Austrian Plant Phenotyping Network

APPN Workshop on Modern Plant Microscopy

Looking at a dead slice of cork through the ocular of his self-built compound microscope, Robert Hooke could for the first time see that there is an odd, yet somewhat periodic, underlying order. He decided to call the little building blocks he was seeing “cells”, and chronicled them along with sketches of many other natural objects in his now classic treatise on the subject – Micrographia. This was over 350 years ago, and optical microscopy techniques for studying plants have grown and flourished in many diverse directions since. Driven by our insatiable desire for understanding the inner molecular and structural workings of plants, aided by some “clever” tricks that allow us to squeeze out hidden information, and supported by the latest technological advances, optical microscopy is almost an eternity from where it was during Hooke’s fateful first experiments.

The “Modern Plant Microscopy” meeting gives center stage to six leading European researchers in the field of optical microscopy and spectroscopy to present their latest findings and how they have helped us gain a deeper insight into the complex processes underlying the biology of plants.

 

Date: February 16th, 2018

Venue: Vienna Biocenter, IMBA/GMI lecture hall, Dr. Bohr Gasse 3, 1030 Vienna

Deadline for registration: February 4th, 2018

 

Program:

 

9:30 – 10:00            Registration infront of the IMBA/GMI lecture hall

10:00 – 10:45           Malgorzata Baranska, Jagiellonian University (PL)

10:45 – 11:30           Miroslav Ovecka, Palacký University Olomouc (CZ)

Coffee Break

11:50 – 12:35          George Komis, Palacký University Olomouc (CZ)

12:35 – 13:20          Alexander Johnson, ISTA (AT)

Lunch

14:15 – 15:00          Andras Gorzsas, Umeå Universitet (SE)

15:00 – 15:45          Alexis Peaucelle
, INRA, Versailles (FR)

 

16:00-17:00           Lab/facility tours for those interested

 

Scope:

Raman Spectroscopy, Atomic Force Microscopy, Light Sheet Microscopy, Superresolution Microscopy

 

Talks:

Raman microscopy in plant analysis: to get a deeper insight into complex processes of plant biology

Malgorzata Baranska, Jagiellonian University (PL)

Raman spectroscopy is considered as a fast and non-destructive method, which can be a useful and powerful tool for in situ plant analysis.

Information on chemical composition of plant tissue can be obtained using various Raman spectroscopy techniques. The spectrum of Raman measurement consists of several bands, which code for information on chemical composition of the sample. Although plant tissues contain hundreds of various molecules, several of these components, like e.g. polysaccharides, fatty acids, carotenoids, polyphenols, alkaloids or polyacetylenes, can be identified due to their predominant key bands. Further technical improvements enabled Raman spectroscopy to be combined with microscopy (Raman imaging/Raman microscopy). The results can be presented as a two-, or three-dimensional spectroscopic maps and directly compared to the corresponding visual images of the investigated tissue. They are providing detailed information on the distribution of the analyzed molecules occurring in a surface layer of the plant sample or as a depth profile when confocal microscopy is applied. This advanced research tool is suitable for gathering molecular information with a high spatial resolution, even at a cellular level. Raman spectroscopy can be combined with other microscopic methods, i.e. AFM and SNOM, and high resolution images of subcellular structures (e.g. single carotene crystals) can be analysed together with their topography, stiffness etc.

 

Impact of light sheet fluorescence microscopy on plant developmental studies

Miroslav Ovečka, Palacký University Olomouc (CZ)

Plant growth and development is a complex process requiring continuous update of different imaging methods of modern plant microscopy. Classical microscopy methods pose some serious limitations for long-term live cell and developmental plant imaging. These include out-of-focus fluorescence in wide-field microscopy, phototoxicity, photobleaching, restricted temporal resolution and limitations in imaging depth in confocal and spinning disk microscopy. Recently mesoscopic imaging methods and especially light sheet fluorescence microscopy (LSFM) emerged as a new tool for fast and long-term imaging of animal and plant development. LSFM is based on sample illumination by thin sheet of light (excluding out-of-focus fluorescence), while fast optical sectioning captured by camera within milliseconds ensures minimal phototoxicity to the living sample. Importantly, plants growing in culture medium are positioned vertically in the microscope. All these parameters favour LSFM for long-term developmental plant imaging at the subcellular, cellular, tissue, organ, and whole-organism levels in a natural orientation maintaining undisturbed plant growth in near-environmental conditions. We developed protocols for preparation of living plants for long-term LSFM imaging at diverse scales, ranging from subcellular compartments up to whole seedlings. We will present examples from LSFM imaging of individual plant organs such as roots, hypocotyls and cotyledons, different tissues and cell types as well as observation and tracking of different subcellular organelles.

