Radiant energy – Inspection of solids or liquids by charged particles
Reexamination Certificate
2000-06-29
2003-09-16
Lee, John R. (Department: 2881)
Radiant energy
Inspection of solids or liquids by charged particles
C250S297000, C250S440110, C250S440110, C356S370000, C356S370000, C073S105000, C075S728000
Reexamination Certificate
active
06621079
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an apparatus and method for a near field scanning optical microscope, and more particularly optically recording near field images of living cells in an aqueous solution at a resolution higher than 50 nm, more than ten times greater than the wavelength of the light source used, and which provides a high resolution image and a rapid scan of the specimen (sample) without altering or damaging the specimen.
BACKGROUND OF THE INVENTION
Far field optical microscopy, such as fluorescence microscopy, has become a major research tool in basic biomedical research and an effective diagnosis technique in clinical medicine. However, the need for high spatial resolution and signal sensitivity has so far severely limited the effectiveness of this far field method, and in fact, all optical microscopy related research. Similar limitations are also present when used in clinical applications. Presently, the far field technique has found broad applications in a number of areas, such as cancer cell detection, characterization of cell abnormalities and evaluation of fundamental processes involving the localization and function of membrane associated proteins, such as ion channels and surface receptors.
Unfortunately, further improvement in far field optical microscopy is limited by the diffraction limit of light. Currently available optical microscopes place the specimen on the image plane of the optical system (i.e., located at a distance far exceeding that of the wavelength). Yet diffraction limits the spot size formed in the case of a scanning beam type of microscope or the smallest features that can be resolved in the case of an imaging system. Therefore, the highest resolution of a conventional optical microscope is in the sub-&mgr;m range, even when used with a confocal system.
Far field optical microscopy, regardless of the contrast mechanism being used (i.e., fluorescence, phase contrast or differential interference), is severely limited by the finite wavelength of light due to the unavoidable effect of diffraction. As such, the spatial resolution, either vertical to the plane of focus or lateral in the plane of focus, is roughly in the range of &lgr;/N.A., where &lgr; is the wavelength of the illumination (~0.5 &mgr;m) and N.A. is the numerical aperture which can be up to 1.4. Higher resolution, along with improved signal collection efficiency, would significantly enhance the use of the optical technique.
To overcome this fundamental difficulty, a type of microscopy known as Near Field Scanning Optical Microscopy (NSOM or SNOM) technology has been developed. Rather than placing the specimen at a far distance from the light source, a near field scanning optical microscope (NSOM) places the specimen directly in front of the light source at a distance far smaller than the wavelength of light (i.e.,“near field”). Since the physical size of the light source can be below the wavelength of the light, the spatial resolution would be limited by the size of the aperture. In this case, the illuminated area is no longer limited by diffraction. Therefore, by reducing the size of the light source (aperture), the volume being illuminated can be reduced accordingly. When such a light source is scanned over the specimen surface a two-dimensional image is obtained. Thus the system achieves the functions of a microscope. Since the resolution is directly related to the volume being illuminated, the resolution can be reduced to below that determined by diffraction.
Many techniques have been developed for fabrication of such small apertures. Normally a glass optic fiber can be pulled with laser heating to produce a very sharp apex, which can be coated with metal to make a small aperture at the end of the pulled fiber. Since only a very small volume is illuminated during the operating mode of a NSOM, a scanning mechanism must be used to acquire a two dimensional image. Since the image is obtained sequentially, the entire emission angle of 4&pgr; is available for signal collection in the case of fluorescence imaging, thus improving the collection efficiency. This is an important practical factor to consider when taking into account the effects of photo-bleaching and frame-time for image acquisition.
To make a NSOM instrument useful, the position of the probe aperture must be precisely controlled to avoid crashing the probe into the specimen. It is also important to ensure the image contrast is due to fluorescence rather than variations of the distance between the probe and the specimen (sample) surface. When operated in air, distance control can be achieved using a vibrating probe sensitive to the probe sample interaction or other methods. As such, the NSOM technique and its variations do not satisfy the applications required by biomedical research and clinical medicine. This is because most biologically relevant applications need the specimen to remain fully hydrated, preserving its native structure and function. When a conventional NSOM probe is placed in aqueous solution, the viscosity of the media produces a much lower Q value in the vibrating device, rendering it completely insensitive to the minute probe-specimen interactions. NSOM has therefore had very little success when applied to biological systems.
For some NSOM devices, pulled optical fibers are used as a conduit for the light source. The optical fiber surface is coated with a thin layer of metal to render it optically opaque but the apex of the fiber remains open (acting as the aperture) to allow for the transmission of light. It has been possible to fabricate apertures as small as 10 nm. However, as mentioned above, a critical feature that is required to make the NSOM a practical instrument is the method of maintaining the position of the aperture at a fixed distance from the specimen surface during scanning. Otherwise, the surface topography will have a profound effect on image contrast, creating artifacts that cannot be separated from the optical information. Even though many schemes of controlling the probe position have been demonstrated, the most successful design thus far is based on shear force detection. Here, the probe is driven to laterally oscillate at its resonant frequency. When the aperture is near the specimen, the interaction between the probe and the specimen surface will produce a shift in the resonant frequency, leading to a change in the oscillation amplitude. Therefore, by locking on to a predetermined reduction in amplitude, the distance between the probe and specimen can be controlled. It is intended that the resulting optical signal is independent from surface topography when this separation distance can be precisely controlled. As mentioned earlier, the above-mentioned conventional NSOM devices do not work in solution.
As such, conventional NSOM technology has a number of drawbacks. As mentioned previously, a major application of optical microscopy is in the field of biology and biomedicine, including disease diagnosis and fundamental research. Most of these applications require that the specimen or the sample be completely immersed in aqueous environment in order to retain full specimen functionality. To the detriment of the conventional NSOM technologies, including the conventional shear force technique, when the optical probe is immersed in solution, the probe Q value (a value directly related to the sharpness of the resonance peak or quality of the resonance is diminished. Yet, the shear force technique detection requires a reasonably high Q value so as to prevent the probe from crashing into the specimen before a frequency or amplitude shift could be detected. This is a fundamental limitation of most mechanically based NSOM detection schemes. Therefore, truly high resolution NSOM imaging of biological specimens in aqueous solution have not been known despite many years of effort by numerous research groups and industrial laboratories. For these reasons, the NSOM technology has not had a major impact on the fields of biology and biomedical research. Without sol
Mannelqvist Anders
Shao Zhifeng
Szabo Gabor
Decker Robert J.
Gill Erin-Michael
Lee John R.
University of Virginia Patent Foundation
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