Electron microscopy is developed from a set of techniques and equipment that allow imaging, using electron beams, the intracellular content of samples (ultrathin sections in transmission electron microscopy) or the extreme surface of samples (scanning electron microscopy) with high to very high resolution (resolving power of a few tens of nanometers, potentially reaching 0.1 nm).
The observations that must be carried out in a very low-pressure environment (10-3-10-4 Pa) often involve significant and sometimes drastic conditioning and sample preparation in a large portion of our case studies (biological samples) to manage water content and sample conduction into the beam.
The new generations of electron microscopes (controlled pressure) and the use of proven cryo-techniques within the microscope chambers allow for observations of biological samples "without preparations" or observations in cryo-genic mode (sample frozen and kept as is under high vacuum and at a temperature of -140°C in the microscope chamber).
Subjected to electron beams or X-rays, matter re-emits photons by fluorescence that are characteristic in Energy and Wavelength of atomic transitions and thus of the interacting atoms. Based on this principle, the collection and counting of these photons using spectrometers placed in detection on the microscope enclosure allows for the qualification and, under strict conditions of sample type and preparation, the quantification of the chemical elements composing this material at the scale of observation magnification (EDS-WDS microanalyses. EDX in the case of sample irradiation with X-rays).
Tungsten Emission Scanning Electron Microscope
Model: LEO (Zeiss) 1452VP
Characteristics:
- Scanning Electron Microscopy Controlled pressure imaging
- EDS, WDS, EDX Micro-Analyses
This device allows for imaging with very good resolution a very large number and type of sample surfaces from a tungsten filament emission gun. It can operate in controlled pressure mode (1-133Pa) to avoid any preparation (drying, metallization,…) before acquiring digital images. The electronic signals resulting from the scanning of the primary beam with the sample surface are collected by 3 specific detectors and processed by a high-performance digital chain, direct imaging on screen, synchronous with the beam scanning:
High-resolution secondary electron detector of the Everhart-Thornley surface type (acquisitions exclusively in high vacuum mode).
Backscattered Electron Detector (4 silica sectors): chemical contrast + topographic contrast during acquisition. (operation in both vacuum modes, high vacuum and degraded vacuum).
Secondary Electron Detector in degraded vacuum mode (controlled pressure). Surface imaging in controlled pressure mode.
The optimal use of the device in controlled pressure mode may require cooling partially hydrated samples (<40% water) to permanently preserve this water (ice) in the tissues and allow them to be directly imaged under degraded vacuum. For this, this microscope is equipped with a sample holder cooled (or heated) by the Peltier effect (DEBEN Peltier effect stage -50°C; +50°C).
Micro-Analyses EDS, WDS, EDX
The research applications developed on this microscope are largely carried out thru X-ray microanalysis of the scanned surfaces.
On two ports of the device's chamber are installed two X-ray spectrometers specifically for the collection and counting of photons resulting from the electron-matter interaction in the SEM (X-ray Fluorescence):
- SDD Spectrometer (Silicon Drift Detector) XMAX 80mm² (Oxford Instruments)
Energy-Selective Detection (EDS) of photons resulting from energy transitions within the atoms that make up matter (spectral detection range from Boron to Uranium).
Continuous acquisition of spectra across the entire energy scale.
- INCA WAVE Spectrometer (Oxford Instruments)
Wavelength Dispersive Spectroscopy (WDS) detection of photons resulting from energy transitions within atoms. High-resolution acquisition, high sensitivity, and low detection threshold of peaks in the emission wavelength of photons (emission during energy transition by a single selected atom).
Measurement principle (X-ray diffraction on the Rowland circle) based on the focusing of photonic radiation with a monochromator crystal (diffraction) and a proportional counter (counting the diffracted X-rays at the exact emission wavelength by the atom of interest).
- Software for managing EDS+WDS micro-analyses INCA ENERGY+ (Oxford Instruments)
The installed interface offers acquisition (simultaneous EDS+WDS acquisition possible) EDS and WDS and spectrum processing (spectral deconvolution and mass fraction calculation) according to various integrated software 'suites' that optimize the selection of areas of interest (SEM control via INCA), allow elemental mapping (distribution of elements on the scanned surface), automation of these acquisition tasks (macro-programmed routines), and signal threshold detection and elemental micro-analysis (EDS) of particles (feature interface from GSR of the scientific police).
- X-ray generator guided by polycapillary optics ('focusing' of X-rays) iMOXS (lfG GmbH Modular X-ray Source)
This X-ray generator (rhodium tube) guided by polycapillary optical fibers directly adapted to the SEM chamber allows for the irradiation of imaged surfaces (in coincidence with the electron beam) with a relatively small diameter X-ray spot (100-150 µm) and the collection of photons from X-ray fluorescence from a significant volume of the sample with the energy-selective spectrometer (volume EDX µanalysis).
The physicochemical properties of these interactions are different from those generated by primary electronic radiation: integration into the sample volume, reduction of matrix background noise (improvement of detection and spectral deconvolution), reduction of the spectral investigation range (magnesium-uranium), analysis reduced in spatial resolution (spot size).
Field Emission Electron Microscope
Model: CRYO-MEB-FEG (Zeiss, Oxford Instruments, Leica)
Characteristics:
- Scanning Electron Microscopy
- Very high-resolution imaging
- Controlled pressure imaging
- Cryo-microscopy
- Correlative microscopy
- Transmission microscopy
This microscope is equipped with:
- electron detectors for imaging in both modes (backscattered electrons, secondary electrons in controlled pressure mode, secondary electrons in secondary vacuum mode)
- high-resolution detector Inlens transmission electron detector (STEM)
- energy-selective X-ray analysis spectrometers (EDS-SDD 80mm2)
- cryopreparation and cryoobservation module (-120 °C), Peltier effect stage (-50°C/+50°C)
This equipment is dedicated to high-resolution and low-voltage observations of the extreme surface of primarily plant-based biological samples in multiple modes (controlled pressure, secondary vacuum, transmission imaging, high-resolution imaging of cryo-fixed samples…) with the possibility of X-ray micro-analyses (elemental mapping).
An integrated module and graphical interface allow for observations and images correlated with other images and sites of interest acquired from other equipment (confocal laser microscope...).
Other internal developments allow for the precise correlation of observations made at different scales: Graphic scanner/ Photonic imaging/ Electronic imaging.
A specific detector integrated into the FEG allows for scanning transmission electron microscopy (STEM) observation of ultrafine anatomical sections placed on transmission electron microscopy grids.
Contact:
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