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Description

Specific fluorescent probes (chemical dyes or fluorescent protein technology) allow temporal and spatial co-localisation of your (bio)molecule of interest, for simultaneous, multicolor, multidimensional , time laps or FRAP imaging.

Information obtained by this technique

(Co)-localisation
Specific fluorescent probes (chemical dyes or fluorescent protein technology) allow temporal and spatial co-localisation of your (bio)molecule of interest. A range of fluorescent probes can be combined for simultaneous, multicolor, multidimensional image acquisition. Specific applications like time laps imaging or FRAP can be performed.

3D reconstruction
Optical sections from the confocal microscope can be achieved using piezo stage (nm resolution) for 3D reconstruction of (bio)molecules, organelles and tissues. 3D reconstruction can also be performed using multi colored images.

Dynamics using FCS (fluorescence correlation spectroscopy)
The MM-CLSM has a Single Molecule Detection (SMD) module that allows fluorescence correlation spectroscopy for the quantification of biomolecules either in vitro or in vivo. Furthermore, the diffusion coefficient can be derived for dilute solutions. The interaction between differently labeled molecules can be detected by applying fluorescence cross-correlation spectroscopy (FCCS).

Fluorescence lifetime imaging microscopy
The second feature of the MM-CLSM is to perform Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM is a unique and robust technique for the detection of protein interactions in living cells. This FLIM set up allows monitoring multiple fluorescence lifetimes simultaneously.

FCS-FLIM
Combination of FCS and fluorescence lifetimes allows the detection of interacting molecules at single molecule level. This technique is mostly applicable for in vitro studies.

Technical Details

The Leica SP8X-SMD high-tech fluorescence microscope is a multi-mode confocal microscope. It combines confocal imaging with single molecule detection (SMD) and advanced (time-resolved) fluorescence spectroscopy. The MM-confocal SMD microscope has a unique configuration including:

• Variable excitation with a supercontinuum tunable white light laser (WLL) source
• WLL confocal imaging using up to eight excitation wavelengths simultaneously
• WLL tunable pulsed excitation with two wavelengths simultaneously for FLIM applications
• Confocal microscope using hybrid detector technology allowing for complete, filter free, and spectral freedom for imaging
• Confocal microscope and time resolved SMD controlled by confocal software platform
• Automatic 3D-FLIM acquisition using a third parameter like wavelength, time or volume.
• FLIM acquisition having time resolution (TTS) of 100 ps or better, and short detector dead time in combination with a high dynamic range

Applications

The MM-CLSM can be using in different research projects, varying from signal transduction pathways in planta to protein folding aspects or detection of fast dynamics in photosysnthesis.  Furthermore, specific applications comprise protein interactions (in vitro or in vivo), signal transduction, food processing, single molecule biophysical studies and colloid chemistry.

Complementary Techniques

Next to the supercontinuum laser, this microscope is also equipped with a “standard” argon laser (458, 477, 488, 514 nm) and a pulsed diode laser (440 nm). The microscope operates extreme flexible by the incorporation of an AOBS, AOTF and a 5 channel filter free, spectral HyD detectors.

Publications

The role of plasmodesma-located proteins in tubule-guided virus transport is limited to the plasmodesmata. den Hollander PW, Kieper SN, Borst JW, van Lent JW. Arch Virol. 2016 Sep;161(9):2431-40. doi: 10.1007/s00705-016-2936-2.

Molecular Characterization of Arabidopsis GAL4/UAS Enhancer Trap Lines Identifies Novel Cell-Type-Specific Promoters. Radoeva T, Ten Hove CA, Saiga S, Weijers D. Plant Physiol. 2016 Jun;171(2):1169-81. doi: 10.1104/pp.16.00213.

Active 5′ splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes Katarzyna Knop1, Agata Stepien, Maria Barciszewska-Pacak, Michal Taube, Dawid Bielewicz, Michal Michalak, Jan W. Borst, Artur Jarmolowski,* and Zofia Szweykowska-Kulinska

Reporters for sensitive and quantitative measurement of auxin response. Liao CY, Smet W, Brunoud G, Yoshida S, Vernoux T, Weijers D. Nat Methods. 2015 Mar;12(3):207-10, 2 p following 210. doi: 10.1038/nmeth.3279.

A bHLH-Based Feedback Loop Restricts Vascular Cell Proliferation in Plants. Vera-Sirera F, De Rybel B, Úrbez C, Kouklas E, Pesquera M, Álvarez-Mahecha JC, Minguet EG, Tuominen H, Carbonell J, Borst JW, Weijers D, Blázquez MA. Dev Cell. 2015 Nov 23;35(4):432-43. doi: 10.1016/j.devcel.2015.10.022.

A set of domain-specific markers in the Arabidopsis embryo. Wendrich JR, Möller BK, Uddin B, Radoeva T, Lokerse AS, De Rybel B, Weijers D. Plant Reprod. 2015 Dec;28(3-4):153-60. doi: 10.1007/s00497-015-0266-2.

Encapsulation of GFP in Complex Coacervate Core Micelles. Nolles A, Westphal AH, de Hoop JA, Fokkink RG, Kleijn JM, van Berkel WJ, Borst JW. Biomacromolecules. 2015 May 11;16(5):1542-9. doi: 10.1021/acs.biomac.5b00092.

Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors. Boer DR, Freire-Rios A, van den Berg WA, Saaki T, Manfield IW, Kepinski S, López-Vidrieo I, Franco-Zorrilla JM, de Vries SC, Solano R, Weijers D, Coll M. Cell. 2014 Jan 30;156(3):577-89. doi: 10.1016/j.cell.2013.12.027.

Plant development. Integration of growth and patterning during vascular tissue formation in Arabidopsis. De Rybel B, Adibi M, Breda AS, Wendrich JR, Smit ME, Novák O, Yamaguchi N, Yoshida S, Van Isterdael G, Palovaara J, Nijsse B, Boekschoten MV, Hooiveld G, Beeckman T, Wagner D, Ljung K, Fleck C, Weijers D. Science. 2014 Aug 8;345(6197):1255215. doi: 10.1126/science.1255215.

Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Bücherl CA, van Esse GW, Kruis A, Luchtenberg J, Westphal AH, Aker J, van Hoek A, Albrecht C, Borst JW, de Vries SC. Plant Physiol. 2013 Aug;162(4):1911-25. doi: 10.1104/pp.113.220152. PMID: 23796795 Free PMC Article Similar articles Select item 23415953 15.

A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis. De Rybel B, Möller B, Yoshida S, Grabowicz I, Barbier de Reuille P, Boeren S, Smith RS, Borst JW, Weijers D. Dev Cell. 2013 Feb 25;24(4):426-37. doi: 10.1016/j.devcel.2012.12.013.