pathways to deliver the nanosensors to specific locations inside the cells. In this talk, I will describe such a targeting nanosensor. The ability to develop a gold-core, silver-shell surface enhanced Raman scattering (SERS) nanosensor that has both a targeting component as well as a sensing component is demonstrated. The nanosensor is capable of targeting through the Fc(epsilon}RI receptor-mediated endocytic pathway due to the adsorption of 2,4-(epsilon}-dinitrophenol-L-lysine (DNP) ligand on the nanoparticle surface. The nanosensor is also sensitive to pH changes in the endocytic vesicle within the pH range of 4.5–7.5 by 4-mercaptopyridine (4-MPy) adsorbed to the particle surface. Vibrational bands at frequencies of 1580 cm-1 and 1612 cm-1 display these proportional ratiometric effects dependent upon pH, based on protonation and deprotonation of the ring N atom in the 4-MPy molecule. The targeting and sensing moieties do not significantly interfere with each other’s function. The sensing component of the nanosensor is calibrated with in vivo measurements of rat basophil leukemia (RBL-2H3) cells in standard buffer solutions using the ionophore nigericin, which serves to equilibrate the external [H+] concentration with that of the cell compartments. The targeting of the nanosensor is verified with a (beta}-hexosaminidase assay. It is also demonstrated that the targeted nanosensor is capable of making accurate cellular pH measurements in RBL-2H3 cells, to at least ninety minutes after targeted nanosensor addition. Whole-cell, time-lapse, hyperspectral Raman image cubes demonstrating endocytic vesicular pH changes during Fc(epsilon}RI receptor mediated endocytosis are demonstrated. Hyperspectral Raman time lapse maps are acquired at 3-4 minute time resolution. At each pixel, entire Raman spectra are acquired, which relate to the pH at each specific location. Use of these techniques allows for dynamic changes in the ensemble pH distributions inside a single complete cell to be recorded. In addition, with this capability, the ability to record the cellular pH changes in response to drug based stimuli (amiloride or bafilomycin) when H+ flux across endosomal membranes is affected. Bafilomycin, an H+ ATPase pump inhibitor, blocks H+ pumping into the endosomal vesicle, and is shown to increase vesicle pH and inhibit molecules from progressing to late endosomes and lysosomes. Amiloride, a Na+/H+ exchanger inhibitor, blocks Na+ transport into the endosomal vesicle and further exit of H+ out of the vesicle. Endocytic vesicle trafficking, both temporally and spatially, is made possible by monitoring/imaging the vesicles pH environment with the use of the IgE targeted SERS pH nanosensor.