Guillaume A. Lessard

An apertureless near-field microscope for fluorescence imaging

We describe an apertureless near field microscope for imaging fluorescent samples. Optical contrast is generated by exploiting fluorescent quenching near a metallized atomic force microscope tip. This microscope has been used to image fluorescent latex beads with subdiffraction limit resolution. The use of fluorescence allows us to prove that the contrast mechanism is indeed spectroscopic in origin.

Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments

We demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three-dimensional Brownian motion (diffusion coefficient approximately 0.5 microm(2)/s) in environments with a signal-to-background ratio as low as 2:1, significantly worse than observed in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to determine changes in the emission lifetime as a function of position and positively identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full three-dimensional volume of a live cell for durations up to 15 s.

Scanning apertureless fluorescence microscope

We describe a near-field apertureless fluorescence microscope, capable of imaging fluorescent latex beads with subwavelength precision. The instrument is based on a home- built tapping-mode atomic-force microscope, to which an inverted optical microscope was added. The fact that the wavelength of the fluorescence that we observe is different from the wavelength of the illumination allows for a relatively straightforward detection mechanism. Sample images are presented, along with evidence that the observe effect is of optical origin.

Tracking Single Quantum Dots in Three Dimensions

We describe new instrumentation developed in our lab (essentially a confocal microscope with feedback) capable of following 3 dimensional motion of single quantum dots moving at rates comparable to intracellular protein traffic (microns/sec).

Apertureless near-field fluorescence microscope for biological imaging

We measured the optical response of fluorescent beads to sharp metallic and semiconducting probes, revealing several underlying near-field interactions. Our results suggest that /spl sim/10 nanometer optical resolution with spectroscopic chemical sensitivity is possible, and bear strongly on molecular-scale biological imaging.

Fluorescence-enhancement microscopy at 10 nm resolution

The historical importance of optical microscopy to biological study is difficult to overstate. Optical techniques are relatively non-invasive and minimally perturbative both during sample preparation and measurement, enabling temporally resolved imaging of dynamically evolving samples in vivo or at near physiological conditions. Spectroscopic contrast provided by fluorescence and Raman imaging, in particular, provides superior molecular addressability and chemical differentiability. Although spectroscopic techniques are widely utilized in molecular and cellular biology, typically the spatial resolution is limited by light diffraction to 200 – 500 nm, well above the length scale where individual molecules or complexes interact (<10 nm). Alternate microscopy techniques, such as X-ray crystallography, high-resolution nuclear-magnetic-resonance tomography, electron microscopy, scanning-tunneling microscopy, and atomic-force microscopy (AFM), can provide exquisite, sub-nm spatial resolution but do so at the price of chemical differentiability and/or temporal resolution. This gap in imaging functionality has been addressed by scanning near-field optical techniques and recently 25-30 nm resolution for fluorescence [1] and Raman [2] microscopy has been demonstrated using so-called apertureless approaches which rely on enhancement of the optical signal near a sharp (metal) probe. Although this resolution is well below the diffraction limit, it is only incrementally better than what can be obtained with standard near-field techniques where an apertured illumination source (or detection region) is scanned in close proximity to the sample.

Fluorescence apertureless near-field microscope: a step toward imaging information in DNA

Single molecule imaging with optical methods has become an important tool in biophysical studies. However, when imaging molecules at room temperature using far field optics, one can only resolve molecules that are separated by a distance greater than the diffraction limit of the microscope, about 220 nanometers. Near field techniques have allowed researchers to image with resolutions on the order of 30-50 nanometers. However, there are numerous reasons to try to push the resolution limit further. One that particular concerns our group is the \notion to try to image information in DNA in order to measure sequence information. To that end, we have developed a new type of near field microscope, the fluorescence apertureless near field microscope.

An apertureless near field microscope for fluorescence imaging

Summary form only given. We demonstrate an apertureless near-field scanning microscope for fluorescence imaging with λ/20 resolution. The use of fluorescence allows artifact free imaging and provides a stringent test that the contrast mechanism is optical in origin.