Quantified Fluorescence Microscopy

A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising:

• a polymer layer comprising at least one fluorescent agent, wherein said device has minimal local and global nonuniformities;
• and a transparent substrate;
• wherein said device is dimensioned for placement in an optical scanner.

A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising:

• a polymer layer comprising at least one fluorescent agent, wherein said device has minimal local and global nonuniformities;
• and a substrate;
• wherein said device is dimensioned for placement in an optical scanner.

A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising:

• a polymer layer comprising at least one fluorescent agent, wherein said device is dimensioned for placement in an optical scanner, and wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.

A method for manufacturing a calibration tool for a fluorescent microscope that is useful to image an array, comprising

• providing a support, providing a solid surface layer including a fluorescent material, and
• fabricating a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of the surface layer;
• forming the solid surface layer by depositing it onto the support, comprising depositing an adhesion promoting layer onto the support for forming the solid surface layer including the fluorescent material, the deposition includes evaporating or sputtering, the depositing includes spin coating to form the solid surface layer including the fluorescent material.

A method for using a fluorescent microscope to image a bioploymer array, comprising exciting the array at 473 nanometers, producing ½ volt at a photomultiplyer tube in the microscope, using a fluorophore having a fluorescent efficiency that is approximately 1×10−6 or higher, and using a calibration tool.