The ability to follow and observe single molecules as they function in live cells represents a major milestone
for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in
three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative
microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum
dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE
antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior
studies of two dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008). In addition, the
microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to
the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of
following single molecule dynamics on live cells with three dimensional spatial resolution.
Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650 nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Förster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.
Single molecule measurements are generally made in conditions that depart from physiological conditions, such as with molecules excised from cells or even immobilized on surfaces. Such departures can easily cause measurements on biomolecules to be inexact. A tracking instrument to follow a single molecule's path in three dimensions inside a living cell would be a major step towards enabling single-molecule observations in physiological conditions. We describe an instrument that will extend the state of the art in single-molecule
tracking technology, allowing extended observations of single particles as they diffuse and are transported. Computations show that our approach should be capable of tracking a protein-sized object diffusing at intracellular speeds for average times of over two seconds - long enough to track a typical fluorescent molecule
from capture to photobleaching.
KEYWORDS: Molecules, Microscopes, Near field, Luminescence, Near field optics, Atomic force microscopy, Diffraction, Image resolution, Molecular lasers, Photons
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.
KEYWORDS: Atomic force microscopy, Luminescence, Near field, Photons, Near field optics, Near field scanning optical microscopy, Microscopes, Photodetectors, Optical amplifiers, Sensors
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.
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