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Constructing advanced fluorescence systems for measuring phytoplankton biomass, physiology, and photosynthesis in laboratories and oceans

1. Research purpose and content

Research Objective

The purpose of this project is to construct a small desktop instrument called the F-fluorescence I-induction and R-relaxation (mini FTIR) system for discrete sample analysis and continuous measurement of the abundance and physiological status of phytoplankton in the ocean. Unlike the previous generation FRRF and FIRE fluorescence instruments invented and developed by the Rutgers team, the new instrument will exhibit enhanced sensitivity (about 10 times) and provide more physiological parameters in real-time. new instrumenttallSensitivity makes them of great value for field work in the open sea.

Research Content

Evaluation of photosynthetic activity of phytoplankton and other photosynthetic organisms using variable fluorescence technology-The rapid and non-destructive assessment of the physiological status of photosynthetic organisms relies on the use of rapid repetition rate fluorescence optics (FRRF) and its subsequent fluorescence induction and relaxation (FIRE) techniques. This technology was invented and developed by the Rutgers team. The basic method for evaluating the survival ability of photosynthetic organisms relies on the measurement and analysis of the 'variable fluorescence' profile of chlorophyll, which is a characteristic of the photosynthetic mechanism (reviewed by Falkowski et al. in 2005). The 'variable fluorescence' technology relies on the relationship between chlorophyll fluorescence and the efficiency of photosynthesis processes, and provides a comprehensive set of fluorescence and photosynthesis parameters for organisms. Optical measurement is sensitive, fast, non-destructive, and can be completed in real-time and in situ.

This method and the implemented principles of instrumentation were established in peer-reviewed literature (Falkowski and Kolber 1995; Kolber at al., 1998; Gorbunov et al., 2000, 2001; Gorbunov and Falkowski 2004). Originally developed for studying phytoplankton in the water column, FRR technology providedaccurateProvide information on the functioning of phytoplankton communities and the impact of environmental factors controlling marine primary productivity (e.g. Falkowski and Kolber 1995; Falkowski and Raven 2007; Behrenfeld et al., 1996; Coale et al., 2004; Falkowski et al., 2004). The use of desktop and submersible FRR and FIRE fluorescence meters has become an integral part of most biological oceanography projects in the United States and around the world.

We have developed F fluorescence I induction and R relaxation (FIRe) technologyTo measure a comprehensive set of photosynthetic and physiological characteristics of photosynthetic organisms (Gorbunov and Falkowski 2005). The FIRE technology is based on the recording and analysis of fluorescence transients caused by a series of excited flashes, with precise control over their intensity, duration, and interval (Figure 1 and Gorbunov and Falkowski 2005). This technology provides a comprehensive set of parameters characterized by the photosynthetic daylighting process, photochemistry in photosystem II (PSII), and the transfer of photosynthetic electrons to carbon fixation. Due to the sensitivity of these processes to environmental factors, FIRE technology provides a basis for identifying and diagnosing natural (nutritional limitations, photochemical and photostimulation, thermal stress, etc.) and anthropogenic stress factors (such as pollution).

Figure 1Example of FIRE fluorescence transience. The kinetic record of fluorescence yield is recorded with microsecond time resolution and includes four stages:（oneStage, 100 ms) A strong short pulse of 100 ms (known as single turn flash, STF) is used to accumulate saturated PSII and measure fluorescence induction from Fo to Fm (STF): (Stage 2, 500ms) Weak modulation light is used to record the relaxation kinetic energy of fluorescence production on a 500ms time scale; (Stage 3, 50 ms) A strong long pulse of 50ms duration (known as multi turn flash, MTF) is used to saturate PSII and PQ libraries; (Stage 4, 1 s) Weak modulation light is used to record the kinetics of PQ library re oxidation on a 1-second time scale. The analysis in Phase 1 provides: minimum and up tobigFluorescence yield (Fo, Fm); Quantum efficiency Fv/Fm (STF) of PSII photochemical charge separation; The functional cross-section of PSII, σPSII; And the connectivity factor (p). The second stage provides a time constant for electronic transfer to the PSII receiver (i.e. Qa receptor side re oxidation). The third stage provides Fm (MTF) and Fv/Fm (MTF). The fourth stage revealed the electron transfer time constant between PSII and PSI (re oxidation of PQ library).

The biophysical background of variable fluorescence technology-At room temperature, chlorophyll fluorescence is mainly generated by PSII. When the PSII reaction center is in an open state (Qa oxidation), the fluorescence yield is extremely low, Fo. When Qa is reduced (e.g. by exposure to strong light), the reaction center closes and the fluorescence yield increases to its high level Fm. To detect Fo and Fm, the FIRE technique recorded fluorescence induction caused by strong saturated pulsed light (~100 μ s, known as single turn flash, STF) (Figure 1, stage 1). The fluorescence induction rate is directly proportional to the functional absorption cross-section of PSII, and the relative amplitude of fluorescence increase Fv/Fm is defined by the quantum efficiency of PSII photochemistry. The shape of fluorescence induction is controlled by the excitation transfer between individual photosynthetic units and defined by the 'linking factor' (Kolber et al. 1998). Therefore, in the absence of energy transfer (p=0), fluorescence induction exhibits an exponential trend, and when p increases to a maximum value of~0.5 to 0.7, it becomes an inverse curve.

