In biological research, it is often necessary to assay samples for content of various chemicals, hormones, and enzymes. Spectroscopy, which is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules, or atoms, of a sample move from one energy state to another, is widely utilized for this purpose. Currently, the most common spectroscopic techniques pertain to measurements of absorbance, fluorescence, and luminescence.
Chemical analyses with absorption spectroscopy allow one to determine concentrations of specific components, to assay chemical reactions, and to identify individual compounds. Absorbance measurements are most commonly used to find the concentration of a specific composition in a sample. According to Beer's law, for a composition that absorbs light at a given wavelength, the total absorbed quantity of such light is related to the quantity of that composition in the sample.
Fluorescence, in turn, is a physical phenomenon based upon the ability of some substances to absorb and subsequently emit electromagnetic radiation. The emitted radiation has a lower energy level and a longer wavelength than the excitation radiation. Moreover, the absorption of light is wavelength dependent. In other words, a fluorescent substance emits light only when the excitation radiation is in the particular excitation band (or bands) of that substance.
For fluorescence measurements, fluorescent dyes called fluorophores are often used to "tag" molecules of interest, or targets. After being irradiated by an excitation beam, fluorophores, bonded to the targets, emit light that is then collected and quantized. The ratio of the intensity of the emitted fluorescent light to the intensity of the excitation light is called the "relative fluorescence intensity" and serves as an indicator of target concentration. Another useful characteristic is the phase relationship between the cyclic variations in the emitted light and the variations in the excitation light, i.e., the time lag between corresponding variations in the emission and excitation beams.
As noted above, luminescence measurements can also be employed for analyzing biological samples. Luminescence is the property of certain chemical substances to emit light as a result of a chemical change; no excitation from a light source is necessary. Moreover, luminescence can be produced by energy-transfer mechanisms that take energy of a high intensity, e.g., a radioactive emission, and transform it to energy of a low intensity, e.g., a flash of light
At the present time, a variety of spectroscopic instruments are commonly used in the art. A number of these instruments are designed to be utilized in conjunction with multi-site analyte receptacles called "microplates", which usually comprise one-piece structures having multiplicities of wells for holding analyte samples. Microplates are beneficial since they allow simultaneous preparation of a large number of test samples. Moreover, microplates are inexpensive, safe, sturdy, and convenient to handle. They are also disposable and can be cleaned easily when necessary.
One instrument currently available for fluorescent analysis of samples in microplate wells is the Cytofluor 2300 fluorometer, distributed by Millipore Corporation, Bedford, Mass. This fluorometer includes a scanning head that resides underneath the microplate and moves along the bottom face thereof to scan the sample sites. The scanning head interfaces with the optical system of the device via a bundle of optical fibers that transmits excitation and emission radiation.
However, the capabilities of the Cytofluor 2300 fluorometer are limited in that it cannot perform absorbance measurements. Furthermore, the movement of the scanning head from one microplate well to another continuously alters the geometrical configuration of the optical-fiber bundle that is attached to the head. Consequently, curvatures of the light-transmitting fibers change, introducing variations in their optical properties. These variations create inconsistencies in readings between different wells and adversely affect the repeatability, and thus, accuracy of measurements. Moreover, continuous bending of the fibers produces stresses that cause mechanical failure of the fiber cores.
Additionally, to allow unrestricted movement of the scanning head, flexible plastic fibers are employed, as opposed to less pliable quartz fibers. On the down side, plastic fibers cannot efficiently transmit radiant energy in the ultraviolet (UV) region of the spectrum. Accordingly, the fluorometer is unable to perform measurements, such as binding studies of certain proteins, e.g., tryptophan, since fluorescence analyses of this type require the use of UV radiation. Furthermore, the deformation resistance of the optical-fiber bundle slows the movements of the scanning head, thus limiting the ability of the apparatus to perform kinetic measurements.
Another spectroscopic apparatus utilizing microplates is disclosed in U.S. Pat. No. 4,968,148 to Chow et al., 1990. Chow's device uses an optical distributing element to selectively direct radiant energy to specified microplate sites. One drawback of this instrument is its inability to perform fluorescence measurements. Moreover, the large number of fibers unnecessarily complicates the apparatus and increases production costs. Also, the light-delivery system of the instrument has a fixed geometry that can only accommodate a microplate with one particular well layout. Chow's apparatus does not have the versatility to be utilized with microplates having different configurations of wells.
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