Micro array biochips are being used by several biotechnology companies for scanning genetic DNA samples applied to biochips into computerized images. These chips have small substrates with thousands of DNA fragments that represent the genetic codes of a variety of living organisms including human, plant, animal, and pathogens.

They provide researchers with information regarding the DNA properties of these organisms. Experiments can be conducted with significantly higher throughput than previous technologies offered by using these biochips. Biochip technology is used for genetic expression, DNA sequencing of genes, food and water testing for harmful pathogens, and diagnostic screening. Biochips may be used in pharmacogenomics and proteomics research aimed at high throughput screening for drug discovery.

DNA fragments are extracted from a sample and are tagged with a fluorescent dye having a molecule that, when excited by a laser, will emit light of various colors.

Typically, the DNA fragments are tagged with more than one dye. Each dye includes fluorescent spectral band that is excitable by a laser of a particular wavelength. These fluorescently tagged DNA fragments are then spread over the chip. A DNA fragment will bind to its complementary (cDNA) fragment at a given array location. A typical biochip is printed with a two-dimensional array of thousands of cDNA fragments, each one unique to a specific gene. Once the biochip is printed, it represents thousands of specimens in an area usually smaller than a postage stamp.

A microscope collects data through a scanning lens by scanning one pixel of a specimen at a time. The scanning lens projects emitted light from the specimen onto a scanner that is manipulated along a predetermined pattern across the chip scanning an

entire biochip one pixel at a time. The pixels are relayed to a controller that sequentially connects the pixels to form a complete, computerized biochip image.

Inaccurate computerized biochip images are obtained when the spectral band of a first dye overlaps with the spectral band of a second dye. Often, a laser having a wavelength formulated to excite the spectral band of the first dye will excite the overlapping spectral band from the second dye. When this occurs the pixels relayed to the controller will be blurred potentially rendering the connected pixels unreadable.

Therefore, a need exists for a method of reading the genetic code from a specimen having more than one dye from a biochip and yet still be able to compensate for overlapping spectral bands.

SUMMARY OF THE INVENTION AND ADVANTAGES The present invention provides an improved method for scanning a specimen with an optical instrument. A first dye having an excitation wavelength with multiple fluorescence bands that are excited when contacted by a first optical signal is applied to the specimen. A second dye having an excitation wavelength with multiple fluorescence bands that are excited when contacted by a second optical signal is also applied to the specimen. The overlapping spectral bands of the first dye and of the second dye are predetermined and programmed into a computer. The first optical signal and the second optical signal are directed onto the specimen. The specimen is scanned for emitted wavelengths from the first dye with a first detector for creating a first data set. The first data set is relayed to the computer to create the biochip image. The computer is programmed for removing from the data set the band of wavelength from the second dye excited by the first optical signal.

By predetermining the spectral bands that overlap between the two dyes the computer can be programmed to remove the overlap from the relayed data sets.

Therefore, the problem of producing unreliable computerized biochip images resulting from overlapping spectral bands from multiple dyes is addressed.

BRIEF DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: Figure 1 is a detailed perspective view of an optical instrument of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The optical instrument assembly of the present invention is generally shown in Figure 1 at 10. The assembly includes a transmitter 12 for emitting an optical signal 14.

In the preferred embodiment, the transmitter 12 comprises a laser. Figure 1 shows two transmitters 12a, b, each emitting an optical signal 14a, b having a different wavelength.

Additional transmitters 12 may be introduced to the assembly 10 as needed.

A reflector 30 directs the optical signal 14 onto a specimen 90. The reflector 30 includes a plurality of turn mirrors 32. Figure 1 shows two turn mirrors 32a, b corresponding to the same number of transmitters 12a, b. Each optical signal 14a, b is reflected by the turn mirrors 32a, b into corresponding beam combiners 34a, b. The beam combiners 34a, b, known as dichroic filters, transmit light of one wavelength while blocking other wavelengths. The beam combiners 34a, b collect the individual optical signals 14a, b into a combined beam along a single path and direct the beam towards a beam splitting mirror 20. The beam splitting mirror 20 includes an opening 22 through which the combined optical signals 14a, b travel. Subsequently, the combined optical signals 14a, b reflect off a ninety degree fold mirror 36 located immediately above a scanning objective lens 52, which focuses the combined optical signals 14a, b onto a section of the specimen 90. A first drive mechanism 50 varies the position of the combined optical signal 14a, b on the specimen 90 as will be explained further herein below.