 

Superresolution imaging of the plant cytoskeleton

George Komis, Palacký University Olomouc (CZ)

The plant cytoskeleton is an intricate intracellular structure comprising of microtubules and actin microfilaments. Both elements are considerably spread, spanning the entire three dimensions of plant cells and they are involved in key functions including cell division, growth and differentiation. Our understanding of cytoskeletal organization in plants walked in pace with advances in microscopic imaging. Such advances include nowadays superresolution imaging, an ensemble of conceptually different methods that lower the diffraction limits of traditional far-field microscopy. Superresolution microscopy is already counting 40 years from conception, development and finally dissemination in the cell biology community. Although the plant community adopted superresolution imaging quite late, there is a steady increase of applications of structured illumination, photoactivation localization and stimulated emission depletion microscopies. Superresolution studies of plants span from fixed and immunolabeled samples to living tissues expressing some fluorescent biomarker. We will focus on the imaging of actin microfilaments, microtubules and some selected microtubule-associated and signaling proteins and examine the potential and the limitations of the principal superresolution methods in 2D, 3D, time lapse and multichannel imaging.

 

High Spatial and Temporal Resolution Imaging of Clathrin Mediated Endocytosis Accessory Proteins in Arabidopsis.
Alexander Johnson, ISTA (AT)

Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. In plants, endocytosis has been implicated in a wide range of key physiological functions; ranging from growth and development to pathogen defense. Lack of direct visualization of the plant cell surface, and examination of single events of endocytosis, has greatly hampered our ability to precisely characterize endocytosis. We therefore developed a reliable protocol for the use of total internal reflection fluorescence microcopy (TIRF-M) in intact plants to allow the direct examination of cell surface lifetimes of endocytosis accessory proteins (EAPs). We developed a nonbiased and high throughput automated particle detection and tracking algorithm to precisely determine cell surface lifetimes of EAPs. It allowed dynamic studies using pharmacological agents to unravel the mechanisms of plant endocytosis. Use of dual color TIFR-M allowed examination of single events of endocytosis, where clathrin departure from the cell surface was used as a physiological marker for endocytosis. Using this ‘departure assay’, we were able to generate temporal recruitment profile of proteins to single sites of endocytosis. Thus, providing a powerful tool to begin characterization of plant endocytic pathways.

 

Vibrational Microspectroscopy in Plant Sciences

Andras Gorzsas, Umeå Universitet (SE)

Vibrational (Fourier transform infrared, FTIR, and Raman) spectroscopy provides information about the chemical composition of a wide range of samples, including plants. Combined with a microscopy accessory, the chemical landscape of different plant tissues can be mapped, at cellular and subcellular spatial resolution in situ, without extraction or external markers (such as labels, dyes, etc). The techniques are rapid, inexpensive and non-distractive, providing information about all chemicals present in the sample simultaneously. Example applications from plant sciences will be presented to illustrate the power and versatility of vibrational microspectroscopy in plant sciences.

 

 

Atomic force microscopy: from mechanical measurements to sonocytology and beyond

Alexis Peaucelle
, INRA, Versailles (FR)

After a brief presentation of the challenges of mechanic measurements on living matter, I will present how we used AFM to measure elasticity and relate it to growth. I will present the challenges and limits of AFM and discuss complementary approaches. Finally I will show preliminary results on sonocytology of plants showing the versatility of AFM. Finally I will discuss the power of the FluidFM to go beyond ordinary AFM mechanical measurements.

 

Download flyer (.pdf)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

for recommended overnight stay, refer to http://www.appn.at/registration/hotel/

for contact details, refer to http://www.appn.at/contact/

 

Looking forward to meeting you at the VBC!

Yours, organizing committee (Kareem Elsayad, Jan Hejatko, Stefanie Koemeda, Klara Wuketich & Jakub Jez)

Contact: 

Kareem  Elsayad

Vienna Biocenter Core Facilities

Head, Advanced Microscopy Facility

kareem.elsayad@vbcf.ac.at

+43 664 80847 7110

 

Jan Hejátko

CEITEC Brno

Jan Hejátko Research Group

jan.hejatko@ceitec.muni.cz

+420 54949 4165

 

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