Multiple protocol fluorometer and method
First Claim
1. A method of stimulating and analyzing a fluorescence signal in a sample, the fluorescence signal being measured to analyze parameters of the sample, said method comprising the step of:
- (i) exciting the sample with a sequence of flashlets having a duration of about 0.5-1 μ
s each and time intervals ranging from about 0.5 μ
s to 2.0 μ
s, to saturate PSII reaction centers in the sample within about 40-80 μ
s.
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Abstract
A multiple protocol fluorometer measures photosynthetic parameters of phytoplankton and higher plants using actively stimulated fluorescence protocols. The measured parameters include spectrally-resolved functional and optical absorption cross sections of PSII, extent of energy transfer between reaction centers of PSII, F0 (minimal), Fm (maximal) and Fv (variable) components of PSII fluorescence, photochemical and non-photochemical quenching, size of the plastoquinone (PQ) pool, and the kinetics of electron transport between Qa and PQ pool and between PQ pool and PSI. The multiple protocol fluorometer, in one embodiment, is equipped with an excitation source having a controlled spectral output range between 420 nm and 555 nm and capable of generating flashlets having a duration of 0.125-32 μs, an interval between 0.5 μs and 2 seconds, and peak optical power of up to 2 W/cm2. The excitation source is also capable of generating, simultaneous with the flashlets, a controlled continuous, background illumination.
85 Citations
34 Claims
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1. A method of stimulating and analyzing a fluorescence signal in a sample, the fluorescence signal being measured to analyze parameters of the sample, said method comprising the step of:
(i) exciting the sample with a sequence of flashlets having a duration of about 0.5-1 μ
s each and time intervals ranging from about 0.5 μ
s to 2.0 μ
s, to saturate PSII reaction centers in the sample within about 40-80 μ
s.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of stimulating and analyzing a fluorescence signal in a sample, the fluorescence signal being measured to analyze parameters of the sample, said method comprising the steps of:
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(i) exciting the sample with a sequence of between 40-80 flashlets having a duration of about 0.5-1 μ
s each and time intervals ranging from about 0.5 μ
s to 2.0 μ
s, to saturate PSII reaction centers in the sample within about 40-80 μ
s;(ii) exciting the sample a second time with between 40-80 flashlets having a duration of about 0.125-0.5 μ
s each and time intervals exponentially increased from about 50 μ
s to 20 ms; and(iii) repeating steps (i) and (ii) of exciting about 15-30 times with about 40-80 flashlets, wherein total time interval of each repeated step (ii) of exciting ranges between about 200 μ
s to 10 ms.
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13. A method of stimulating a fluorescence signal in a sample, the fluorescence signal being measured to determine parameters of the sample, said method comprising the step of:
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exciting the sample with about 2000-10000 flashlets; and individually controlling each flashlet length and each time interval between the flashlets so that energy output of each flashlet is maintained at a constant level.
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14. A method of stimulating a fluorescence signal in a sample, the fluorescence signal being measured to determine parameters of the sample, said method comprising the step of:
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exciting the sample with a sequence of N flashlets; measuring the fluorescence signal of the excited sample; and continuously adjusting one of N, flashlet length and time intervals between flashlets to maintain the fluorescence signal at a predetermined level.
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15. A fluorometer for stimulating and analyzing a fluorescence signal in a sample, comprising:
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an excitation source for producing flashlets; a flash control circuit for controlling number, length and time interval of the flashlets; and a fluorescence detector for measuring the fluorescent light generated by the sample in response to flashlets produced by the excitation source. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A system for stimulating and analyzing a fluorescence signal in a sample comprising:
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an excitation source that generates both a sequence of flashlets and a steady state background irradiance, wherein the protocol includes flashlets having a duration of about 0.125 μ
s-2.0s and time intervals between the flashlets ranging from about 0.5 μ
s-100 μ
s;an excitation control circuit that controls both the sequence of flashlets and the steady state background irradiance; an excitation/emission optical system that guides the flashlets to the sample and guides an emitted fluorescent light from the sample; a fluorescence detector that detects the emitted fluorescent light and generates a fluorescence signal; a data acquisition circuit that processes the fluorescence signal and generates a digitized fluorescence and reference signal; and a controller that processes the digitized fluorescence and reference signal to calculate photosynthetic parameters of the sample. - View Dependent Claims (30, 31, 32, 33, 34)
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Specification