Focus

Single-Molecule Spectroscopy (SMS) is a technique where the fluorescence emission from spatially isolated molecules are collected and analyzed over a course of time in order to retrieve new information about individual molecules in a condensed media. Using SMS, rare events that are normally obscured by ensemble measurements can be observed and the inhomogeneity in specific molecular properties can be evaluated. In the past decade, SMS has been shown to be a very powerful tool to elucidate complex mechanistic pathways and a unique probe for the local nano-environment around isolated molecule. Our plan is to take a multidisciplinary approach towards the detection and imaging of Single-Molecules (SM) that would incorporate physical chemistry with nano-materials and biomolecular sciences. We plan to focus on research fields related to fluorescent behaviors of Single-Molecules or their Aggregates in unique local environments in contrast to that of bulk. Using a specialized technique called Total Internal Reflection Fluorescence Microscopy (TIRFM) as an experimental tool, we want to visualize and study real-time dynamic processes occurring near the interface to answer fundamental questions pertinent to chemistry, biology, polymer science and nano-materials.

Principal Technique

Single-Molecule Detection and Imaging using TIRFM: We are in the process of building an optical microscopy setup that uses evanescent waves generated from Total-Internal-Reflection (TIR) to excite fluorescent molecules near the interface. During total internal reflection of light, an evanescent field (near-field standing waves) is generated very close to the interface, the intensity of which decays exponentially from the surface. This physical principle can be exploited to selectively excite fluorescent molecules near the interface and can be imaged by collecting their fluorescence using a microscope objective lens/CCD camera. Microscopy using evanescent waves has been shown to be a very powerful technique since the molecules in the bulk phase are not excited by the evanescent wave, resulting in a significant enhancement of the signal-to-noise ratio. Since SM detection and imaging requires very high photon collection efficiency and a reduced background noise in order to properly analyze the behavior of photoemission from spatially isolated molecules, this near-field imaging technique, known as Total Internal Reflection Fluorescence Microscopy (TIRFM), serves as one of the most advanced methods to do so. Being a wide-field epi-fluorescence technique, it provides a relatively large field of view and hence tens to hundreds of single-molecules can be detected simultaneously, making TIRFM a truly high-throughput technique. TIRFM has also gained increasing popularity due to the efficacy in monitoring real-time dynamic processes because intensity fluctuations over time for all the SMs can be probed with ease down to the milliseconds timescale. Details of our TIRFM setup to detect, image and monitor behaviors of SMs in real-time can be found here.

Current and Future Research Plans

We are developing a versatile optical microscopy laboratory in which fluorescent molecules and particles very near an interface can be detected and imaged down to the Single-Molecule level. We would specifically like to visualize and analyze dynamic processes in chemical and biological systems - either due to spatial movements, or due to electronic structure changes – occurring in the timescale of milliseconds to seconds. On the aspect of electronic structure changes, we are currently investigating charge-trasnfer (CT) processes in aggregates and single-molecule--single-nanoparticle conjugates, blinking intermittency and spectral diffusion of fluorophores and custom-made semiconductor nanoparticles in unique environments, and also developing photoinduced electon-transfer (PET) based single-molcule sensor that can detect analytes at ultra-low concentrations.The aspect part of our research focus on physical (spatial) changes in conformation and/or position of single-floorescent molecules and Quantu-Dots in restricted and confined environemtns and the real-time dynamics of flexible polymers and biomolecules such as DNA in solution. In the long run, our intention is to branch off into more interdisciplinary fields of research, such as monitoring intra- and extra-cellular dynamics in live-cells, in the light of Single-Molecule Microscopy and in collaboration with materials chemists and biologists.

Some of our current and future research goals include

(i) Understand electronic delocalization and charge-transfer dynamics in nanoscale materials such as self-assembled organic fluorophores and individual semiconductor nanoparticles-dye conjugates using single-molecule emission dynamics and spectroscopy.

(ii) Develop and design fluorescent probes for high-throughput sensing and screening of specific toxic, hazardous, and biologically relevant chemicals down to the ultimate level of sensitivity.

(iii) Probe the glass-transition and plasticization behavior in polymer thin films using single-molecule dynamics and spectroscopy

(iv) Probe real-time dynamic processes in bio-molecular recognition events with particular interest in target-specific DNA-binding proteins, nucleic acids and small molecules [Future]

(v) Probe the dynamics of single isolated flexible and semi-flexible polymers in solution using real-time single molecule imaging. [Future]

(vi) Image live cells using fluorescent reporter molecules to understand mechanistic, physiological, and biochemical pathways [Future]

We are also involved in Instrumentation and software-development to detect and analyze dynamic behaviors of isolated molecules and particles in various matrices. [Projects page]

 

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