Electron microscopes and sample preparation equipment – Divided Cont Toggle

Electron microscopes

  • FIB-SEM DualBeam microscope
  • High resolution scanning electron microscopy with focus ion beam

Scanning electron microscope with focused ion beam milling equipped for cryo-imaging and correlative light and electron microscopy (CLEM)

Applications

  • 3D scanning electron microscopy (FIB-SEM)
  • high resolution scanning electron microscopy (embedded, dried or frozen samples)
  • elemental analysis (EDS)

Specifications

Software
AutoSlice&View 4.1, Amira 6 softwares for image analysis
Electron and Ion sources
Electron gun with Schottky thermal field emitter LMIS Ga3+ ion source
Electron column/optics
specifications
  • Available accelerating voltage: 350 V – 30 kV
  • Probe current: 0,8 pA – 100 nA
  • Imaging modes: field free, XHR immersion, EDS optimized
Ion column/optics
specifications
  • Ion column type: Tomahawk
  • Available accelerating voltage: 500 V – 30 kV
  • Probe current: 0,1 pA – 65 nA
Detectors available
  • In-lens detector (TLD – SE, BSE)
  • In-column SE detector (ICD)
  • In-column BSE detector (MD)
  • Everhart-Thornley SE detector (ETD)
  • Retractable high contrast solid-state backscatter electron
    detector (CBS)
  • Retractable STEM detector with BF/DF/HAADF segments
  • Secondary electrons/ions detector (ICE)
  • Energy dispersive spectrometer (EDS)
  • Others: IR camera for viewing sample/column, Chamber
    mounted Nav-Cam+, Integrated beam current measurement

Resolution
Top ResolutionConditions
0,6 nm30 kV (STEM)
0,6 nm15 – 2 kV
0,7 nm1 kV
1,0 nm500 V (ICD)
4,0 nm30 kV (i-beam)
Stage
High precision 5-axes 16 inch piezo motorized stage Cryo-stage Leica VCT100
Others
Gas Injection System – platinum and carbon depostion, Plasma cleaner
(mounted on the chamber), Micromanipulator EasyLift EX (FEI)
  • Transmission Electron Microscope equipped with cryo-pole piece and on axis TVIPS TemCam–XF416 4K CMOS camera with frame rate up to 24 f/s and high sensitivity

Transmission Electron Microscope equipped with a cryo-pole piece, fast and sensitive TVIPS XF-416 4kx4k camera and side-entry cryo holder Gatan 626.

Applications

  • Transmission electron microscopy (TEM) at room temperature
  • Advanced sample mapping
  • Electron tomography (ET)
  • Diffraction at cryo and room temperature
  • Cryo-electron microscopy
  • SerialEM software for automated image acquisition, montage mapping, tomography in ambient and cryo conditions

Specifications

JEOL manufacturer specifications (https://www.jeol.co.jp/en/products/detail/JEM-2100.html)

Software
TEM Center, EM Menu 5, SerialEM
Electron sources
Single crystal LaB6 cathode with a current range of at least 4pA to 40nA
Acceleration voltage (min. step)
80kV - 200kV (50 V)
Stability
Acc. VoltageOL Current
2 x 10-6 /min1 x 10-6 /min
Magnification range
(MAG/Low MAG mode)
50x – 1.000.000x
Bottom Mount High-sensitivity
CMOS Camera
  • TVIPS TemCam–XF416
  • 4k x 4k (4096 x 4096 pxl)
  • Signal/noise ratio 15:1 (200 kV)
  • Frame rate up to 24 f/s
  • Physical pixel size 15.5 um
  • Field of view 63.5 um
Camera length
SA DIFF (mm) : 100 to 2,500
Resolution at 200 kV
Point resolution 0.27 nm/Line resolution 0.14 nm
Optical paremeters for
objective lenses
Focal Length Spherical aber.coeff.Chromatic aber.coeff.Minimal focal step
2.8 mm2.0 mm2.1 mm2.0 mm
Goniometer tilt
From 70° to - 70°
Holders
  • Single tilt holder (1 grid)
  • Specimen quartet holder (4 grids)
  • High-tilt holder (1 grid)
  • Cryo-holder Gatan 626
Others
  • Minimal Dose System (MDS)
  • Anti-contamination device (ACD)

