Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2002-09-23
2003-09-16
Font, Frank G. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S306000
Reexamination Certificate
active
06621575
ABSTRACT:
The invention concerns a method and apparatus for the analysis of molecular reaction products in relation to biological cells.
BACKGROUND OF THE ART
Structures below 0.2 &mgr;m can no longer be detected with a laser scan microscope. Light sources involving dimensions in the nanometer range are used for optical near-field microscopy. In that case the light sources are delimited by the apertures of tips of tapered optical fibers or micropipettes involving dimensions in the range of some ten nanometers. Conversely however the apertures can also be used to collect in light from the object. Two-dimensional registration of absorption or fluorescence of the nano-scale cell structures is achieved by serially scanning the object being investigated by the near-field probe. For that purpose the probe must be moved two-dimensionally (x-y-plane) and the near-field condition (probe-object spacing<50 nm) must be embodied at each x-y-position (image point).
In that way, it is in principle possible to investigate attachment mechanisms in relation to individual cells and cell membranes with near-field microscopy. When selecting a suitable wavelength for the lighting device particularly when the absorption of the sample molecules is known—it is possible to observe and/or measure attachment distribution on the surface of a cell membrane. With a modified arrangement and a suitably adapted method fluorescence-optical determination is also a possibility. In that case the emphasis lies on determining binding specificity.
Near-field microscopy in accordance with the state of the art however suffers from two serious limitations. On the one hand the attachment efficiency is to be determined only at one respective cell sequentially by way of series of tests which are tedious in terms of the time involved while on the other hand those investigations cannot be carried out, or can be carried out only in specific cases, in a humid nutrient medium. High-resolution (nano-scale) optical investigations of biological samples are possible at the present time with a sufficient degree of accuracy and reliability, only in the fixed or dry state. On the one hand because the needle probe influences the object to be investigated (spacing control by lateral force) and, in the case of cells in a nutrient solution, the approach for the purposes of observing the near-field condition is not guaranteed. On the other hand, in a procedure involving serial scanning by displacement of the probe (raster time for the entire image region: 30-300 s) a large part of the reaction kinetics on biological membranes is not detected (the attachment dynamics of antibodies on cell membrane proteins embraces the time range of ms to some seconds). High-resolution microscopy processes such as raster electron microscopy (REM) and transmission electron microscopy (TEM) in principle make it possible to measure off nano-scale structures. As their operating principle requires an evacuated environment and special sample preparation investigations in vivo or in vitro respectively are not possible. The samples are investigated generally in the dried or fixed state. Admittedly, on the basis of measurement of binding forces, atomic force microscopy permits investigation of the attachment of molecules to different cell constituents with sub-cellular resolution, but in this case also a severe influence on the cells and thus obscure results in investigations in a nutrient solution cannot be avoided.
Clarification, which is important from scientific and technological points of view, of sub-cellular transport mechanisms—time-dynamic, locationally resolved and under physiological conditions—cannot be achieved with equipment available at the present time.
SUMMARY OF THE INVENTION
Therefore the object of the present invention is to provide an apparatus and a method with which a large number of cells can be evaluated in terms of their reaction state with molecular reactants under near-field optical conditions in a measuring and evaluation procedure. The object of the invention is in particular to determine the attachment efficiency of the molecular reactants in respect of attachment density in the cell partial areas in which the target proteins are disposed and in regard to attachment specificity which is given by virtue of the fact that the carrier or effective substance molecules bind only to special target proteins. In addition the object is characterised in that the determining operation must be implemented in a time-efficient manner on a large number of living cells—that is to say also in a nutrient medium—and that the determining operation is carried out on samples in the micro- and nano-scale range.
In accordance with the invention that object is attained in that a sample platform is provided, which is characterised in that fitted therein are light sources of different aperture diameters in the nano- and micro-range respectively. In accordance with the invention the geometrical arrangement thereof can be both unordered and also structurally ordered. The arrangement thereof relative to each other and the distribution functions in respect of number/aperture size groups are then to be presupposed to be known.
In accordance with the invention the two-dimensional nano-light sources array is embodied by a plurality of near-field light sources which are arranged in mutually juxtaposed relationship in a raster configuration and which are excited jointly or in succession. A semiconductor material, preferably silicon or GaAs, is used as the carrier material. The individual near-field light sources each have a respective hollow passage, wherein the individual hollow passages are used directly as nano-apertures for the exciting radiation or to produce secondary radiation (for example fluorescence or exciton radiation) are filled with a fluorescence- or exciton-active material. This nano-light source arrangement in accordance with the invention is covered by a 2-20 nm thick cover layer so that a large amount of sample of cells/cell membranes can be bound with a low degree of movement on the nano-source arrangement under a near-field condition for the time of the measurement procedure. It has surprisingly been found that biological objects (for example cells) can be fixed stably to the surface over a relatively long period of time by virtue of a biocompatible adhesion layer. In addition that layer guarantees that the near-field condition is constantly maintained. It is now possible without any problem to add a nutrient fluid which at the same time permits transport of the carrier and/or effective substance molecules to the membranes of the samples.
Upon registration of the overlap of the individual nano-light sources with the cell samples by counting off the bright points or measurement of the levels of light intensity it is surprisingly found that statistical evaluation of the distribution of intensity permits assessment of the lateral extent of the cells or active cell constituents. For that purpose the diameter and the distribution of the nano-light sources and the density in relation to surface area of the samples must be known. In that respect the aperture and the spacing of the nano-light sources are to be selected in a manner suited to the extent of the cells/cell nuclei belonging to a species.
The relative number of the nano-sources which overlap with objects depends on the known size and distribution density of the sources, and the size, shape and distribution density of the objects, in which respect shape has only a very slight influence. The overlap distribution can be determined by simple intensity measurement in a situation involving serial source excitation. The measured intensity distribution function (fluorescence or transmitted light) makes it possible to determine the object size, when the source size and distribution are known.
Attachment of the carrier and/or effective substance molecules to different proteins in the cell membrane, upon optical excitation of the resulting complexes by virtue of different binding energies, results in different abso
Beuthan Juergen
Eberle Hans-Georg
Helfman Juergen
Mueller Gerhard
Font Frank G.
Grant Stephen L.
Hahn Loeser + Parks LLP
Laser- und Medizin Technologie GmbH Berlin
Lauchman Layla
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