Confocal Microscopy

 

The Confocal Laser Scanning Microscope Zeiss LSM 510 META and the Spinning Disk Laser Microscope System Andor Revolution® XD are valuable tools for obtaining high resolution images and 3-D reconstruction at the resolution of the light microscope. The key feature is its ability to produce blur free images by thin optical sectioning. The use of 3-D reconstruction of a Z-series enables the visualization of larger structures or compartments. Gene technology with the possibility to fuse fluorophores to proteins and a great variety of specific fluorescence antibodies and dyes offer CLSM a wide field of application. 

 

Plant tissue is characterized by various autofluorescent signals (chlorophyll, lignin). The overlapping emission spectra with a number of widely used fluorescent dyes, causes very often problems for the signal detection. With Emission Fingerprinting of the Zeiss LSM 510 META, the highly efficient optical grating onto 32 channels of the META detector, allows the separation of complex fluorescence signals - even with widely overlapping emission spectra - is an easy, three-step task. Additional technical features as multitracking, lamda-scan or Acousto-Optical Tunable Filter (AOTF) improve the signal detection and therefore cell viability of examined tissue. Finally this increases the efficiency of optical sectioning, detection of weak fluorescence signals, monitoring of cell dynamic processes by using single – or time laps imaging or cell biological methods like FRET or FRAP (Fluorescence Recovery after Photobleaching). Other aspect of confocal microscopy are gene expression analysis and the monitoring of a cellular ph shift or ion concentrations. 

 

One of the major features in plant research is the monitoring of cell dynamic processes and the localization of fluorescent labeled molecules in living cells and tissues by the use of sophisticated techniques to answer open questions about plant morphology or the mechanism of primary and secondary metabolic pathways. To visualize distance dependent protein-protein interactions Fluorescence Resonance Energy Transfer (FRET) or “tools“ like Cameleon and “splitting GFP“ are used. 

 

The detection of fast or weak fluorescent signals in high spatial resolution as a function of time is still a challenge in modern cell biology. The increased speed afforded by array-scanning the specimen opens the door to acquiring images at previously unattainable rates. Thus, Spinning Disk confocal microscopes enable imaging with a high temporal resolution. The major advantage  over conventional optical microscopy, including wide field and laser scanning confocal microscopy is manifested by the ultra rapid acquire of images up to 1,000 frames per second. Typically a pair of rotating disks with thousands of pinholes in a spiral Archimedes is used.  In the Yokogawa CSU unit of our Andor Revolution® XD Spinning Disc system,  the second disk is fitted with microlenses which enable multiple point excitation of the sample. As the whole field of view is scanned in this way during a single camera exposure a high quality confocal image is formed very quickly, allowing the capture of quality confocal images at incredibly high speed.  The acquisition of images at very high frame rates with minimum illumination of samples,  allow imaging of weakly expressing samples with reduced photobleaching. These qualities make Spinning Disk confocal microscopy particularly well suited to high speed 3D imaging of living systems.

 

With the help of fluorescent markers or the expression of fusion proteins in  tissue or cell compartments , the complememtary use of both confocal systems offers various conceptional starting points for the investigation of the primary and secondary metabolism in plants.  

 

 

Microscopes

 

Confocal Laser Scanning Microscope Zeiss LSM 510 META equipped with 8 laser lines (351nm, 361nm, 458nm, 477nm, 488nm, 514nm, 543nm, 633nm) and a Zeiss AxioCam HRc.     

 

 Confocal Laser Scanning Microscope Zeiss LSM 780 eqipped with 6 laser lines (405nm, 458nm, 514nm, 543nm, 633nm).

 

Spinning Disk Laser Microscopy System Andor Revolution® XD equipped with 6 laser lines (405nm, 488nm, 514nm, 561nm, 640nm) and 3 CCD cameras (Andor iXon DU-897-BV, Andor Clara, Andor Neo sCMOS)