Theory of Operation

The LI-COR 6400 (LI-6400) utilizes gas exchange principles to measure the photosynthesis rates of plants. Net photosynthesis rates are expressed as rates of CO2 uptake (umolCO2 m-2 s-1). Gas exchange in the LI-COR 6400 is measured in an open-mode design. Previous LICOR’s models (6200, 6000) were based on a closed mode design. In the closed mode, air is kept in the chamber without renewal until leaf photosynthesis drops the original CO2 levels to a prefix value. In the open mode, on the contrary, an air-flow is moved through a controlled atmosphere surrounding a plant leaf enclosed in an assimilation chamber. Thus, the CO2 level of the air is maintained steady- state.

LI-6400 open system chart

Leaf chamber and CO2 analyzer

Description of the Instrument

The hardware of the sytem is the console and the leaf chamber (the sensor head). The leaf chamber has tightly sealed gaskets that do not interact with H2O or CO2, nor are deformed excessively by the leaf midribs. It also houses a PAR light sensor parallel to the leaf plane, a thermocouple, and a speed- variable mixing fan. The sensor head encloses a leaf surface of up to 6 cm2 and has integrated sensors for monitoring light, temperature, H2O and CO2 levels.

LI-6400 console

More importantly, these parameters can be precisely controlled to create the environmental conditions desired. For example, the temperature of the chamber block is controlled by Peltier coolers to any level within +- 6 C of the air temperature. Light can be adjusted at any level from 0 to more than 2000 umol m-2s-1, and CO2 from 0 to 2000umolm-2s-1 as well. Artificial light is optionally supplied by a “cold” lamp (LED type, 670 nanometers). The selected levels of CO2 are supplied by a CO2 injector system. Any combination of light and CO2 level can be selected while the rest of the variables are held constant. Visible light external to the chamber can also be measured with an optional sensor.

LED light source

One distinctive aspect of the LI 6400 is that the CO2 and H2O infrared analyzers are located in the sensor head instead of the console. The minimum distance between the leaf atmosphere and the analyzer allows fast measurements of both CO2 and H2O , which can thus be displayed in real time. The two infrared analyzers, whose readings are averaged, measure the absolute CO2 and H2O concentrations at both the reference flow and the sample itself. CO2 and H2O are discriminated from other IR-absorbing gasses by making reference measurements at 4.1 microns (CO2) and 2.40 microns. Thus, the analyzers avoid background noise.

LI-6400 chamber diagram

All the environmental parameters can be monitored and controlled from the console. Instructions are typed in by means of a ASCII type keyboard. The console is really a datalogging computer that includes an Intel 80C188 processor. The data generated can either be stored temporarly in the Ram memory of the system (1 Mbytes) or stored permanently in a hard disk, or unloaded through a RS-232C port at a very high speed -up to 115,200 bps . The system is powered by four-12V batteries that supply 4-8 hours of operation. Additional sensors can be plugged to the console computer. For doing that, both analog and digital input/output channels are available. Measured and calculated variables, such as photosynthesis rate, conductance and internal CO2 concentrations are displayed in real time.

The task of measuring, control and calculation of variables is performed by a software program termed “Open”, which has an open software architecture. If so desired, the user can either modify the software or write his own programs. Other applications, such as word processors, spreadheets and graphics can be loaded and run in the LI-COR computer as well.

“Open” already includes a program for displaying plots of variables in real time. For example, it can quickly display - during a measurement run- plots of photosynthesis versus internal CO2 concentrations, curves of light response, and curves of photosynthesis and conductance as related to the observations, among others. Most importantly, “Open” includes the so-called “Autoprogram” routines. “Autoprograms” automatically modify the levels of CO2, light and temperature in the chamber accordingly to a set of values pre-selected by the user to generate light and CO2 response curves. The software also averages analyzer signals to increase sensitivity.

There is also optional software for the LI 6400. The “File Exchange” software speeds the transfer of files -up to 115,200 bps- between the LI-6400 and any external computer. Additional software enables the user not only to write and test new programs out of the LI-6400, but also to simulate LI 6400 performance in the external computer itself. Besides, the LI-6400 can be controlled and its information accesed from remote locations.

Functioning of the LI-6400

The LI-6400 has an open-mode design. In the first stage, the CO2 and H2O concentrations of the incoming air are modified by a CO2 scrubber (Mg(ClO4)2) and a desiccant placed in the console. The air flow entering the chamber can be either dry or moist. Humidity control of air is critical since stomata respond markedly to humidity.

Light and temperature sensors

There are two alternatives for achieving the desired CO2 concentrations. One is by reducing the CO2 levels in the incoming air until they reach the selected values. The other, by using the optional CO2 injector system. With the latter alternative, the rate of CO2 injected can be either set as a function of the demand of the leaf (the null-ballance method) or adjusted to keep a constant CO2 concentration entering the chamber.

After being conditioned, the air is pumped in and its flow controlled. Further flow restrictors partitions the air stream, allocating 75% of the flux to the sample (leaf chamber) and the remaining 25% to the reference. The dual IR analyzers monitor the CO2 and H2O of the two air streams and, by difference, calculate CO2 and H2O balance.

Calibration of the LI-6400

Calibration needs are minimal. Both infrared analyzers and photodiodes come already calibrated from factory. Photodiodes provide feedback control for the artificial light source and are calibrated in the 0-2000 umol m-2 s-1 range. Infrared analyzers come calibrated for 0-3000 umol mol-1 (CO2) and 0-75 mmol mol-1 (H2O).

The user needs to fix only the zero and the span of the analyzers. The span is adjusted by passing a standard CO2 concentration through the analyzer. The frequency of span adjustments is reduced by the presence of a barometric sensor, which compensates pressure from one day to the next. Temperature compensation of the analysers is made automatically. The zero is also set automatically after generating a zero reference gass by passing it through the desiccant and the CO2 scrubber.


photographs reproduced with written (E-mail) permission from LI-COR Inc.