Sample preparation equipment

  • The EM GP2 plunge freezes fluid or extremely thin samples spread on an electron microscopy grid into liquid ethane after removing excess fluid by automatic blotting.
  • Prior to freezing the sample is maintained in a temperature and humidity controlled environmental chamber, adjustable between +4°C and +60°C and room humidity to 99 %

Specifications

Leica EM GP2 on Leica Microsystems webpage (https://www.leica-microsystems.com/products/sample-preparation-for-electron-microscopy/p/leica-em-gp2/)

Leica EM GP2 Flyer (https://www.leica-microsystems.com/products/sample-preparation-for-electron-microscopy/p/leica-em-gp2/)

Operating Conditions
FeatureMin/MaxIdeal
Room Temperature+15 to +30 °C+21 to +24 °C
Room Rel. HumidityMax. 55 %Around 40 %
Storage Temperature+5 to +35°C+21 to +24 °C
Storage HumidityMax. 55 %Around 40 %
Room Ventilation is required at all times during operation.
Environmental Chamber
FeatureTemperature ControlHumidity Control
Min/Max+4 to +60 °CRoom Humidity to 99%
Chamber Features: Light (LED), Anti-Fog Heating
Dewar
LN2 Dewar VolumeDewar TemperatureDewar bake-out TemperatureLN2 Consumption
1 l-196 °CMax. +110°C~ 1 l/h1
Preparation Chamber
Allowed Secondary CryogenSecondary Cryogen Container TemperatureSecondary Cryogen Container Volume
Ethane / Propane0 to -196 °C2.5 ml
  • cryofixation of samples for EM

High pressure freezing system for cryo-fixation (vitrification) of samples for EM. There are several advantages over chemical fixation. Sample is freeze in a range of milliseconds, which immobilize all macromolecular components simultaneously. This technique also improves preservation of biological samples.

Types of sample
  • Cell suspension
  • Cell monolayer
  • Tissue
  • Hydrogel

Size limitation is to 6 mm carrier: height 200 µm, diameter 5 mm
Application
  • Freeze fracturing
  • Cryofixation for SEM, TEM FIB-SEM
  • Sample preparation for correlative light and electron microscopy
Preparation toolkit
  • Cell suspension, tissue, hydrogel
    • 3 mm and 6 mm aluminium coated carriers A and B type
    • 3 mm gold coated copper membranes
    • freeze fracture 4,6 mm system
    • copper tube system for cell suspension
    • microbiopsy toolkit
  • Cell monolayer
    • 3 mm and 6 mm sapphire disks
    • 3 mm sapphire disks labelled for light microscopy
    • aclar foil
  • precise coating of samples foe subsequent examination with an electron microscope
  • glow discharge
  • freeze fracture cryo set

Application

  • Metal coating of sample to cerate a conductive layer which inhibits charging, reduces thermal demage and improves the secondary electron signal in the SEM
  • Making the carbon layers to be transparent to the electron beam but conductive (needed for X-ray microanalysis), to support films on grids
  • Glow discharge to render grids surface hydrophilic
  • Freeze-Fracture of frozen samples

Main components

The Leica EM ACE600 includes the following main functional units:

  • High vacuum chamber
  • Rotation sample stage, 24 positions for 12,7 mm SEM stubs
  • Quartz thickness measurement
  • Carbon thread source or sputter source (platinum and gold targets)
  • Freeze-Fracture cryo set includes vacuum cyro transfer Leica EM VCT100 and cryo stage
  • drying of samples with CO2 in critical point for SEM

Automated Critical Point Dryer

Application

  • The drying of biological specimens such as pollen, tissue, plants, insects for SEM analysis
  • The drying of industrial samples , for example MEMS (Micro Electro Mechanical Systems)

Method

Crytical point drying is a method which use of CO2 as transitional fluid because its critical point is at 31 °C and 73,8 bar. Technically these temperature and pressure requirements of the CO2 can be implemented relatively easily then for water (critical point is 374 °C and 228,5 bar). The water in the cell is replaced by an acetone which is very soluble with liquid CO2. This procedure is follow by substitution with liquid CO2 through serial dilution steps. The increasing the temperature and pressure will transfer the CO2 through its critical point into a subcritical state. By controlled depressurization and constant temperature is subcritical CO2 convert into its gaseous phase without crossing the phase boundary between liquid and gas. This technique of preparing sample for scanning electron microscope is more gentle and better preserve structure than air drying sample.

