MICROSCOPE ACCESSORY AND MICROPLATE APPARATUS FOR MEASURING PHOSPHORESCENCE AND CELLULAR OXYGEN CONSUMPTION
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.
1 . A system for measuring and imaging phosphorescence, comprising:
an inverted microscope having a movable stage and a condenser holder above the motorized stage;
a sample holder positioned on the motorized stage; and
a phosphorescence detecting device having a mounting bracket releasably engageable with the condenser holder, the phosphorescence detecting device being sized to be positioned between the condenser holder and the sample holder.
2 . The system of claim 1 , further including a computer in electrical communication with a power source.
3 . The system of claim 2 , wherein the phosphorescence detecting device further includes:
a substantially vertical frame coupled to the mounting bracket;
a first light source and second light source each coupled to the frame and emitting excitation pulses of light that substantially converge at a point on or within the sample holder, the sample holder re-emitting at least a portion of the excitation light;
a first lens and second lens each coupled to the frame, each lens having a plane and focal point, and the first and second lenses and the movable stage being positionable in relation to each other so as to reposition the sample holder within the focal plane; and
a photon counting apparatus coupled to the second frame and above the first lens, the second lens, and the light convergence point, the photon counting apparatus and focal point of the second lens being substantially aligned with a vertical axis and positioned to receive light re-emitted from the sample holder.
4 . The system of claim 3 , wherein the photon counting apparatus includes:
a sensitive light detector;
a peripheral component interconnect board in electrical communication with the sensitive light detector, the first light source, the second light source, and the computer.
5 . The system of claim 4 , wherein the first and second light sources are either light-emitting diodes or laser diodes, and the sensitive light detector is at least one of an avalanche photodiode or photomultiplier tube.
6 . The system of claim 5 , wherein the first and second light sources are each aligned at an angle from the vertical axis.
7 . The system of claim 6 , wherein the angle is approximately 15 degrees.
8 . The system of claim 5 , wherein the first and second light sources emit excitation pulses of light having a peak wavelength of at least one of approximately 370 nm and approximately 530 nm.
9 . The system of claim 4 , wherein the photon counting apparatus further includes an emission filter substantially blocking the pulses of light emitted from the first and second light sources from passing into the photon counting apparatus and substantially allowing light re-emitted from the sample holder to pass into the photon counting apparatus.
10 . The system of claim 2 , wherein the phosphorescence detecting device further includes:
a substantially vertical frame coupled to the mounting bracket;
an optical device coupled to the frame;
a light source coupled to the frame and generating light that is substantially directed toward the optical device, the optical device redirecting at least some of the light toward the sample holder;
a first lens and second lens each coupled to the frame; and
a photon counting apparatus coupled to the frame above first lens, the second lens, and the optical device, the photon counting apparatus, the first lens, the second lens, and the optical device being substantially aligned with a vertical axis and positioned to receive light re-emitted from the sample holder.
11 . The system of claim 10 , wherein the photon counting apparatus includes:
a sensitive light detector;
a peripheral component interconnect board in electrical communication with the sensitive light detector, the light source, and the computer.
12 . The system of claim 11 , wherein the phosphorescence detecting device further includes:
a fast photodiode coupled to the frame opposite to the light source, the optical device being located between the fast photodiode and the light source;
an analog-to-digital converter in electrical communication with the fast photodiode and the computer; and
an emission filter mounted to the frame between the second lens and sensitive light detector.
13 . The system of claim 12 , wherein the light source is at least one of a light-emitting diode or a laser diode, the light source emitting excitation pulses of light having a peak wavelength of at least one of approximately 370 nm and approximately 530 nm, the excitation pulses of light being between approximately 1 μsec and approximately 10 μsec in duration.
14 . The system of claim 13 , wherein the sensitive light detector is at least one of an avalanche photodiode and a photomultiplier tube.
15 . The system of claim 1 , wherein the sample holder includes a plurality of microwells.
16 . The system of claim 15 , wherein the sample holder further includes a plurality of sensors.
17 . The system of claim 16 , wherein the sensors are selected from the group consisting of: pH sensors, oxygen sensors, fluorescent/phosphorescent probes, phosphorescent fluorescent temperature sensor, nitric oxide sensor, and standard fluorescent probe.
18 . The system of claim 17 , wherein the oxygen sensors are Pt-porphyrin sensors.
19 . A system for measuring and imaging phosphorescence, comprising:
an inverted microscope in electrical communication with a power source and having a movable stage and a condenser holder above the movable stage;
a sample holder positioned on the movable stage;
a computer in electrical communication with a power source and the inverted microscope; and
a phosphorescence detecting device having a mounting bracket that is releasably engageable with the condenser holder, the phosphorescence detecting device including:
a substantially vertical frame coupled to the mounting bracket;
an optical device coupled to the frame;
a light source coupled to the frame and emitting light that is directed along a substantially horizontal axis directed toward the optical device, the optical device redirecting at least some of the light toward the sample holder;
a first lens and a second lens each coupled to the frame and each having a focal point;
a sensitive light detector coupled to the frame above first lens, the second lens, and the optical device, the sensitive light detector, optical device, and focal point of the second lens being substantially aligned with a vertical axis;
a fast photodiode coupled to the frame opposite the light source and in substantially the same horizontal axis as the optical device and light source; and
a peripheral component interconnect board in electrical communication with the sensitive light detector, light source, and computer,
wherein the phosphorescence detecting device is sized to be positioned between the condenser holder and the sample holder.
20 . A device for measuring phosphorescence from a biological sample, the device including:
an accessory bracket;
a substantially vertical frame coupled to the mounting bracket;
a first light source and second light source each coupled to the frame and generating excitation pulses of light that are substantially directed toward a light convergence point;
a first lens and second lens each coupled to the frame and having a focal point and focal plane;
a sensitive light detector coupled to the frame and above the first lens, the second lens, and the light convergence point, the sensitive light detector and focal point of the second lens being substantially aligned with a vertical axis;
a peripheral component interconnect board in electrical communication with the sensitive light detector, first light source, and second light source; and
an emission filter coupled to the frame between the sensitive light detector and second lens,
wherein the peripheral component interconnect board is programmed to control the excitation light pulses and the sensitive light detector.