The specimen 90 is treated with dyes having fluorescent properties when subjected to the optical signal 14a, b. In the preferred embodiment, at least a first dye having an excitation wavelength with multiple fluorescence spectral bands that are

excitable by the first optical signal 14a is applied to the specimen. Additionally, a second dye having an excitation wavelength with multiple fluorescence spectral bands that are excitable by the second optical signal 14b is applied to the specimen 90. The dyes are selected to examine different specimen 90 properties. Utilizing multiple dyes allows different properties of the same specimen 90 to be examined simultaneously.

Frequently, the first and second dyes will have at least one spectral band in common. Therefore, it is possible for the first optical signal 14a to excite a spectral band from the second dye causing inaccurate fluorescence to occur. Likewise, the second optical signal 14b can excite a spectral band from the first dye. The spectral bands that are in common between the two dyes are predetermined, the reason for which will be explained further hereinbelow.

The assembly 10 includes a detector 40 with a sensor 42 for detecting an emitted optical signal 44 from the specimen 90. The emitted optical signal 44 reflects off the opposite side of the beam splitting mirror 20 through a plurality of beam splitters 38a, b to separate the emitted optical signal 44 into individual signals 44a, b corresponding to the different dyes. Each individual signal 44a, b passes though an emission filter 46a, b and is focused by a detector lens 48a, b into a pinhole. The individual signals 44a, b proceed through the pinhole to contact the individual sensors 42a, b.

The sensors 42a, b are in communication with a computer 80. The first sensor 42a relays a first data set emitted from the first dye and the second dye to the computer 80.

The second sensor 42b relays a second data set emitted from the second dye and the first dye to the computer 80.

The computer 80 is programmed for removing from the first data set the spectral band from the second dye excited by the first optical signal 14a. The computer is also programmed for removing from the first data set the spectral band emitted from the first dye that is excited by the second optical signal 14b.

Further, the computer 80 is programmed for removing from second data set the spectral band from the first dye that is excited by the second optical signal 14b.

Additionally, the computer is programmed for removing from the second data set the spectral band emitted from the second dye that is excited by the first optical signal 14a.

An algorithm is programmed into the computer 80 to facilitate the filtering of the unwanted spectral band that are emitted from the various dyes. First, a ratio of the total spectral bands from the first dye excited by the first optical signal to the band of wavelength from the second dye excited by the first optical signal is calculated. Further, a ratio of the total spectral bands from the second dye excited by the second optical signal to the spectral band from the second dye excited by the first optical is calculated.

A first algorithm is programmed into the computer 80 to remove from the first data set the spectral band from the second dye excited by the first optical signal and to remove the spectral band from the first dye excited by the second optical signal.

Dyel (scan) = Scanl (total)- (Scan2 (total)-Scanl (total) x Ratiol) x Ratio2 wherein: Dyel (scan)-the spectral band from the first dye excluding the spectral bands detected from the second dye; Scanl (total)-the total bands of wavelength detected by the first detector; Scan2 (total)-the total bands of wavelength detected by the second detector; Ratiol-the ratio of the total spectral bands from the first dye excited by the first optical signal to the spectral band from the first dye excited by the first optical signal detected by the second detector; and Ratio2-the ratio of the total spectral bands from the second dye excited by the second optical signal to the spectral band from the second dye excited by the first optical signal detected by the first detector A second algorithm is programmed into the computer 80 to remove from the second data set the spectral band from the first dye excited by the first optical signal and to remove the spectral band from the second dye excited by the first optical signal.

Dye2 (scan) = Scan2 (total)- (Scanl (total)-Scan2 (total) x Ratio2) x Ratio 1 wherein: Dye2 (scan)-the spectral band from the second dye excluding the spectral bands detected from the first dye; Scan2 (total)-the total bands of wavelength detected by the second detector; Scanl (total)-the total bands of wavelength detected by the first detector; Ratio2-the ratio of the total spectral bands from the second dye excited by the second optical signal to the spectral band from the second dye excited by the second optical signal detected by the first detector; and Ratiol-the ratio of the total spectral bands from the first dye excited by the first optical signal to the spectral band from the first dye excited by the second optical signal detected by the second The algorithms perform concurrent calculations utilizing both the predetermined values and the detected wavelength bands to remove unwanted wavelength bands that blur the computerized image of the specimen 90.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, wherein reference numerals are merely for convenience and are not to be in any way limiting, the invention may be practiced otherwise than as specifically described.