Fluorescence Excitation And Detection - Walz MINI-PAM Manual

CHAPTER 8
Fluorometer. These include the optics for fluorescence excitation and
detection, the selective amplifier, the data acquisition and storage
system, an actinic light source for saturation pulses and continuous
illumination, a large rechargeable battery and the user interface, with
the LC-display and keyboard. Details on some of these components
are given in the following sections.
8.1.1

Fluorescence excitation and detection

In the standard version of the MINI-PAM fluorescence is excited
by pulse modulated red light from a light-emitting-diode (LED). The
pulse-width is 3 µs and pulse frequency is 0.6 or 20 kHz. In the so-
called "burst-mode" pulse trains of 0.2 s are alternating with 0.8 s
dark-intervals. The LED-light is passed through a cut-off filter
(Balzers DT Cyan, special) resulting in an excitation band peaking at
650 nm, with a very small "tail" at wavelengths beyond 700 nm.
Fluorescence is detected with a PIN-photodiode at wavelengths
beyond 700 nm, as defined by a long-pass filter (type RG 9, Schott).
The effective intensity of the measuring light at the level of the
sample is an important parameter for correct determination of the
minimal fluorescence yield, Fo, of a dark-adapted sample. Its
absolute value depends on
• intensity setting (menu point 50, preset value 8),
• measuring frequency (menu point 4, preset at 0.6 kHz),
• burst mode status (menu point 5, preset to be off),
• distance between fiberoptics and sample (standard 12 mm).
At the standard distance of 12 mm between fiberoptics and
sample, and at measuring light intensity 8, the quantum flux density
of photosynthetic active radiation typically amounts to 0.15 µmol
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COMPONENTS OF THE MINI-PAM