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

a microplate apparatus and accessory technology, applied in the field of microscopy, can solve the problems of contributing significantly to the cost of the system, not working well with very small wells, and not providing the necessary sensitivity to measure single-cell oxygen consumption

Inactive Publication Date: 2012-06-21
UNIV OF MIAMI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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.

Method used

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

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first embodiment

[0023]Referring now to FIGS. 1A (front view) and 1B (side view), schematic representations of a system 10 and device 12 in accordance with the principles of the present invention are shown. The system 10 may generally include a phosphorescence measurement device 12, a sample holder 14 having a plurality of sample wells (or “microwells”) 16 in which one or more samples 18 may be contained, and an inverted microscope 20 for imaging the samples 18. The sample holder 14 may further include one or more sensors 22 for measuring oxygen levels, changes in pH, or other parameters.

[0024]The inverted microscope 20 may further include a condenser holder 23, a movable (for example, motorized) x-y and / or x-y-z stage 24 at a predetermined or variable distance from the condenser holder and in electrical communication with a stage controller 26. The inverted microscope 20 may further include one or more microscope objective lenses 28 for imaging the samples 18. Additionally, the inverted microscope ...

second embodiment

[0037]Referring now to FIG. 3, a device that is sized to fit into the condenser holder is shown. The device 12 shown in FIG. 3 may be representative of the configuration of a device 12 according to FIG. 2 (second embodiment). For example, the device 12 shown in FIG. 3 may include a mounting bracket 23, an avalanche photodiode 36, an emission filter 41, a relay lens 46, a light source 34, an optical device 48, a fast photodiode 54, a lens mounting bracket 47, and a main lens 44. The device 12 may further include other elements shown and described in FIG. 2.

[0038]As an example of the phosphorescence-measuring functionality of the system 10 as shown in any of FIGS. 1A, 1B, and 2, a phosphorescence measurement cycle begins with a short (1 to 10 μsec) excitation light pulse from the one or more light sources 34 directed toward the samples 18 within the sample holder 14. The light may be sent directly to the samples 18 either through one or more lenses 44, 46 (as in FIGS. 1A and 1B) or th...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N21/64
CPCG01N33/84
Inventor BARRETT, JOHN
Owner UNIV OF MIAMI
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