Introduction to Tip-Enhanced Fluorescence Microscopy (TEFM)

Our lab has developed a nanoscale optical imaging technique called tip-enhanced fluorescence microscopy (TEFM), which combines fluorescence microscopy and atomic force microscopy (AFM). Our TEFM instrument is the highest-resolution fluorescence microscope in the world (as far as we know). It is capable of imaging structures with better than 10 nm resolution, which is roughly a factor of 30 below the optical diffraction limit for the light wavelength used (~600 nm).

TEFM schematic

In TEFM, a laser beam illuminates a sharp AFM probe that is held in close proximity to a sample surface (see figure above). If the excitation laser is polarized along the vertical axis of the probe, the tip concentrates the light intensity at its apex. We use either a beam mask or a radial polarization converter to generate axial polarization at the AFM tip apex. When a sample feature is scanned through the region of enhanced near-field intensity, the local fluorescence rate increases, and this increase is measured in the far-field using an objective lens and a photodetector. An image is generated by correlating the measured fluorescence signal with the relative position of the sample and tip.

TEFM of DNA

The resolution of TEFM is limited by the sharpness of the AFM tip instead of the wavelength of light used. We demonstrated this by imaging short strands of DNA with a single dye molecule attached at each end, establishing a resolution of ~10 nanometers, as shown in the figure to the left. In this experiment, end-labeled DNA oligomers 60 base-pairs (bp) in length were dried onto a glass coverslip and imaged with TEFM. The length of the "DNA dumbells" is expected to be 12-15 nanometers, which agrees with measurements of more than 100 molecules.

Because the tip-enhanced near field requires illumination of the probe (and sample) with a diffraction-limited light source, the resulting fluorescence image is the superposition of a diffuse (far-field) and high-resolution (near-field) signal. The far-field signal becomes stronger as more fluorophores are illuminated, and thus the near-field contrast will decrease for high-density samples, as shown in the figure below. Since high molecule densities characterize many of the samples we are interested in studying, such as biological membranes, it is critical to find ways to increase near-field contrast as much as possible. Toward this end, we have focused on both improving signal acquisition and data analysis techniques, and investigating various tip materials. Our approach is completely general in that it can be applied to any tip geometry, including plasmon-resonant optical antennas, and also to any optical process, such as Raman scattering, Rayleigh scattering, and two-photon fluorescence.

Decreasing Contrast
Prof. Jordan Gerton | James Fletcher Building | Room 314 | 115 South 1400 East | Salt Lake City, UT | 84112
Office: +1-801-585-0068 | Lab: +1-801-581-5078 | Email: jgertonphysics.utah.edu