Microscopy Facilities

Electron Microscopy

Samples of biological specimens infected with class 3 pathogens can be prepared for observations by Transmission Electron Microscopy, including: 

– Morphological analyzes, using high-resolution images (0.4 nm) of viruses, bacteria, organelles, cells. Pre-observation techniques are adapted to the nature of the specimen.

– Immuno-labeling (with labeling before or after inclusion in coating resins) enabling precise localization of one or more specific epitopes inside a virus, organelle or a cell

These techniques allow to follow a particular molecule from its synthesis to the sites of storage and secretion or on the contrary its entry and its intracellular path.

– Observations in conditions of negative staining, reserved for small biological objects (less than 100 nm) which are deposited directly on a grid covered with a very thin layer of plastic or carbon. Several dyes can be used and the choice depends on the nature of the sample (viruses, bacteria etc.)

Super-Resolution Microscopy

The CEMIPAI platform proposes a unique opportunity to image biological samples through Photoactivation Localization Microscopy (PALM) installed in the BSL3 during 2017. 

PALM achieves nanometre scale resolution by exploiting the properties of photoswitchable fluorophores.  Exposing the fluorophores to a certain wavelength results in a change in emission spectra – photoactivatable and reversible photoswitachable fluorophores become fluorescent, and reversible photoswitchable fluorophores change from one fluorescent state to another.

The basic principle behind PALM is that a low-power activating laser beam will ‘stochastically’ convert the fluorophores, so that only a few will be in their “active” or “on” state. These molecules are then imaged by a high-power illuminating laser beam to immediately convert them back to their “inactive” or “off” state, e. g. by photobleaching. This process of activation and inactivation is then repeated over thousands of frames to ensure all molecules have been imaged. 

Multiple images are taken with total internal reflection fluorescence (TIRF) microscopy, and then combined to yield a single, high-resolution image with a high density of features.

Equipment

TIRF-PALM microscope Model Nikon Ti-E

Diodes Laser: 405nm, 488nm, 561nm, 641nm, Objectives: 20x, 40x, 100x

EMCCD Camera IXON 897 (512×512 chip, pixel 16µm)

TIRF arm
Motorized stage XYZ with autofocus system PFS

Atomic Force Microscopy (Bio AFM) 

 courtesy from M. Baclayon et al., 2010. DOI: 10.1039/B923992H

 

Planned for late 2017 or early 2018

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Equipment
AFM configuration
  • JPK NanoWizard® 4 AFM head, specifically designed for safe use in BSL3 environment (extra sealing around cantilever holder,  no sharp edges, head completely sealed against vapor and liquids). 980 nm light source.
  • JPK Vortis SPM control station (high speed data capture,  1 high speed fully digital Lock-in amplifier (60MSamples/s), 1 mid speed fully digital Lock-in amplifier (800kSamples/s), Built in Phase locked loop (PLL) feedback, 4x 24bit DAC scan signal generation, 1x 60MHz 16bit, 6x 18bit ADC channels, 1x 24bit high precision ADC, Built-in signal access with 2 analog inputs and 4 analog outputs, 2 digital IO channels
  • Fast scanning mode >70Hz line rate for scan ranges up to 2µm
  • Supported imaging modes in air and liquid :
    •  JPK Quantitative Imaging QI™
    • Contact mode with lateral force (LFM)
    • AC mode
  • Force-distance spectroscopy, fast force mapping
  • scanner specifications:
    • Advanced closed loop control
    • Z axis mechanically decoupled from XY scanner
    • XY scan range: 100µm x 100µm
    • Z scan range: 15µm
  • JPK Manual Precision Stage AFM
  • PetriDish holder specifically designed for safe use in BSL3 environment. Temperature range: room temperature up to 60°. Compatible with 35x10mm Petri dishes from WPI and TPP (plastic or plastic with glass bottom) 
  • DirectOverlay module for AFM scan selection in the optical background image 
  • Benchtop Active vibration isolation system Halcyonics i4
  • 1560 x1065x1160 mm³acoustic enclosure

 

Optical Microscope configuration

  • Nikon Eclipse Ti2-U Inverted optical microscope, manual Long distance condenser (WD 65mm, NA 0.3, Brightfield, PhL, Ph1, Ph2), LED light source
  • 20x Objective Nikon CFI Achromat LWD ADL-20x Ph1
  • 40x Objective Nikon CFI Achromat LWD ADL-40x Ph2
  • 100x Objectif Nikon, CFI APO VC 100x oil (A.N. 1.4 DT 0.13)
  • Epifluorescence light source C-HGFL Intensilight 100w DC, manual intensity control
  • Camera JENOPTIK Fluorescence ProgRes MFcool (2/3” 1.4 MegaPixel CCD, resolution 1350×1024 pixel, 14bit)
  • Filter Cubes :
  • GFP (Ex466/40; DM 495; BA 525/50)
  • mCHERRY, mRFP (Ex562/40; DM 593; BA 640/75)
  • CY5, APC, DiD, Alexa Fluor 647, Alexa fluor 660 (Ex628/40; DM 660; BA 692/40)