Monitoring temperature with fluorescence

A method of measuring a temperature of a sample, comprising:

• providing a sample that includes:
• a nucleic acid;
• a temperature-sensitive fluorescent dye that, in response to a stimulus, emits a temperature-sensitive fluorescent signal that:
• in the presence of the nucleic acid, is not directly proportional to an amount of the nucleic acid present in the sample, is not affected by dsDNA denaturation, or is substantially independent of the amount of the nucleic acid present in the sample;
• and
• changes as a function of temperature in a known manner;
• and
• a second fluorescent dye that produces a second fluorescent signal,
• wherein the second fluorescent signal is indicative of an amount of double-stranded nucleic acid present in the sample;
• exciting the temperature-sensitive fluorescent dye to induce emission of the temperature-sensitive fluorescent signal;
• measuring the temperature-sensitive fluorescent signal emitted from the temperature-sensitive fluorescent dye;
• determining a fluorescence-determined temperature of the sample based on the temperature-sensitive fluorescent signal emitted by the temperature-sensitive fluorescent dye while melting the nucleic acid and measuring the signal indicative of the amount of double-stranded nucleic acid present;
• and
• generating a melting curve based on the second fluorescent signal, the melting curve being adjusted based on the fluorescence-determined temperature, wherein the melting curve is generated using the formula
• T
• =
• 1
• ln
• ⁡
• (
• I
• ⁢
• /
• ⁢
• I
• ref
• )
• C
• +
• (
• 1
• ⁢
• /
• ⁢
• T
• ref
• )
• wherein I is a measured fluorescent intensity of the temperature-sensitive reagent at T temperature, Iref is a fluorescent intensity at a reference temperature Tref, and C is a calibration constant equal to
• ln
• ⁢
• ⁢
• (
• 1
• I
• ref
• )
• /
• (
• 1
• T
• -
• 1
• T
• ref
• )
• .

A method of calibrating a sample heating device, comprising:

• disposing a sample in the sample heating device, the sample including a temperature-sensitive fluorescent dye that, in response to a stimulus, emits a temperature-sensitive fluorescent signal that:
• in the presence of a nucleic acid, is not directly proportional to an amount of the nucleic acid, is not affected by dsDNA denaturation, or is substantially independent of the amount of the nucleic acid;
• and
• changes as a function of temperature in a known and predictable manner;
• stimulating the temperature-sensitive fluorescent dye to induce emission of the temperature-sensitive fluorescent signal therefrom;
• determining a fluorescence-determined temperature of the sample based on the temperature-sensitive fluorescent signal emitted by the temperature-sensitive fluorescent dye;
• determining a device-determined temperature for the sample;
• and
• adjusting a temperature setting of the sample heating device based on at least the fluorescence-determined temperature.

A thermocycling system configured to employ temperature-dependent fluorescence as an indication of internal sample temperature, the system comprising:

• a sample vessel having an aqueous sample disposed therein, the aqueous sample comprising an amount of a nucleic acid and a temperature-sensitive fluorescent dye that, in response to a stimulus, emits a temperature-sensitive fluorescent signal that:
• in the presence of a nucleic acid, is not directly proportional to the amount of the nucleic acid present in the sample, is not affected by dsDNA denaturation, or is substantially independent of the amount of the nucleic acid present in the sample;
• and
• changes as a function of temperature in a known manner;
• a sample temperature controlling device configured to manipulate a temperature of the sample;
• a sample temperature control mechanism configured to utilize the sample temperature controlling device to regulate the temperature of the sample;
• wherein the sample temperature control mechanism comprises a sample temperature raising mechanism and a sample temperature lowering mechanism;
• and
• a sample fluorescence measuring element configured to quantify fluorescence of the sample;
• wherein the sample temperature control mechanism regulates the temperature of the sample based on the fluorescence of the sample.

A PCR mixture, comprising:

• an aqueous fluid;
• and, disposed in the aqueous fluid:
• a nucleic acid;
• and
• a temperature-sensitive fluorescent dye that emits, in response to a stimulus, a fluorescent signal that:
• in the presence of a nucleic acid, is not directly proportional to an amount of the nucleic acid present in the sample, is not affected by dsDNA denaturation, or is substantially independent of an amount of the nucleic acid present in the sample;
• and
• changes between 95° C. and 50° C. as a function of temperature in a known manner.

A method of controlling thermal cycling through a thermocycling profile of a sample using feedback control, the method comprising:

• providing a sample at a first temperature, wherein the sample includes a temperature-sensitive fluorescent dye that, in response to a stimulus, emits a temperature-sensitive fluorescent signal that:
• in the presence of a nucleic acid, is not directly proportional to an amount of the nucleic acid present in the sample, is not affected by dsDNA denaturation, or is substantially independent of an amount of the nucleic acid present in the sample;
• and
• changes as a function of temperature in a known manner;
• stimulating the temperature-sensitive fluorescent dye to induce emission of the temperature-sensitive fluorescent signal therefrom;
• detecting the temperature-sensitive fluorescent signal emitted by the temperature-sensitive fluorescent dye;
• and
• changing to a next phase in the thermocycling profile upon detecting a predetermined value of the temperature-sensitive fluorescent signal,
• wherein the predetermined value of the temperature-sensitive fluorescent signal indicates an appropriate time to initiate the change to the next phase in the thermocycling profile.