Optics: measuring and testing – Standard – Surface standard
Reexamination Certificate
2000-01-10
2001-07-10
Pham, Hoa Q. (Department: 2877)
Optics: measuring and testing
Standard
Surface standard
C436S008000
Reexamination Certificate
active
06259524
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention pertains to the preparation and use of thin, photobleachable luminescent layers for calibration and standardization of optical imaging devices, such as optical or Raman microscopy. For the quantitative application of optical and Raman microscopy, it is essential that the intensities in the images acquired with these microscopic techniques are determined only by the spatial distribution of the concentration, absorption, and emission characteristics of the luminophores in the specimen under investigation. If this is not possible, the image intensities should at least be proportional to these parameters. Generally, however, image intensity variations are not only determined by the specimen, but also by spatial non-uniformities of the optical system of the microscope across the field of view, so that only qualitative investigations can be performed. In order to realize the images required for quantitative microscopy, the microscope must be calibrated and standardized. The thus obtained images allow the comparison of different samples obtained on the same microscope, also at a different point in time, or the comparison of images obtained on different microscopes, provided that the different microscopes have been calibrated in the same way.
In earlier work, calibration and standardization of an optical microscope was attempted by an approach using images of a uniform luminescent layer (K. R. Castleman,
Digital Image Processing.
Prentice-Hall, Englewood Cliffs, N.J., 1979, and Z. Jericevic, B. Wiese, J. Brayan & L. C. Smith, “Validation of an Image System,” in
Luminescence Microscopy of Living Cells in Culture, Part B, Quantitative Luminescence Microscopy
-
Imaging and Spectroscopy,
edited by D. Lansing Taylor and Y. Wang, Academic Press, San Diego, Calif., 1989). Such an approach has three disadvantages. Firstly, in the case of an image of a luminescent layer, the product of the illumination and detection efficiency distributions is measured, and no information on the separate distributions is available. Secondly, completely uniform luminescent layers are difficult to obtain. Thirdly, the results of calibration and standardization based on this approach are affected by luminescence photobleaching of the layer. For general calibration and standardization in optical microscopy, it would be preferable to have an approach which does not suffer from these disadvantages. Jericevic et al. (Z. Jericevic, B. Wiese, J. Brayan & L. C. Smith, “Validation of an Image System,” in
Fluorescence Microscopy of Living Cells in Culture, Part B, Quantitative Fluorescence Microscopy
-
Imaging and Spectroscopy,
edited by D. Lansing Taylor and Y. Wang, Academic Press, San Diego, Calif., 1989) attempted to do away with the first disadvantage by using luminescence photobleaching techniques for the determination of only the illumination distribution. In his method, at least 20 images of a uniform, photobleaching luminescent layer were required. They showed that by numerically fitting the luminescence intensity decrease in each pixel of the first image with an exponential function, it was possible to determine only the excitation intensity distribution in the field of view of the used microscope (Z. Jericevic, D. M. Benson, J. Bryan, & L. C. Smith, “Rigorous Convergence Algorithm for Fitting a Monoexponential Function with a Background Term Using the Least-Squares Method,”
Anal. Chem.,
59 (1987), 658-662). There are several drawbacks to this method and experimental approach. Firstly, a luminescent layer has to be prepared by spreading an FITC-IgG mixture on a microscope slide. With such a procedure, it is very difficult to obtain a uniform luminescent layer. Secondly, the method provides only the illumination distribution; no information about the detection distribution is obtained. Thirdly, the determination of the illumination distribution is based on numerically fitting routines, which renders the method relatively slow.
BRIEF SUMMARY OF THE INVENTION
This invention describes the preparation and use of thin, photobleachable luminescent films for the calibration and standardization of an optical or Raman microscope in the wavelength region of 250 nm-1700 nm, preferably 250 nm-1100 nm, and more preferably 350 nm-900 nm.
DETAILED DESCRIPTION OF THE INVENTION
This is achieved according to the invention by the preparation of a photobleachable luminescent calibration layer and its subsequent use for the determination of excitation intensity and detection efficiency distributions in the field of view of the used microscope. The term photobleaching comprises all processes which result in the reduction of the intensity of luminescence light generated at the wavelength of excitation. Excitation may be done by laser or by a focused light source in the wavelength ranges defined above. Examples of such processes are photo-oxidation, photo-reduction, photo-isomerization or photo-addition reactions, or light-induced electron transfer processes.
It is sufficient for the effectiveness of the invention that the prepared calibration layer is approximately uniform, luminescent, and photobleachable, preferably approximately uniform, luminescent, and mono-exponentially photobleachable in a certain regime. The calibration layer should satisfy the following requirements.
(i) The luminescence intensity of luminophore in the calibration layer should be proportional to the excitation intensity, the luminophore concentration, and the illumination duration.
(ii) The rate of photobleaching of the luminophore in the calibration layer should be proportional to the illumination intensity and independent of the luminophore concentration.
(iii) The luminescence quantum yield, the absorption cross-section, and the bleach factor—defined as the ratio of the rate of photobleaching to the excitation intensity—of the luminophore in the calibration layer should be independent of the micro-environment within the layer.
The first two requirements already suffice for qualitative calibration of the measurement. The third requirement in combination with the first two allows absolute measurement of an image in optical or Raman microscopy.
The calibration layer is applied on an optically flat and transparent substrate by spin-coating, dip-coating or rod-coating (doctor blading) of a, preferably, 1-30 wt % solution of an optically transparent polymer containing an amount of photobleachable luminescent material present in such a way that the final polymer film contains less than 10 wt % of luminophore and has an optical attenuation of less than 0.3 absorption units in the wavelength region of interest, or of a solution of a sidechain polymer with an amount of photobleachable luminescent groups covalently attached to it, in such a way that the relative molar content of the sidechains is lower than 10% and the optical attenuation of the calibration layer is less than 0.3 absorption units in the wavelength region of interest. The useful concentration region is determined by the necessity to prevent intermolecular interactions (energy transfer) and inner filter and concentration quenching effects, which may lead to deviations tom simple mono-exponential decay. Optical attenuation more than 0.3 absorption units is possible, but mathematical corrections are required. Such attenuation is therefore less preferred. Suitable polymeric materials, which are transparent across the wavelength region of interest, are polyacrylates, polymethacrylates, polycarbonates, polyolefins, polyethers, polyurethanes, polyetherketones, polyesters, polystyrenes, polysiloxanes, and the like, or copolymers thereof. Suitable polymeric sidechain materials are based on the same optically transparent building blocks as applied in the transparent polymer types mentioned above and a suitable luminescent and photobleachable molecule which is equipped with a functional group so that it either may be attached to said polymer or may react with other functional monomers to form a luminescent sidechain-main chain polymer. Alternatively, thin films m
Brakenhoff Godefriedus J.
Ghauharali Rick I.
Hofstraat Johannes Willem
Knoble & Yoshida LLC
Pham Hoa Q.
The University of Amsterdam
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