Fluorometer for Analysis of Photosynthesis in Phytoplankton

Measurements could be made in situ and in vivo.

Stennis Space Center, Mississippi

An instrument that measures the characteristic lifetime of fluorescence of chlorophyll has been invented for in situ, real-time oceanographic studies of photosynthesis in phytoplankton. The basic design and principle of operation lend themselves to development of the instrument as a relatively inexpensive, sensitive, compact, rugged, portable, low-power-consumption, hand-held, shipboard unit. Similar units with designs adapted to agricultural applications (e.g., assessment of physiological statuses of crops) are also envisioned.

The need for this or a similar instrument arises because fluorescence lifetimes are robust measures of physical and chemical mechanisms that affect photosynthesis and of the photosynthetic productivity of phytoplankton. The fluorescence lifetimes of particular interest are those associated with fluorescent de-excitation in photosystem II. ("Photosystem II" denotes a series of photosynthetic reactions in which a pigment absorbs light at wavelengths up to 680 nm and absorbed light energy causes the splitting of water molecules, giving rise to oxygen and to a high-energy reductant.) These lifetimes range from about 2 ns down to as little as hundreds of picoseconds. Therefore, in order to exploit the full potential of fluorescence-lifetime measurements for detecting small changes in photosystem-II physiological status, this or any similar instrument would have to exhibit an error <50 ps and a resolution 10 ps.

The present instrument does not measure fluorescence lifetimes directly; instead, it is based on a principle of phase fluorometry, which can be implemented more easily. In phase fluorometry, a sample is excited with light modulated sinusoidally at an angular frequency w. The resulting fluorescence emitted by the sample is modulated at the same frequency but, because of the finite lifetime of the excited state, is delayed in phase by an angle f relative to the excitation. The phase angle Ø is measured and used to calculate a phase lifetime (_p) according to _p = tan Ø/w. In order to be able to determine t_p accurately by this method, one must choose a modulation frequency comparable to the rate of decay of the fluorescence.

In the instrument (see figure), a baseband signal of frequency f₀ (typically 600 Hz) is generated for use both as a sample signal and as a reference signal for the phase measurement. Using a single-sideband technique, the sample signal is up-converted by combining it with a carrier signal of frequency f (typically 70 MHz). The up-converted signal (at frequency f₀ + f) is used to modulate a light-emitting diode (typical wavelength 670 nm), the output of which is used to excite the sample.

The fluorescence emitted by the sample acquires a phase delay that corresponds to a frequency-dependent weighted average of the lifetimes of the fluorophores present in the sample. The fluorescence, which is modulated at frequency f₀ + f, is detected by a photomultiplier tube. The electrical output of the photomultiplier is down-converted to f₀ by mixing it with the carrier signal and low-pass filtering the product signal. The phase information acquired by the up-converted signal through interaction with the sample is preserved in the down-converted signal. The phase-shifted down-converted f₀ sample signal and the reference f₀ signal are captured by an analog-to-digital converter, and the output of the analog-to-digital converter is stored and processed to determine Ø and _p.

This Instrument Measures the Phase Difference between sinusoidally modulated optical excitation and the resulting fluorescence. The fluorescence lifetime is then calculated from the phase difference and the modulation frequency.

 

This work was done by Salvador M. Fernandez, Ernest F. Guignon, Robert Kersten, and Ernest St. Louis of Ciencia, Inc., for Stennis Space Center. For further information, access the Technical Support Package (TSP) free on-line at www.nasatech.com under the Physical Sciences category.

In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to

Ciencia, Inc.

111 Roberts Street

Suite K

East Hartford, CT 06108

Refer to SSC-00110, volume and number of this NASA Tech Briefs issue, and the page number.

 

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