Related Experiment Video
Updated: Jun 3, 2026
12:27
Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging
Published on: November 25, 2009
An array of planar apertures for near-field fluorescence correlation spectroscopy
Christopher V Kelly1, Barbara A Baird, Harold G Craighead
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA. ck462@cornell.edu
Biophysical Journal
|April 6, 2011
Summary
We developed a novel near-field fluorescence correlation spectroscopy method using 50 nm apertures for high-resolution analysis of live cells. This technique achieves 1 μs and 60 nm resolution without complex equipment or sample penetration.
Area of Science:
- Biophysics
- Optical Spectroscopy
- Nanotechnology
Background:
- Fluorescence correlation spectroscopy (FCS) is a powerful tool for studying molecular dynamics.
- Traditional FCS is limited by diffraction, restricting spatial resolution.
- Achieving sub-diffraction-limited resolution in FCS typically requires complex setups like scanning probes or pulsed lasers.
Purpose of the Study:
- To develop a method for near-field fluorescence correlation spectroscopy (NFFCS) with enhanced spatial resolution.
- To demonstrate sub-diffraction-limited illumination and analysis of proximal samples, including live cells.
- To create a robust and accessible NFFCS platform using an array of nanometric apertures.
Main Methods:
- Fabrication of an array of millions of 50 nm diameter circular apertures in an aluminum thin film on a coverslip.
- Planarization of the aperture array to ensure a smooth surface for sample adhesion.
- Performing near-field fluorescence correlation spectroscopy on supported lipid bilayers and live cell plasma membranes adhered to the substrate.
Main Results:
- Demonstrated sub-diffraction-limited illumination and analysis using the fabricated aperture array.
- Achieved high spatiotemporal resolution of 60 nm and 1 μs.
- Successfully applied the technique to proximal samples, including live cells, without invasive procedures.
Conclusions:
- The developed NFFCS method offers a significant advancement in high-resolution molecular analysis.
- This technique provides a simpler and more accessible approach to sub-diffraction-limited spectroscopy.
- The platform holds potential for studying cellular dynamics and membrane properties with unprecedented detail.
Related Concept Videos
01:16Confocal Fluorescence Microscopy
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
01:05Total Internal Reflection Fluorescence Microscopy
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
01:37Super-resolution Fluorescence Microscopy
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
01:29Atomic Fluorescence Spectroscopy
Atomic fluorescence spectroscopy (AFS) is an analytical technique that involves the electronic transitions of atoms in a flame, furnace, or plasma being excited by electromagnetic (EM) radiation. When these atoms absorb energy, they become excited and subsequently release energy as they return to their original state. This emitted light, or "fluorescence," is observed at a right angle to the incident beam. Both absorption and emission processes transpire at distinct wavelengths, which are...