Microscope accessory and microplate apparatus for measuring phosphorescence and cellular oxygen consumption

Abstract

A device and system that allows an inverted microscope with a movable x-y stage to also function as a time-resolved plate reader. The device, which is sized to fit into the condenser holder of an inverted microscope, not only provides plate-reading function at very low cost compared to that of purchasing a plate reader, but it also allows the correlation of plate reader measurements with images of a specimen. The device and system is also able to measure other parameters associated with cell metabolism, such as pH changes.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is related to and claims priority to U.S. Provisional Patent Application Ser. No. 61 / 423,670, filed Dec. 16, 2010, entitled PLATEREADER DEVICE FOR MEASURING SINGLE CELL OXYGEN CONSUMPTION, and U.S. Provisional Patent Application Ser. No. 61 / 546,692, filed Oct. 13, 2011, entitled MICROSCOPE ACCESSORY FOR WHOLE WELL FLUORESCENCE AND TIME RESOLVED PHOSPHORESCENCE MEASUREMENTS, the entirety of both of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]n / aFIELD OF THE INVENTION[0003]The present invention relates to the field of microscopy and, in particular, to adapting a traditional microscope system to read fluorescence / phosphorescence signals with high accuracy. It also relates to the field of using phosphorescent sensors to measure cellular oxygen consumption and to platereader technology with small microwells.BACKGROUND OF THE INVENTION[0004]Fluorescence and time...

AI Technical Summary

Benefits of technology

The present invention provides a system and device for measuring and imaging phosphorescence using an inverted microscope with a movable stage and a phosphorescence detecting device. The system includes a computer, an optical device, a light source, and a phosphorescence detecting device. The phosphorescence detecting device includes a mounting bracket that is releasably engageable with the condenser holder, a sample holder positioned on the movable stage, a first lens and a second lens each coupled to the frame, a sensitive light detector, and a peripheral component interconnect board in electrical communication with the computer. The system can also include a fast photodiode and an emission filter. The sample holder can include a plurality of microwells and sensors for measuring pH, oxygen, fluorescent / phosphorescent probes, phosphorescence, and standard fluorescent probe. The system can also include a phosphorescence detecting device that includes a mounting bracket, a sample holder, a first lens, a second lens, a sensitive light detector, and a fast photodiode. The device is sized to be positioned between the condenser holder and the sample holder. The technical effects of the invention include improved accuracy and efficiency in measuring and imaging phosphorescence, as well as the ability to measure and image other biological processes using the same equipment.

Problems solved by technology

Although plate reader systems, such as those used with enzyme-linked immunosorbent assay (ELISA) reader, may efficiently and accurately measure biological parameters of a sample, they have associated disadvantages.

Also, most platereaders do not work well with very small wells, for example, of less than 500-micron diameter.

However, the larger well sizes of standard microtitre plates (which are often as large as 100 to 200 microliters) fail to provide the necessary sensitivity to measure single-cell oxygen consumption.

Finally, typical methods for measuring oxygen consumption with very small samples use charge coupled device (CCD) cameras for detecting phosphorescence, which can contribute significantly to the cost of the system.

Not only are CCD cameras costly, but they can also be bulky and have a limited effective dynamic range (typically 8-10 bits).

However, an inverted microscope is not capable of taking the biological activity measurements of a plate reader, such as time-resolved phosphorescence measurements or very high accuracy quantitation of fluorescence changes with time.

One problem with relying on fluorescence, however, is that body tissue may contribute a significant amount of background fluorescence noise.

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