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.

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Principle 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.

 

 

Current and Future Research Plans

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• Imaging, spectroscopy and dynamics of single fluorescent molecules/luminescent nanocrystals.

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• Develop high-throughput spectrally, polarization and time-resolved fluorescence microscopy techniques in order to probe chemical, materials and biological systems.

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• Photoluminescence and electroluminescence microscopy/spectroscopy of carrier localization centers in quantum-well/-rod based solid-state light emitting devices (ss-LEDs).

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• Carrier dynamics of individual semiconductor nanocrystals (undoped/doped/alloyed) using spectrally-resolved photoluminescence imaging.

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• Understand spatial and temporal inhomogeneities in soft matter matrices using rotational and translational mobility of single fluorescent molecules/quantum-dots.

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• Understand origins of blinking, and carrier mobility and recombination processes in all-inorganic and hybrid (organic-inorganic) perovskite micro- and nano-crystals for solar PV applications.

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• Develop sensing methods for analyte distribution in heterogeneous media such as biological cells. We are also involved in Instrumentation and software-development to detect and analyze dynamic behaviors of isolated molecules and particles in various matrices.

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Aim: Understand spatiotemporal heterogeneity in semiconductor nanocrystals/heterostructures, soft-materials and biological systems using sub-ensemble and single-molecule fluorescence spectroscopy and diffusion dynamics

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Systems of current interest to probe extent of heterogeneity:

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• Carrier localization in (In,Ga)N quantum-wells (QWs) used for solid-state LEDs

• Luminescent quantum-dots (QDs), doped/alloyed nanocrystals and nanoaggregates

• Hybrid/doped perovskite micro-/nano-crystals for solar photovoltaics (PVs) and LEDs

• Soft matter hydrated networks/gels such as polymers/ionomers and polymersomes

• Spatial distribution of analytes (ions/molecules) in intracellular media (single-cells)

• Structural heterogeneity of amyloid fibrils relevant for neurodegenerative diseases

• Liquid liquid phase separation of proteins and peptides

• Biosensors

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Techniques and methodologies used

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• Laser epifluorescence and total internal reflection fluorescence (TIRF) microscopy

• Single-emitter (molecule/nanocrystal/defect) spectroscopy and blinking (emission intermittency)

• Super-resolution single-molecule/particle tracking (translational and reorientational dynamics)

• Spectrally resolved fluorescence microscopy (spatially-resolved luminescence spectroscopy)

• Spectrally resolved sensitization imaging

• Polarization-resolved fluorescence anisotropy imaging

• Polarization-resolved single-molecule tracking (PR-SMT)

• Fluorescence correlation spectroscopy (FCS)

• Fluorescence lifetime imaging microscopy (FLIM)