Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-08-07
2002-05-14
Font, Frank G. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S246000, C356S317000, C250S458100
Reexamination Certificate
active
06388746
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an optical method for high-sensitivity detection of fluorescent molecules based on the use of a highly focused light beam and light-induced fluorescence spectroscopy, to an apparatus for high-sensitivity detection of fluorescent molecules comprising a light source and a fluorescence detector, to a method for the production of a flow cell for high-sensitivity detection of fluorescent molecules, as well as to use of said method, apparatus or flow cell in combination with a microscope.
BACKGROUND ART
Techniques based on miniaturised chemical separation have made possible the analysis of the contents of individual cells (O. Orwar, H. A. Fishman, N. Ziv, R. H. Scheller, R. N. Zare,
Anal. Chem.
, 67, 4261 (1995)), and individual subcellular organelles (D. T. Chiu, S. J. Lillard, R. H. Scheller, R. N. Zare, S. E. Rodriguez-Cruz, E. R. Williams, O. Orwar, M. Sandberg, J. A. Lundqvist, Science in press). However, there is a need to analyse the contents of ever smaller sample volumes and even monomeric units cleaved off from single biopolymers such as RNAs, DNAs, and proteins. In order to render this possible, it is necessary to develop techniques with sensitivities approaching the inverse of Avogadro's constant, N
A
(6.0221×10
23
mol
−1
)
The use of microcolumn separation techniques, such as capillary electrophoresis, capillary electrochromatography, and microcolumn high performance liquid chromatography, for the compositional analysis of various types of samples, especially in the area of biomedical research and in the pharmaceutical industry, has experienced tremendous growth during the last twenty years. Since these techniques are particularly useful for the analysis of ultra-small sample volumes, 10
−6
to 10
−21
litres, that often contain trace amounts of analytes, severe demands are placed on detection sensitivity.
The possibility to detect very small quantities of biologically important molecules is of great interest in many fields, such as molecular biology, medical diagnosis, drug development and forensic analysis. Of particular interest is often the detection of antibodies, antigens, hormones, enzymes, proteins, peptides, amino acids or nucleic acids present in a sample. However, these samples often contain very small amounts of the molecules in question and they are therefore difficult to detect adequately. It is often necessary to amplify the material to obtain greater quantities before detection. In the case of e,g. DNA, this amplification is most frequently made by means of polymerase chain reaction (PCR), which duplicates DNA sequence of interest However, amplification of the molecules to be detected is not always desirable since it may, for example, lead to the introduction of substances contaminating the sample. Hence, there is a demand for techniques enabling direct detection of small amounts of a given substance. There are already some techniques available, and most of these are based on optical detection methods and on the use of different spectroscopy methods.
In 1961 came the first report on single-molecule studies in solution (B. Rotman,
Proc. Natl. Acad. Sci., USA
, 47,1981 (1961)). This study also has biological significance since the presence of a single enzyme molecule could be detected using a fluorogenic substrate. In 1976 a single antibody tagged with 80-100 fluorescein molecules could be detected using evanescent-wave excitation (T. Hirschfeld,
Appl. Opt
. 15, 2965 (1976)). Since then, much has been done in this field. One of the most promising techniques for sensitive detection is laser-induced fluorescence, mainly applied in two different set-ups: detection within a focused laser beam and detection in a near-field scanning optical microscope. Other techniques, such as nuclear magnetic resonance, electrochemistry, cavity ring-down spectroscopy have also been proposed for single molecule studies. Also, the use of biosensors in chemical separations have made it possible to distinguish single biomolecules (O. Orwar, K. Jardemark, I. Jacobson, A. Moscho, H. A. Fishman, R. H. Scheller, R. N. Zare,
Science
, 272, 1779 (1998)).
Methods based on laser-induced fluorescence have been demonstrated to have the ability to detect a single fluorescent molecule in solution. However, the known methods are diffusion-limited and can be employed only for samples containing a large amount of fluorescent molecules. Therefore, the sampling efficiency, i.e. the number of fluorescent molecules detected over the total amount of fluorescent molecules present in the solution, is extremely small, on the order of 10
−6
or even less. In one commonly employed embodiment of single-molecule detection in solution, a drop containing the fluorescent molecules is placed on a coverslip (S. Nie, D. T. Chiu, R. N. Zare,
Anal. Chem.
, 67, 2849 (1995) and R. Riegler, U. Mets, J. Widengren, P. Kask, Eur. Biophys. J., 22, 169 (1993) and S. Nie, D. T. Chiu, R. N. Zare,
Science
, 266, 1018 (1994)). Single-molecule fluorescence is then collected and detected in a confocal fluorescence microscope set-up. With this technique it is, however, difficult to accomplish detection of molecules separated by a microchemical fractionation technique.
Detection of single molecules has also been achieved in capillary structures, both coupled to separation devices and as stand-alone flow cells (Y-H Lee, R. G. Maus, B. W. Smith, J. D. Winefordner,
Anal. Chem.
, 64, 4142 (1994)). Also in these cases, however, detection has been performed in solutions containing a large excess of the fluorescent molecule over the actual detected number of molecules. Typically, 10
−9
to 10
−12
M of fluorescent solutes are introduced into the system in solution volumes of from 10
−6
to 10
−3
1. Thus, again sampling efficiencies on the order of 10
−6
to 10
−12
are obtained.
In the last decade, there has been rapid development in high-resolution optical and electro-optical techniques, driven by the need to understand biochemical and biophysical processes in greater detail. For example, confocal microscopy and two-photon microscopy have provided striking images on the workings of cellular machinery, such as the dynamics of intracellular calcium ion and the localisation of single serotonin-containing granulae in RBL cells (see egg. B J. Bacskai, P. Wallen, V. Lev-Ram, S. Grillner, R. Y. Tsien,
Neuron
, 14, 19-28 (1995) and S. Maiti, J. B. Shear, R. M. Williams, W. R Zipfel, W. W. Webb,
Science
, 275, 530-532 (1997)). Higher optical resolutions—as high as 12 nm—are obtained in near-field spectroscopic probes, wherein it is possible to reach, or even bypass the Abbe diffraction limit (E. Betzig, J. K. Trautman, T. D Harris, J. S. weiner, R. L Kostelak,
Science
, 251, 1468 (1991)). The manipulation of single organelles and even single biomolecules has been made possible by optical trapping, and this technique has been applied to a wide range of interesting biological problems (A. Ashkin,
Phys. Rev. Lett.
, 24 (4), 156 (1970) and K. Svoboda, S. M. Block,
J. Annu. Rev. Biophys. Biomol. Struct.
, 23, 247-285 (1994) and D. T. Chiu, A. Hsiao, A. Gaggar. R. A. Garza-Lopez, O. Orwar, R. N. Zare,
Anal. Chem.
, 69, 1801-1807 (1997)).
As stated above, techniques that can detect a single molecule rapidly moving in solution are based almost exclusively on optical methods. By using lasers which produce spatially and temporally coherent bundles of monochromatic light, a tightly focused diffraction-limited laser spot can be obtained with appropriate optics.
If detection is made through a pinhole or a narrow slit, in a confocal detection arrangement, an extremely small laser probe volume can be created on the order of about 5×10
−16
1. In this way, an extremely narrow depth-of-focus is obtained. The confocal advantage includes extremely low background scattering from Rayleigh and Raman events, where the intensity has an inverse quadruplicate dependence on laser wavelength, a linear dependen
Chiu Daniel T.
Eriksson Peter
Orwar Owe
Cellectricon AB
Lauchman Layla
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