  • An automatic reagent handling system for AFS2

Automatic freeze substitution (AFS) connects cryo-fixation (e. g. high pressure freezing) and resin embedding. It is a process of dehydration in low temperatures without crystal formation. In the beginning of freeze substitution, amorphic water contained in sample is dissolved by an organic solvent (e.g. acetone, ethanol), which also includes chemical fixatives. After dehydration temperature rises to point where is resin embedding possible. Freeze substitution processor (FSP) is an automatic reagent handling system combined with the Leica EM AFS2, dispenses reagents for freeze substitution. It also provides UV lamp for photopolymerization of acrylic resins.

Application

Cryo-substitution and resin embedding

Types of sample
  • Cell suspension
  • Cell monolayer
  • Tissue
  • Hydrogel
Resins
  • Epoxy resins: Embed 812, Araldite, Durcupan, Spurr, Ultrabed
  • Acrylic resins: Lowicryl, LR white
  • Cryo-substitution for samples frozen in HPM100

Automatic freeze substitution (AFS) connects cryo-fixation (e. g. high pressure freezing) and resin embedding. It is a process of dehydration in low temperatures without crystal formation. In the beginning of freeze substitution, amorphic water contained in sample is dissolved by an organic solvent (e.g. acetone, ethanol), which also includes chemical fixatives. After dehydration temperature rises to point where is resin embedding possible. Freeze substitution processor (FSP) is an automatic reagent handling system combined with the Leica EM AFS2, dispenses reagents for freeze substitution. It also provides UV lamp for photopolymerization of acrylic resins.

Application

Cryo-substitution and resin embedding

Types of sample
  • Cell suspension
  • Cell monolayer
  • Tissue
  • Hydrogel
Resins
  • Epoxy resins: Embed 812, Araldite, Durcupan, Spurr, Ultrabed
  • Acrylic resins: Lowicryl, LR white
  • sample preparation for EM

Microwave Processor containing Pelco ColdSpot®Pro cooling system

Application

Sample preparations for EM (fixation, contrastation, dehydratation, embedding, polymerisation)

Types of sample
  • Histological tissues
  • Suspension cells
  • Hard-to infiltrate biological specimens
Adjustable features
  • Timing
  • Wattages (0 – 750 W)
  • Temperature (0 – 100°C)
On/Off systems
  • Vacuum
  • Bubbler
  • Stirrer
Other features
  • Temperature check probe
  • Manual or Automatic running mode
  • Data recording
  • Protocols storage
  • EM UC7 Basic Instrument with eucentric 180° swivelling microscope carrier
  • Built in anti-vibration system
  • Cutting window adjustment from 0.2-15 mm
  • Knife block 360° rotatable, dual sided self locking precision drive with ±30° graduation, clearance angle drive with 1° scale from -2° to over 15°
  • Knife holder for 6-12mm glass and diamond knives
  • 360° rotation of specimen with self locking drive

Specification

  • Eucentric movement of the stereo microscope carrier (+ 5° / – 8°) with patented designated positions for specimen approach, for glass and diamond knives
  • Multi-LED-illumination for standard illumination, back light illumination, specimen
    transillumination and spot light illumination
  • Dual automatic advance system with stepping motor for 200μm specimen advance, stepping motor for 10mm knife N-S movement
  • Stepping motor for 25mm E-W movement
  • Reflexomat II -peristaltic water pump; water spout in magnetic holder
  • Syringe, special tubing for Reflexomat II
  • Segment arc with specimen transillumination, self locking precision drives with ±22° eucentric movement, transferable stem for longitudinal and cross sectioning