Areas of expertise: microscopy; histo/cytology; histo/cytochemistry; plant anatomy
The years spent at university allowed me to specialize and gain experiences of a wide variety of techniques in the field of microscopy applied to study of plants.
Techniques listed below have been partly developed to respond to the needs of various projects in which I was involved in laboratories of the Department of Botany and Plant Biology (Geneva University) but also for collaborations with other laboratories in Switzerland or in France.
Among these techniques I will cite the localization of gene expression by in situ hybridization on paraffin section using a non-radioactive method (DIG) and the comparison at the tissue level (light microscopy) and cellular level (light and electron microscopy) of plant phenotypes (see Research).
The different techniques were also made available to master and PhD students participating in my curses and practical curses : “Histochemistry and Cytochemistry” and “ Introduction to Electron Microscopy” (see Teaching).
Light Microscopy
In this field, various techniques for preparing plant samples have been developed and were used routinely in view of their observation and of their histological and cytological study. They involve sectioning very different plant samples: flowers, siliques, stems, roots, seeds, root nodule. The sections were made in (1) fresh material with either a razor blade or a hand microtome or with a vibratome; (2) frozen samples, with a cryostat; (3) samples fixed and included un different media: paraffin, technovit 7100, epoxy resins (Epon, Spurr) and acrylic resins (LR White) using a rotary microtome.
The thickness of the sections varies according to the technique, from 100 µm (freehand and vibratome sections) to 1 µm (sections in resins). The fixation and inclusion methods vary, of course, depending on the objective.
According to the type of sections, protocols for staining and for cell study have been developed and were used routinely. They are listed here.
- Routine topographic staining: carmine – iodine green, hematoxylin, hemalun eosin, toluidine blue, safranin FAST green, safranin- alcian blue, basic fuchsin & astra blue, naphthol yellow, methylene blue – basic fuchsin.
- Histochemical staining to demonstrate various cellular constituents: DNA (Feulgen, DAPI, Acridine Orange, Gomori), polysaccharides (PAS, calcofluor, propidium iodide), applied to paraffin sections and staining of starch with lugol applied to fresh sections.
- Localization of peroxidase activity on fresh sections (vibratome) or in whole organs with different substrates: DAB (diaminobenzidine), TMB, chloronaphthol, syringaldazine.
- Immunolocalization on paraffin sections and freezing sections by fluorescence (FITC, rhodamine, AlexaFluor).
- Localization in situ of enzymatic activities on frozen sections: peroxidases (different substrates), dehydrogenases, phosphatases.
- Localization of gene expression by in situ hybridization on paraffin sections (non-radioactive method, DIG, RNA probes).
- Localization of GUS expression on Technovit sections.
Microscopes used: Leica DMI R2; Leica Laborlux S; Nikon Eclipse 80i equipped with digital CCDs cameras.
Confocal Microscopy
– Detection in situ of Activated oxygen forms in roots of young Arabidopsis plants using a fluorescent indicator e.g. fluorescent HPF (hydroxyphenyl fluorescein).
– Preparation of Arabidopsis roots according to the mPS-PI (pseudoschiff Propidium iodide) staining protocol. This technique is perfectly appropriote to the architectural cellular study in roots and embryos of Arabidopsis.
– Localization of cellular compounds by fluorescence: DAPI for DNA, calcofluor for polysaccharides (see above histochemical staining).
Microscopes : confocal Leica SP2 at the photonic bioimaging center of Science Faculty – Geneva University.
Transmission Electron Microscopy
– Chemical fixation of various plant samples (tissues, organs, cellular fractions) and their inclusion in resins (Epon; Spurr; LR White) for cytological studies, ultrastructural observations and immunolocalizations.
– Ultramicrotomy: ultrathin sections 60 – 90 nm thick using ultramicrotomes, Reichert OMU2 and Leica Ultracut UCT.
– Production of support films on grids (Formvar, Collodion).
– Classical staining of ultrathin sections with Reynold’s lead citrate and uranyl acetate.
– Functional investigations: cytochemical staining of polysaccharides using PATAg procedure and of enzymes (peroxidases, phosphatases, ATPAse); immunostaining with gold for the localization of pectins, peroxidases, membrane proteins.
– Freezing of root and shoot meristems which are then fractured at low temperature with the objective to produce a replica of their surface. The technique is that of freeze fracture.
– Protocols for the preparation of various plant samples have been developed and are made available (see Protocols).
A prerequisite for making ultra-thin sections is most often essential e.g., the realization of semithin sections 1 µm thick and their staining. The objective of this prerequisite is to select under light microscope the part of the sample on which we will focus the observations under an electron microscope.
Microscopes used for observations and micrographs: Philips EM410 (photographic support), FEI Technai G2 Sphera (digital acquisitions) from Photonic bioimaging Center, Sciences Faculty – Geneva University
Protocols of preparation of different plant samples in view of their observation under this kind of microscope.
Microscope used: Microscope Jeol 6510 Lv microscope at the Photonic Bioimaging Center – Sciences Faculty – Geneva University.
The atomic force microscope belongs to the family of microscopes known as near-field microscopes or local probes microscopes. They make possible to obtain an image of the sample, biological or not, by scanning its surface with a probe or tip. The atomic force microscope allowed to get topographic images of the surfaces of numerous dry biological samples but above all in physiological conditions with high spatial resolution (0,5 – 1 nm vertical: 0,1 nm in liquid medium).phto