The kinetic energy of electron transport on the PSII receptor side (i.e. Qa re oxidation) was evaluated through fluorescence pre kinetic analysis after STF (stage 2 of Figure 1). Fluorescence kinetics consists of several parts, as the rate of Qa re oxidation depends on the state of the second electron acceptor Qb, which works as a mobile double electron acceptor:

Qa- Qb → Qa Qb- (150 - 200 ms) (1)

Qa- Qb- → Qa Qb= (600 - 800 ms) (2)

Qa- _ → Qa- Qb → Qa Qb- (~ 2000 ms) (3)

Reaction (3) corresponds to the conditions under which Qb initially dissociates from the D1 protein binding site. In addition, a small number of deactivated reaction centers with impaired electron transport may contribute to the slowest components in the pre kinetic process. The FIRE software uses three components to analyze and process the pre kinetic behavior, in order to retrieve the time constant of electron transport (i.e., Q oxidation tQa).

The time constant tPSII-PSI for electron transfer between PSII and PSI was retrieved from fluorescence pre kinetic analysis after multi turn flash (MTF, stages 3 and 4 in Figure 1). Under most physiological conditions, this time constant is determined by the rate of re oxidation of the plastid quinone (PQ) library, and is one order of magnitude slower than tQa.

Measuring a series of FIRe fluorescence parameters of environmental light intensity can reconstruct the rate of electron transport in photosynthesis, Pf， As a function of light intensity (photosynthesis and light intensity curve) (Kolber and Falkowski, 1993). Pf is directly proportional to the photochemical quantum yield measured under illumination products and ambient light (DF '/Fm'). The analysis of these photosynthesis and light intensity curves provides information on the rate of high electron transfer (Pmax) and light saturation coefficient (Ek) during photosynthesis. Photosynthesis and radiation measurements are carried out using a FIRE photochemical light source (ALS), which is controlled by a computer through FIRE data acquisition software.

Research and development background and professional knowledgeMembers of the Rutgers team have accumulated over 20 years of experience in the development of variable fluorescence technology and methods. They have invented and developed over 10 instruments for biophysical research (see Appendix Reference List in relevant peer-reviewed publications).

2. Instrument Introduction

Mini FTIR is based on the same biophysical principles as the previous desktop FIRE instrument (Gorbunov and Falkowski 2005), but the new instrument is three times more compact and has 10 times higher sensitivity. The lower limit of chlorophyll concentration is as low as~0.005 mg/m3, which makes mini FIRE very valuable for on-site sampling in the open sea.

Here, the Rutgers team proposes to build a mini FTIR (Figure 2) instrument that will be used for discrete sample analysis (e.g. samples collected from Niskin bottles at stations) and/or continuous sampling in the ocean. The instrument will be equipped with a flowing sample chamber for continuous plotting of phytoplankton biomass and photosynthetic characteristics. The following is a list of physiological parameters recorded by mini FTIR and the technical specifications of the instrument mini FTIR (Figure 2). This instrument will be used for discrete sample analysis (such as samples collected from Niskin bottles at stations) and/or continuous sampling in the ocean. The instrument will be equipped with a flowing sample chamber for continuous plotting of phytoplankton biomass and photosynthetic characteristics. The following is a list of physiological parameters and instrument technical specifications recorded by mini FTIR.

Figure 2: Mini FTIR fluorescence analyzer with enhanced sensitivity.

Measurement parameters:

Minimum and maximum fluorescence production after dark adaptation (Fo, Fm)

Effective, minimum, and maximum fluorescence production under light adaptation (F ', Fo', Fm ')*

Photochemical to high effective quantum yield (Fv/Fm and DF '/F m) in photosystem II and PSII

Functional PSII absorption cross-sectional area at three wavelengths (sPSII)

Energy transfer efficiency between photosynthetic units ('connection factor ')

PSII receptor side electron transfer time constants (Qa to Qb, Qa to Qb -)

The time constant of photosynthetic electron transfer between PSII and PSI

● Electronic transfer rate, ETR， As a function of light intensity*

Photochemical quenching coefficient (qP) and non photochemical quenching coefficient (NPQ)*

● Maximum photosynthetic rate, initial slope, and photosynthetic turnover time (obtained from the F and E curves)

These parameters were measured using an actinic light source (ALS) and recorded as light intensity curves.

Technical specifications of mini FTIR system:

Sensitivity: 0.005-100 mg/m3 chlorophyll a (sampling concentration can be increased by adding a neutral density vacuum filter)

● Excitation light source: blue (peak wavelength 450 nm, 30 nm bandwidth), green (peak wavelength 530 nm, 40 nm bandwidth), orange (peak wavelength 590 nm, 30 nm bandwidth), used to selectively excite different functional groups of phytoplankton.

Emission detection: 680 nm (chlorophyll-a) and 880 nm (bacterial chlorophyll-a), other wavelengths can be selected using replaceable emission filters.

● Size: 10 x 5 x 12 inches