Turning a Simple Optical Microscope into a Powerful Tool

Small modifications to an ordinary optical microscope have helped turn it into a powerful instrument that can be used for studying complex cellular biology in real time at the nanoscale level. And such is the resolution that the modified optical microscope can study the cellular biology of a living single cell. All biological events — transport mechanism, protein synthesis — happen at the nanoscale level.The work carried out by an IIT Madras duo — Kamalesh Chaudhari, a research scholar from the Department of Biotechnology and Prof. T. Pradeep of the Department of Chemistry — has made it possible to observe the three dimensional dynamics of tiny nanoparticles with "simpler instrumentation." "Simple optical microscopes have never been used for observing detailed dynamics of simple nanoscale objects," said Prof. Pradeep. The results of the study were published on August 5 in Scientific Reports.Popular methods for studying biological events at nanoscale use fluorescence; the other is tagging a nanoparticle to an object of interest and studying it. "So far people have tried to label fluorescent particles/small molecules and study them. But in those cases the cell is fixed, and if the cell is live, the fluorescence of the small molecules lasts at most for 20 minutes," said Mr. Chaudhari.Optical scattering is one of the other methods used for studying biological events. But it would be useful only if an object produces intense scattering of incident light at a given wavelength. Gold nanorods are excellent scatters of light. So if you observe scattering at a specific wavelength we know a nanoparticle is there," Prof. Pradeep said. But it was the use of a polariser with a small angular tilt that produces an asymmetry in the scattering pattern that has made the technique unique for studying biological events.Since the scattered light from the nanorod will be polarised in a preferential direction, the direction of the polarisation analyser is changed to transmit light of specific polarisation.Unlike a spherical nanoparticle, a nanorod produces two types of scattering — one is along the longitudinal axis and the other is in the transverse axis. While the longitudinal scattering is enhanced in the red region, the transverse scattering is in the green region. "With an optical microscope, we can't see nanorods, but can distinguish the scattering pattern," the senior author said. Based on the colour of the scattered light it becomes possible to say if the rod is perpendicular or horizontal with respect to the incident light."If a particle is spherical then polarisation has no effect because the scattering of an isotropic sphere is independent of orientation," said Prof. Pradeep. But in the case of a rod, the scattering is orientation dependent — whether the rod is vertical or horizontal with respect to the incident light."We get a polar pattern by varying the polarisation angle," the senior author said. In the case of nanorods, the pattern produced by scattering resembles the figure eight. Based on the polar pattern, it is possible to deduce how the nano rods move/rotate inside a cell or if they are sticking to the surface of a cell. According to him, the scattering is very different when the rod is freely moving than when it is sticking to a cell surface."We can see small changes as rods move up and down and also track the vertical and horizontal rotation," said Prof Pradeep. "When the rod is inside a cell, we can track the motion as a movie with an optical microscope. So tomorrow, selective delivery into an organelle inside a cell and how a particle moves inside the organelle can be studied." The team used nanorods of 30 by 10 nanometre In comparison, the size of a cell is 20 microns (20,000 nanometre). The size of a nanoparticle should be much smaller than a cell so it does not alter the chemistry inside the cell.