Optics: measuring and testing – By shade or color – With color transmitting filter
Reexamination Certificate
1999-08-06
2002-08-06
Evans, F. L (Department: 2877)
Optics: measuring and testing
By shade or color
With color transmitting filter
C356S418000, C250S458100
Reexamination Certificate
active
06429936
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the field of optical illumination and detection of biological activity in cells, organs or other samples, and more specifically to a method and apparatus for observing virtually instantaneously, the activity of a sample by illuminating the sample and measuring the light emitted from the sample.
2. Discussion of the Prior Art
Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules or atoms of a sample move from one energy level to another. In biological research, an observation in the changes of the optical absorption and emission of light provide valuable scientific information of what is occurring in the cell, organ or sample, for example, the progress of an ongoing chemical reaction.
Chemical analysis using absorption spectroscopy allows for the determination of concentrations of specific components, to assay reactions and to identify individual compounds while fluorescence is a physical phenomena based upon the ability of some substances to absorb and subsequently emit radiation. The emitted radiation generally has a lower energy level and a longer wavelength than the absorbed radiation which is used to excite the sample. Furthermore, the absorption of the incident light is wavelength dependent. Thus, a sample will only fluoresce when the excitation wavelength of the incident light falls within the excitation band for the substance at that particular time. The phase relationship between variations in the excitation or incident light and the light emitted from the sample is very important in observing changes in a substance as an event or reaction occurs.
One of the deficiencies of the prior art has been the lack of ability to faithfully observe ongoing changes in a sample as a reaction occurs, when the reaction requires detection at several wavelengths of light. With the present state of the art, it is often required in analytical procedures that reactions or assays be monitored at more than one wavelength. For observation systems that can change wavelengths in a little as one second, or theoretically, in {fraction (1/30)}
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of a second, there is high likelihood that a change in a sample occurring in {fraction (1/100)}
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or {fraction (1/1000)}
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of a second will not be detected. Also, in some analytic procedures, it is a requirement to change the wavelength of the excitation and emission light to permit detection at several wavelengths in order for a meaningful measurement to be made.
In newer approaches in the detection of events that occur in cells or other biologically related specimens, meaningful data must be obtained from the compilation of measurements made at more than one excitation and/or emission wavelength. As such, the speed of detection by the detecting device (PMT) is as important as the ability of the instrument to change the excitation and/or detect emission wavelength in a rapid fashion in-between or during the detection events. An instrument that has the ability to rapidly change excitation wavelengths and to rapidly detect a plurality and emission wavelengths in a short period of time, and synchronize events based on detection of light emitted by the sample, can monitor events that occur in the specimen with higher fidelity. Examples of these approaches and procedures for detection are the use of readily available fluorescent chemicals that exhibit spectrum-shifting properties that are dependent on their environment. Fura-2 and indo-1 are two typical chemicals used for the determination of calcium concentration that exhibit spectrum-shifting properties in certain environments.
One device for excitation/emissions measurements invented by Barlow et al. and described in U.S. Pat. No. 5,422,730, sets forth a system which permits for detection by a CCD camera of ongoing reactions in a particular sample. The device set forth below by Barlow et al. uses a pair of filter wheels to select a plurality of filters that transmit pre-selected light wavelengths, but the selection of wavelengths by the two filter wheels are performed in a sequential fashion. The limitations of this device are that it permits detection of events at only one excitation and one emission wavelength. The sequential selection of wavelengths by the filter wheels detrimentally slows the response time of the instrument to obtain meaningful data if more than one excitation or more than one emission wavelength measurement is required for meaningful information.
Other devices for observing reactions in samples can excite a sample using two different wavelengths simultaneously or can measure two different wavelengths at the same time by utilizing beam splitting devices. Some of the more advanced equipment is capable of measuring four different wavelengths emitted by a sample by use of vibrating mirrors, choppers or dichroic mirrors. Each of these devices has the ability to select an excitation or emission wavelength in a rapid fashion, but they do comprise a system in which the excitation and emission wavelengths are changed simultaneously and are synchronized to the detection of light intensity.
There is lacking in the biological field a photometer that is capable of exciting a cell or sample with a plurality of wavelengths, typically more than four, and virtually instantaneous observing the ongoing reaction by analyzing a plurality of wavelengths, typically more than four, emitted by the cell, organ or sample, either in-vitro or in-vivo, that does not interfere with the activity of the cell, organ or sample, and which is inexpensive and easy to operate. While there are instruments available that can make measurements quickly, their ability to do so at multiple wavelengths is limited. Typically, these instruments utilize spinning or vibrating mirrors to change either the excitation or the emission wavelengths (but not both) quickly. Conversely, instruments capable of exciting samples and taking measurements at multiple wavelengths are slow. There is a need for an instrument that can accomplish both excitation and detection quickly.
SUMMARY OF THE INVENTION
Accordingly, an advantage of the present invention is the capability of exciting a cell, organ or sample with a plurality of wavelengths and measuring a plurality of wavelengths emitted by the cell, organ or sample. The measurements are performed hundreds of times per second and may be performed thousands of times per second so that rapidly occurring changes can be observed. Despite the rapid number of measurements made by the device of the present invention, the narrow wavelength band of incident light is always synchronized with a narrow wavelength of emitted light.
Another advantage of the present invention the equipment used for florescent illumination and detection is relatively inexpensive to manufacture and does not require a high degree of skill to use.
Newer analytical procedures require the measurement of light intensity at more than one excitation or emission wavelength in order to get meaningful data. Measurement at one wavelength is not meaningful in the absence of another related measurement at a different wavelength. The results of both measurements are required for a calculation in the assay procedure. An advantage of the present invention is that it provides the capability to rapidly excite an assay or sample at a number of preselected wavelengths and to measure the emission from the assay at a number of preselected wavelengths while correlating the excitation wavelengths to the emissions wavelengths.
Meaningful data from an assay requires the capability to detect and measure a plurality of wavelengths of light emitted from an assay in a very short time frame. An advantage of the present invention is that it has the capability to both excite a sample or assay and detect the emissions from an assay at a plurality of wavelengths in a very short time frame so that meaningful multi-wavelength measurements can be made and recorded.
Still another advantage of the present inventi
C&L Instruments
Evans F. L
Maria Carmen Santa
McNees Wallace & Nurick LLC
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