Optics: measuring and testing – Position or displacement – Position transverse to viewing axis
Reexamination Certificate
1999-08-25
2002-07-23
Evans, F. L (Department: 2877)
Optics: measuring and testing
Position or displacement
Position transverse to viewing axis
C356S244000, C250S458100, C250S459100
Reexamination Certificate
active
06424421
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method and devices for far field microscopy and flow fluorometry for geometric distance measurements between object structures marked with fluorochromes, wherein the distances may be smaller than the half-intensity width of the principle maximum (=full width at half maximum of the intensity peak=FWHM) of the actual point spread function.
BACKGROUND INFORMATION
By employing highly specific markers, such e.g. DNA probes or protein probes, it is possible to mark practically any small (sub-)structures in biological (micro-)objects, in particular in cells, cell nuclei, cell organs or chromosomes (hereinafter also described as objects for abbreviation). Such markers may specifically represent structures in dimensions from several &mgr;m (10−6 m) to a few tens of nm (10−9 m). In these markers normally reporter molecules are integrated, which have a high affinity with complex compounds, to which fluorochromes, but also colloidal microparticles (e.g. gold) are attached. Such fluorochromes/complexes can also be integrated directly into the markers. The available colour emission spectra of fluorochromes stretch from deep blue through green and red to the infrared range of the spectrum. Equally, fluorochromes can be used that do not differentiate in terms of excitation and/or fluorescence emission in their spectrum, but in which the life time of their fluorescence emission is used as a parameter for differentiation.
The latter have the advantage that focal shifts depending on the wavelength do not arise. Fluorochromes can also have a different emission spectrum and thus possess different spectral signatures, and yet be stimulated by the same photon energy, e.g. by means of multiple photon processes. It is also possible in this case to avoid wavelength-dependent focal shifts in the excitation between fluorochromes with different spectral signatures.
The above-named fluorochromes bound to specific (sub-) structures in biological micro-objects are referred to hereinafter as fluorescence markers. Fluorescence will be used below to encompass every photon interaction in which differences arise between a material's stimulation spectrum and its emission spectrum that cannot be attributed to monochromatic absorption or dispersion. This also includes in particular multiple photon interactions in which the stimulation wavelengths may be greater than the emission wavelengths. Furthermore, the term fluorescence is also used here for the closely related phenomenon of luminescence, in particular phosphorescence. This includes in particular longer and medium term fluorescence life time, e.g. fluorescence life time in the range of up to several or many msec (milliseconds). The closely related processes of luminescence, phosphorescence and fluorescence will be treated herein as equally relevant to the invention. If the excitation spectrum and/or the emission spectrum and/or the fluorescence life time of two fluorescence markers agree, they have the same spectral signature based on the parameter in question. If they differ in one or more parameters relevant to the measurement, they have different spectral signatures.
A series of light microscopic measuring methods is used for detecting the fluorescence markers in extended biological objects and for the quantitative localisation relative to defined object points/object structures (distance and angle measurements). This is primarily a case of (a) epifluorescence microscopy, (b) confocal laser scanning microscopy, (c) laser scanning flow fluorometry, (d) the far field microscopy process of “point-spread-function-engineering” and (e) standing wave field microscopy.
a) In the case of epifluorescence microscopy with a classical upright or inverse epifluorescence microscope, the biological object is illuminated by the same lens through which it is detected. The excitation light and the fluorescence emitted are discriminated by appropriate optical filters and conducted into different beam paths. The obtainable resolution, i.e. the smallest distance still measurable between two point-shaped object structures that are marked with fluorochromes with the same spectral signature, is given either by the Abbe criterion (=the maximum 0. order of the diffraction pattern of a point object is localised in the 1st minimum of the diffraction pattern of a second point object) or by the half-intensity width of the principle maximum of the actual point spread function. This depends on the wavelength, on the numerical aperture of the lens used and on the local refractive indices of the objects, of the embedding medium, of any cover slip used and of any immersion fluid applied. (In the case of a higher numerical aperture, its dimension may be smaller than the wavelength of the light used for stimulation).
b) In the case of confocal laser scanning microscopy, unlike epifluorescence microscopy, a laser is focused in the lens and the fluorescence is detected confocally. In order to create a three-dimensional image, the object is scanned in all three directions (x, y, z) with the focus point. As in the case of epifluorescence microscopy, the obtainable resolution is given by the half-intensity width of the principle maximum (FWHM)of the actual point spread function and depends on the wavelengths, on the numerical aperture of the lens used and on the local refractive indices of the objects, of the embedding medium, of any cover slip used and of any immersion fluid applied.
c) In the case of laser scanning flow fluorometry, the objects are conducted for example individually through an appropriate light distribution of the focus by a carrier fluid beam that is free or situated or in an optical cuvette (while in the case of epifluorescence microscopy and confocal laser scanning microscopy, the objects are predisposed in a fixed position on object carriers, i.e. object slides, capillaries, chambers, fluids etc.). The light distribution is normally slit-shaped, i.e. the object is scanned with reference to an axis. The obtainable resolution is determined by the width of the focus of the laser beam used and/or suitably selected detection scans, wherein the variability in the object trajectory (=laminar, usually central “fluid filaments” that carry the object) allows for the focal depth and thus also the minimal focal width, regardless of the carrier medium and method. The advantage of flow fluorometric methods is usually found in the relatively higher detection rate, compared to epifluorescence microscopy and confocal laser scanning microscopy, which can reach several thousand objects per second. The focal width complies with the full half-intensity width of the principle maximum of the actual point spread function of the slit-scan optics in the conditions used.
d) In the case of the far field microscopy technique of “point-spread-function-engineering”, the point spread function is reduced in width optically. This may be achieved by means of coherently overlapping two or more point spread functions (e.g. 4Pi microscopy) or by means of extinguishing the fluorescence of fluorochromes that are situated in the peripheral area of the central point spread function maximum in question (e.g. STED microscopy, ground depletion microscopy). As the resolution of a microscope is given by the full half-intensity width of the principle maximum of the actual point spread function, the half-intensity width is thus reduced and the resolution improved.
e) In the case of standing wave field microscopy according to U.S. Pat. No. 4,621,911, luminescent preparations are illuminated with a standing wave field in an optical microscope (standing wave field fluorescent microscopy, SWFM). The preparations are set in a zone of equidistant wave fronts and stimulated to fluorescence or phosphorescence. The space between the wave fronts and their phases can be varied to generate patterns. The three-dimensional distribution of fluorescent or luminescent object points can be reconstructed from individual optical sections by means of
Bradl Joachim
Cremer Christoph
Hausmann Michael
Rinke Bernd
Davidson Davidson & Kappel LLC
Evans F. L
Ruprecht-Karls-Universität Heidelberg
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