TIRF Microscopy to Detect and Image Single Fluorescent Molecules

Evanescent-Wave Microscopy: Light traveling from a medium of higher (n2) refractive index to that of a lower (n1) one, undergoes total-internal-reflection (TIR) when the incident angle exceeds the critical angle of incidence. Under such conditions, even though most of the light is reflected back into the denser medium, an evanescent field (near-field standing waves) is generated very close to the interface1, the intensity of which decays exponentially with increasing distance from the surface (see scheme below). This basic physical principle can be exploited to selectively excite particular chromophores that lie within very close proximity (~200 nm) to the interface, which can then be imaged by collecting their fluorescence emission. Fluorescence microscopy using evanescent waves has been shown to be a very powerful technique since the molecules in the bulk are not excited by the evanescent-field, resulting in a significant enhancement of the signal-to-noise ratio, enabling the detection of fluorescence emission even from single chromophores.

This schematic shows how the evanescent field selectively excites fluorescent molecules close to the interface

 

Instrumentation and Software-Development to Detect and Analyze Dynamic Behaviors of Isolated Molecules and Nanoparticles

An ideal setup for Single-Molecule Detection and Imaging. We are halfway through....

 

This is what we got so far...

 

Typical "High-Throughput" detection of individual Rhodamine6G, (50ms exposure) obtained in our TIRFM (raw & processed image), embedded in a polymer matrix (PVA) at 293K.

 

What we plan to do with our current SM imaging TIRFM setup

(i) Understand electronic delocalization and charge-transfer dynamics in nanoscale materials such as self-assembled organic fluorophores and individual semiconductor nanoparticles-sensitizer conjugates using Single Molecule 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 behaviors in various polymer thin films using SM 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]

 

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