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Publication Date: 2009-Jul-21

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The Prior Art Database

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The present invention relates, in general, to confocal fluorescence microscopes. More specifically, the present invention relates to determining an optimal focal depth of a confocal fluorescence microscope used in a microarray scanner.

Microarray scanners are known in the art for imaging deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) microarrays. These scanners use confocal fluorescence microscopes for imaging DNA or RNA samples. In a confocal fluorescence microscope, an illumination beam is focused at a point on a fluorescent specimen. The illumination beam reflects from a dichromatic mirror (also known as dichroic mirror) into an objective lens which focuses the illumination beam on the fluorescent specimen. The fluorescent specimen fluoresces due to excitation provided by the illumination beam and emits a fluorescent beam. The emitted fluorescent beam travels along the path of the illumination beam and reaches the dichromatic mirror. The dichromatic mirror allows the passage of emitted fluorescent beam but reflects the illumination beam. Subsequently, a detector detects the emitted fluorescent beam coming from the dichromatic mirror.

A position, at which the illumination beam and the emitted fluorescent beam are mutually focused, is called a focal point of the confocal fluorescence microscope. In confocal microscope systems, where fluorescence arises from a reflective surface (such as a DNA microarray), an autofocus system may use the emitted fluorescent beam to determine a position of the reflective surface with respect to a plane which contains the focal point. However, the autofocus system, used in modern microscopes, needs to be calibrated to establish a relationship between a reflection of the illumination beam and the focal point. For example, if an illumination beam is offset in a predetermined direction, then as distance between the objective lens and the reflective surface is varied, the reflection of the offset illumination beam will move along the offset direction. Using a position sensitive detector (PSD), a user can measure the distance of the reflective surface from the focal point. The confocal fluorescence microscope needs to be calibrated to establish the position on the PSD that corresponds to the focal point.

 Typically, an optimal depth of the focal point is determined in microarray scanners (e.g. Agilent A, B and C scanners) by performing a ‘depth scan’. The depth scan includes scanning a test slide, having a uniform fluorescence region, placed at a series of test focus depths. The test focus depths are incremental distances from the objective lens towards the focal point. The depth scan detects fluorescence signal intensity at multiple test focus depths. The detected fluorescence signal intensity is a maximum for a range...