Steady-State Leser Fluorometry in Sea-Water Phyto – and Zooplankton analysis
Andrey A. Demidov Elina A. Chernyavskaya
Moscow State University, Physics Department
Moscow 119899, Lenin Hills, USSR
Abstract
There is developed the new promising methods for the plankton investigations based on the steady-state He-Cd laser (
l=440 nm). This methods uses the fluorescence of phyto – and zooplankton pigments excited by a continuous laser light. There is detected simultaneously the water Raman signal as a calibration signal. Developed methods was tested in a laboratory as well as in a native sea-water conditions ( South Atlantic). They can be used in analyzing of intact algae in detect chlorophyll a concentration in native intact algae up to 10 nanogram/liter without concentration a probe; b) to estimate the DCMU; c) to detect chlorophyll a and pheophytin a concentrations in acetone extracts of phyto – and zooplankton up to 1 nonogram / liter; d ) to estimate the photosynthetic activity of algae without using of any chemicals like DCMU; c) to detect chlorophyll a and pheophytin a concentrations in acetone extracts of phyto – and zooplankton up to 1 nanogram / liter; d) to control the feed process of individual sample of zooplankton. Created He-Cd fluorosensor has very simple construction. It is cheap and easy in use.
Introduction
Laser remote sensing method are widely use now in the ocean logy practice, particularly, in the phytoplankton chlorophyll analysis [ 1-6]. Commonly, they use the powerful laser pulses to excite the algae pigments fluorescence which carries on the information of a phytoplankton. The most popular lasers in these investigations are YAG: Nd+3 laser ( 8 = 532 nm), nitrogen laser ( 337 nm) and dye lasers (tunable wavelength ). the use of lasers enables one to develop express, distant, non contact and nondestructive methods for a phytoplankton investigations.
In our research we try to use the steady-state He-Cd laser (
l=440 nm). Our methods are based on the principles and ideas developed before for the YAG laser application for the phytoplankton and zooplankton pigments measurements [ 3-7]. The use of YAG laser fluorosensor enables one to measure the intact algae chlorophyll a (chl a ) fluorescence in native water ( without any concentration procedure ) with a high biological productivity down to the mesotrophic waters, but it doesn’t work in the oligotrophic waters because of a not sufficient sensitivity.
We hoped that this problem can be overcomed with the use of the He-Cd laser because this laser can more efficiently excite the chl a fluorescence ( in vivo ) as well as in vitro ) than in the case of YAG laser. The He-Cd laser light is absorbed by chl a in the Soret line, Which has the maximum absorption coefficient. Besides, in this case the Raman signal (I
R) and chl a fluorescence (I
f1) are better separated and as a result the smaller ratio
F=I
f1/I
R can be achieved ( see below ). All these facts increase the sensitivity of our laser fluorometry method {8}.
Materials and Methods
In Our research we have studied a lot of different sea-water plankton objects:
- About 60 probes of a different algae cultures ( Gymnodinium, Sceletonema costatun, Prorocentrum micans, Platymonas viridis ) – laboratory experiments;
- Acetone extracts of marine phytoplankton ( from the same bathometers ) that was prepared and controlled in accordance with standard method [ 9-11] – in vitro experiments;
- acetone extracts of marine phytoplankton ( from the same bathometers ) that was prepared and controlled in accordance with a standard method [9-11] – in vitro experiments;
- hundreds of acetone extracts of a countable number ( up to one sample ) of zooplankton samples that was made in accordance with the work [7].
In our research we used the simplest laser fluorometer constructed by us ( tested in the ‘VITYAZ’ trip, 1990). This fluorometer had the steady-state He-Cd laser ( 8=440 nm, beam power P=20, mWt), the tube with the amplifier, the voltmeter and the photomultiplaying tube with the amplifier, the voltmeter and the plotter. Plotter got us spectra of a probe response signals. Besides, in the laboratory we used the more complex fluorometer based on the optical multichannel analyzer OMA-1 ( PARC, USA).
In this figures the ‘short wavelength ’ signal (I
R) is the Raman scattering of a laser light by water ( Fig. 2) or acetone ( Fig.3) molecules; the ‘long wavelength’ signal (I
f1) is the pigments fluorescence ( for example, ch1 a, fluorescence). In the words [4-6] there were proofed that the ‘fluorescence parameter’
F=I
f1/I
R is independent on the experiment conditions ( the internal calibration principle, I
R calibration signal ) and is linearly connected with the fluorescent pigments concentration C = "M, where " is a parameter. This linear dependence takes place if the because of that the algae functional state can’t influence to the " parameter.
Phytoplankton Photosynthetic Activity Diagnostic
Laser light has enough intensity to influence to the process of a closure of algae photosynthetic reaction centers (RC). and it can be used in the algae photosynthetic activity estimation without involving of any chemicals like DCMU. As we have shown in our research, algae fluorescence behaviors is connected with the laser photo induced driving of a functional states of the algae photosynthetic RC. In turn the RC opf algae ch1 a fluorescence. The physical mechanism of this non-linear phenomena is very simple. When
r<10
-8 ( Fig.1, ‘ low intensity’ region ) the rate of a laser photons absorption by a ch1 a molecules of an algae light – harvesting antenna is less then the rate.
Of Exactions “Utilization” In The RC and then
F=
Fmin. Here,
r=4F, where 4 is the free ch1 a molecules excitation decay time, F is the laser light absorption cross-section, F is the laser light beam photon density, [ photon. sm. s. }. As soon as this balance turns over RC
Fig. 1. Dependence of fluorescence parameter
F on a relative laser beam photon density r; (+) – fluorescence of a Proprocentrum micans, (.) – theoretical calculation, ( _______) – computer spline approximation on the theoretical points.
Would be locked by excitons utilization procedure and reaction centers couldn’t efficiently trap antenna excitions and fluorescence would rise and achieve
M=
M at the
r > 10
-6 ( ‘high intensity’ region).
In the ‘high intensity’ mode the value of a fluorescence parameter (
Fmax) isn’t influenced by a initial algae functional state but in the ‘low intensity’ mode this dependence takes place. So the ration
Fmax /
Fmax will characterize the algae photosynthetic activity.
