for Probing of Cells and Tissue in Microbial Mats and Leaves
The MICROFIBER-PAM employs a very thin optical fiber for fluorescence excitation and detection. This narrow fiber permits probing of small spots of heterogeneous photosynthetic surfaces like soil crusts.
Also, placing the microfiber at different penetration depths permits measurements of photosynthetic gradients within microbial mats and leaves.
Components of the MICROFIBER-PAM: Fiber optic coupler (orange lines with black elongated central part), four different-colored alternative LED light sources (upper left corner), photomultiplier (grey box mounted to stand bar) and PAM-CONTROL unit.
Central part of the MICROFIBER-PAM is a fiber optic coupler consisting of a “beam splitter” to which two pairs of fibers are connected. This “four-port” coupler distributes light incoming from one fiber pair to the other pair where each fiber pair can function as input.
Typically, the fiber optic coupler conducts emission from an LED to the sample, and guides fluorescence from the sample back to a highly sensitive photomultiplier which is shielded against LED light by glass filters.
Fiber optic coupler consisting of a central beam splitter (black elongated central part) and four multi-mode optical fibers (orange lines) with ST (straight tip) connectors. Grey box: Photomultiplier.
Both, light emission of the LED and fluorescence measurement by the photomultiplier are coordinated by the PAM-CONTROL unit.
The PAM-CONTROL unit allows stand-alone operation of the MICROFIBER-PAM but functions also as a physical interface for computer-controlled operation of the MICROFIBER-PAM.
Normally, the MICROFIBER-PAM uses blue LED light for fluorescence excitation but alternative LEDs emitting in the green and red spectral range are available (see Accessories).
Peak emission of the blue LED is 470 nm. The LED emits pulses of several μs duration to elicit the pulse-modulated fluorescence measured by the MICROFIBER-PAM, but also longer-lasting pulses which produce integrated light intensities which can saturate photosynthesis.
The blue light is passed through a short-pass filter transmitting only at wavelengths below 600 nm (MF-L470). The LED is coupled via ST connection to one fiber of the four-port fiber optic coupler, MF-2-2-100. To the parallel fiber of the MF-2-2-100 coupler, the photomultiplier (PM-MF) is attached using an adapter.
The adapter for the photomultiplier is part of the MICROFIBER-PAM adapter set, MF-A. The MF-A adapter set also includes a holder for the blue LED and three additional LEDs.
The photomultiplier is shielded against the blue LED light by long-pass filters transmitting light only at wavelength greater than 640 nm. Without these filters, and face to face orientation of the tips of the two output fibers, the photomultiplier predominantly detects the blue measuring light transmitted between output fibers (compare figures).
Obviously, a lightabsorbing sample placed between the fiber tips reduces the blue light intensity reaching the photomultiplier. Hence, when the MICROFIBER-PAM is configured to detect measuring light, the degree of light attenuation by a sample can by assessed from the photomultiplier signal.
Outline of setup for fluorescence measurements. Measuring and actinic light from the LED is guided via the fiber optic coupler and the sample. Sample fluorescence is conducted via the fiber optic coupler to the photomultiplier which is protected against LED light by a special filter set.
Outline of setup for assessment of light attenuation. Measuring light from the LED (I0) is guided via a fiber optic coupler to both output fibers (I0,path a and I0,path b). The tips of the output fibers are facing each other so that light from one tip can be collected by the other tip. The collected light (I1,path a and I1,path b) is conducted via the fiber optic coupler to the photomultiplier. Placing a light-absorbing sample between the tips of output fibers decreases the measured signal depending on the degree of light attenuation by the sample.
Both, LED and photomultiplier are connected to PAM-CONTROL unit. The PAM-CONTROL unit is delivered with WinControl V.2-Software for operation by Windows computers, an RS 232 cable, a USB-RS 232 adapter (in case that an RS-232 port is not available), a charger MINI-PAM/L, a cable to connect a chart recorder and a transport box.
Further, a stand (ST-101) is included in the BLUE version of the MIRCOFIBER-PAM.
Universal Control Unit
The PAM-CONTROL unit can conduct independently PAM fluorescence measurements but it can also act as physical interface between fluorometer and computer using WinControl V. 2 software.
Universal control unit for ultrasensitive chlorophyll fluorescence measurements in a variety of specialized optical geometries
Schematic representation of Universal Control Unit PAM-CONTROL
The PAM-CONTROL unit is specialized to operate various extremely sensitive setups for chlorophyll fluorescence measurements: the MICROSCOPY-PAM, the MICROFIBER-PAM and the WATER-PAM.
Common to these three setups is the use of the same light-emitting-diodes (LED) as sources of measuring and actinic light, as well as for saturation pulses. Also, all setups use ultrasensitive photomultipliers: as a consequence, they are not suited for operation in the presence of non-modulated background light which would interfere with chlorophyll yield determinations.
The capacity of data storage comprises 4000 data sets. An extensive menu provides full control of instrumental settings and a variety of measuring protocols.
Working Fiber MF-F
For fluorescence measurements, one of the fibers opposite to the LED/photomultiplier fibers is placed in close contact to the sample, providing output of excitation light and input of fluorescence. Also, this fiber can be connected to a “working fiber” (MF-F) which is a single fiber with one free end of 100 μm diameter. The free end can be pulled out to a tapered tip of 10 to 30 μm. By means of a micromanipulator, this tip can advanced into different layers of plant tissues to measure photosynthesis gradients. Similarly, light attenuation within a sample can be assessed using a pointed working fiber.
Green (MF-L520) and Red (MF-L630
and MF-L650) Measuring Light LEDs
Fluorescence excitation by blue light is inefficient in many cyanobacteria. Therefore, we offer two types of red LEDs for studies of cyanobacteria using the MICROFIBER-PAM. Also, green measuring light is available for excitation of some carotenoids and phycoerythrin.
