Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples.

A new light microscope-temperature-controlled chamber (LM-TCC) has been constructed. The special feature of the light microscope-temperature-controlled chamber is the Peltier-element temperature control of a specimen holder for biological samples, with a volume capacity of 1 mL. This system has marked advantages when compared to other approaches for temperature-controlled microscopy. It works in a temperature range of -10 degrees C to +95 degrees C with an accuracy of +/-0.1 degrees C in the stationary phase. The light microscope-temperature-controlled chamber allows rapid temperature shift rates. A maximum heating rate of 12.9 degrees C min(-1) and a maximum cooling rate of 6.0 degrees C min(-1) are achieved with minimized overshoots (<or=1.9 degrees C). This machinery operates at low cost and external coolants are not required. Especially with samples absorbing irradiation strongly, temperature control during microscopy is necessary to avoid overheating of samples. For example, leaf segments of Ficaria verna exposed to 4500 micromol photons m(-2) s(-1) in a standard microscopic preparation show a temperature increase (deltaT) of 18.0 degrees C, whereas in the light microscope-temperature-controlled chamber this is reduced to 4 degrees C. The kinetics of microscope-light induced deltaT are described and infrared thermography demonstrates the dissipation of the temperature. Chloroplasts of the cold adapted plant Ranunculus glacialis show the tendency to form stroma-filled protrusions in relation to the exposure temperature. The relative number of chloroplasts with protrusions is reduced at 5 degrees C when compared to 25 degrees C. This effect is reversible. The new light microscope-temperature-controlled chamber will be useful in a wide range of biological applications where a rapid change of temperature during microscopic observations is necessary or has to be avoided allowing a simulation of ecologically relevant temperature scenarios.