Methods of generating florescence resonance energy transfer (fret) between semiconductor quantum dots and fluorescent dyes/proteins via multi-photon excitation, achieving zero background or direct excitation contributions to the fret signature

A system for sensing physiological conditions in biological applications in a nanoscale sensing system, the system comprising:

• a nanoscale sensing system associated with a plurality of automated measurement instrumentation for preparing a plurality of acceptors in the nanoscale sensing system, wherein one automated measurement instrument of the plurality of automated measurement instrumentation is a photon counting system having a computer processor;
• a pulsed laser source communicatively coupled to the nanoscale sensing system and the plurality of automated measurement instrumentation, wherein the nanoscale sensing system includes a nanocrystal structure;
• an input/output device;
• a data control bus, wherein the data control bus communicatively couples the computer processor of the photon counting system to the pulsed laser source, to the nanoscale sensing system, and to the plurality of automated measurement instrumentation, and to the input/output device;
• and
• a memory, residing in the computer processor, having a dynamic repository and a program unit containing a computer readable and a computer executable program;
• wherein when the computer executable program is executed by the computer processor, the computer executable program causes the system for sensing physiological conditions in biological applications to perform operations including:
• directly optically exciting, with the pulsed laser source, a plurality of donors and the plurality of acceptors in the nanoscale sensing system, wherein the pulsed laser source includes one of pulsed and continuous wave excitation sources;
• transferring energy, in an energy transfer process, between the plurality of donors and the plurality of acceptors;
• investigating, with automated measuring instrumentation, stability of self-assembly of the plurality of acceptors, within the plurality of donors;
• detecting, using a photo detector, changes in the nanoscale sensing system, wherein detecting provides one of a biological specificity for specific targets and compatability with biological environments;
• and
• sensing, using a spectrometer, fluorescence spectra of physiological conditions at a cellular level, wherein sensing, using the spectrometer, fluorescence spectra of physiological conditions includes a transducing of signals into immunoassays, clinical diagnostics and cellular imaging to provide treatment to biological subjects at the cellular level, wherein the transducing of signals by the multi-photon excitation system removes corrupting influences of directly optically exciting the plurality of acceptors, while preferentially directly optically exciting the plurality of donors.

A method of sensing physiological conditions in biological applications using a two photon excitation system having a continuous wave laser source and automated measuring instrumentation in a nanoscale sensing system, the method comprising:

• preparing a plurality of donors and acceptors in the nanoscale sensing system;
• optically exciting, with the continuous wave laser source, the plurality of donors and acceptors;
• transferring energy, between the plurality of donors and acceptors;
• investigating, with automated measuring instrumentation, stability of self assembly of acceptors bound to donors;
• detecting changes in the nanoscale sensing system;
• and
• sensing, using a spectrometer, fluorescence spectra of physiological conditions at a cellular level, wherein sensing, includes a transducing signals into immunoassays, clinical diagnostics and cellular imaging, while removing corrupting influences of optically exciting acceptors, and while preferentially optically exciting donors.