Optics: measuring and testing – By shade or color – With color transmitting filter
Reexamination Certificate
2001-02-26
2003-12-16
Nguyen, Tu T. (Department: 2877)
Optics: measuring and testing
By shade or color
With color transmitting filter
Reexamination Certificate
active
06665072
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed toward instrumentation for biochemical assays, more particularly assays using fluorescence detection with two or more labels in the experiment; or assays requiring a ratiometric measurement of fluorescence intensity at two spectral bands.
2. Description of the Related Art
Fluorescence assays that use multiple probes typically require measuring the fluorescence emission levels in two spectral bands, corresponding to the probes involved. Similarly, one may look in two bands to detect shifts in the spectrum of a single probe, if its emission spectrum is sensitive to environment. Examples of such probes include Indo, for pH sensing; or Acridine Orange to sense binding to RNA. In these cases, one wishes to use spectral ranges whose flux levels will be most sensitive to the spectral shift.
In such measurements, one often seeks to compare the flux in levels between the bands with high precision. In an assay looking for spectral shifts in a single probe, the ability to take precise ratios sets the detection limit for how small of a spectral shift can be detected. In dual-probe experiments, measuring the ratio precisely is valuable as well.
Existing fluorescence readers offer one the means to choose what spectral band will be measured, so it is possible to perform a dual band measurement simply by reading the sample twice in series. However, this is undesirable for several reasons. First, it takes twice as long as a single-band measurement, which can result in sample aging. Second, fluorescent probes exhibit photobleaching so the second reading is diminished in intensity, which distorts the measured ratio of fluxes. Finally, lamp flicker and drift are a significant source of noise in fluorescent measurements, and will degrade the measurement of flux ratios between bands.
Some instruments incorporate two or more detectors viewing the same sample simultaneously. These divide the beam according to wavelength band using partial-mirrors or dicroic elements to split of a sample portion to each detector. This approach is superior in the sense that it affords a higher throughput by reading both bands at once; and since the bands are measured under identical illumination conditions, a ratio of the two is not affected by lamp flicker or drift. However, such instruments bear a cost burden due to the need for two detectors and readout electronics, plus beamsplitters. This cost burden becomes even more significant if one wishes to make an imaging system of this type, using two pixelated detectors such as CCD detectors. In addition to the higher cost of the detectors and readout electronics, the two detectors need to be carefully registered in order that one can relate the image elements seen at a particular pixel on a first detector with a given pixel at the other detector. This registration must be handled carefully and while the approach is simple in theory, building such an instrument in practice tends to be demanding.
There is another problem with existing multiband fluorescence instruments. The use of dielectric filters at non-normal incidence to sample the beam, or indeed anywhere in the instrument, can lead to systematic spectral and polarization errors. In general, the off-axis reflection and transmission properties of dielectric film are different for the S- and P-state of polarization. Simply put, one observes a different spectral band in the S-polarization than one does in the P-polarization. In some cases, the difference is not significant because the spectral width of the overall measurement is determined by some other element in the system, such as a normal-incidence filter elsewhere in the system.
In practice, problems are most likely present when one wishes to observe bands that are spectrally adjacent, or where one band is spectrally close to the excitation wavelength. Yet dual-probe assays typically use one probe with a long Stokes shift, and one with a short Stokes shift (i.e. spectrally close to the excitation wavelength). Such samples are not measured accurately by an instrument that uses dielectric filters to split off the beam to multiple detectors. Thus the deficiencies of the prior art instruments are most likely to be germane, precisely when utilizing their dual-band readout capability to read dual-probe assays.
Such a dual-detector approach also would be less than optimal for measuring fluorescence polarization, due to the polarization sensitivity of the instrument. All existing fluorescence polarization instruments operate on a single band at the same time, so they suffer the throughput and noise penalties discussed above in connection with all single-band instruments.
An instrument described in U.S. Pat. No. 6,160,618 issued to Garner uses an imaging spectrometer to obtain a complete spectrum of the fluorescent emission by means of a dispersive element such as a grating or prism. There are several limitations to this instrument. In addition to the complexity of this approach, the instrument can only collect light from a small region corresponding to the image of the spectrometer slit projected on the sample. Thus its sensitivity is relatively low compared to instruments that can illuminate, and collect light from, extended regions such as sample spots or microtitre plate wells. Further, the efficiency of gratings and prisms depends upon polarization state, which renders a dispersive instrument inherently ill-suited for fluorescence polarization assays
Thus there is no instrument for fluorescence assay measurement which at once provides for simultaneous readout of two bands for high throughput and low-noise assessment of band ratios; with the economy of a single detector; or with high accuracy when used with short Stokes-shift probes or for fluorescence polarization measurements.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide for an ultra-high-throughput measurements at two bands without the need for multiple detectors that add cost or tilted dielectric filters that degrade accuracy.
It is a further object of the present invention to enable making dual-band ratio measurements with high precision suitable for use with Fluorescence Resonance Energy Transfer (FRET) potentiometric assays or environmentally-sensitive probes.
It is a further object of the present invention to enable performing dual label fluorescence polarization assays to detect single nucleotide polymorphism (SNP).
It is a further object of the present invention 1 to provide for fluorescence polarization measurements with high accuracy, for either a single probe or for two probes at once.
It is yet another object of the present invention to provide for reading more than one sample region at a time, to further increase the throughput.
It is yet another object of the present invention to achieve this in a compact, economical design which has no moving parts.
These and other objects are achieved by the present invention, which provides a system and method for separating fluorescent light emitted from a spot on a sample into multiple spots according to wavelength and, in some embodiments, according to polarization state when emitted by the sample as well. The resultant spots are directed to a multi-pixel detector where their flux is measured.
In one illustrative system, the sample is illuminated with a laser spot, and the fluorescence emitted from the spot is first separated into two spots according to polarization state using a double-refractive element. Each of these passes through a birefringent network which changes the state of polarization to its complement, or not, depending on its wavelength. A second double-refractive element further splits each of the two spots by polarization, which now corresponds to wavelength in a predetermined way; to yield four spots, separated according to wavelength and polarization state at the sample.
This arrangement can be used for dual band fluorescence assays of all kinds. It is ideal for dual-probe fluorescence polarization assays, since it simultaneously captur
Cambridge Research & Instrumentation Inc.
Cohen & Pontani, Lieberman & Pavane
Nguyen Tu T.
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