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) 

CHIKv by AFM

CHIKUNGUNYA virus by AFM in liquid in QI mode (data CEMIPAI)

 

Since March 2018, we provide academic and private users with an optimized AFM / Optical Microscope for imaging living cells and pathogens in BSL3 environment. The Atomic Force Microscope is associated with a Nikon Eclipse Ti2-U inverted microscope for the optical approach of complex biological samples such as cells. AFM operates in Contact, High Speed ​​Intermittent Contact (70Hz) or Quantitative Imaging (QI) mode in both air and liquid. It is equipped with an Intensilight 100w lamp (Hg, manual) for epi-fluorescence. The equipment helps to obtain a surface topography and thus to access the height of the structures (z). It does not require an invasive preparation of the sample. It also provides information on the mechanical properties of surfaces such as viscoelasticity.

  • Possible samples: fixed or living cells in their culture medium, macromolecules (nucleic acids, proteins), polymers, fibers, composites, lipid films, lipid vesicles (GUV, LUV), extracellular vesicles (exosomes), virus-like-particles (VLPs), viruses and class 3 bacteria
  • Size of the smallest objects that can be detected on the surface: a few nm X and in Y
  • Height of observable objects: from 0.5nm to 12μm
  • Dimension of square images: from 50nm to 100μm
  • Sizes of the samples that can be observed: 30mm x 50mm in X and Y, 5 to 10mm in Z
  • Ability to work in liquid or air
  • Information provided by the AFM:
    • surface density of objects, surface roughness, thickness
    • Topography in contact mode, intermittent contact and quantitative in air and liquid imaging
    • Nanomechanical properties of the samples thanks to the Quantitative Imaging (QI) mode on a maximum surface of 100μm2

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 mm³ acoustic 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)