Here are the microscopes available on the platform for internal use and external collaborations, from single molecules to small animals

 

 

Nanoscopy / Single Particle Tracking

The nanoscopy technique (also called superresolution) allows sub-wavelength spatial resolution with the use of photo-physical effects (photo-activation, photo-conversion, stochastic emission).
Our homemade setup is designed on a Nikon Ti-u microscope with a 100X, 1.49 NA oil immersion TIRF objective and a Andor Ixon 897 back-illuminated EMCCD camera. The excitation wavelength available are: 405, 458, 476, 488, 496, 514, 568 and 647nm.
We designed a new software dedicated to sigle molecule localisation microscopy (SMLM) called UNLOC (Unsupervised particle LOCalization, see the software part below).
Single Particle Tracking allows to follow diffusion processes and to extract confinement constraints data on targeted cell surface receptors (mostly in the lab with QDots). A homemade algorithm (Multiple target Tracing, MTT, published in 2008 in Nature Methods) has been developed in order to perform SPT analysis at high density (see the software part bellow).
The optical and software developments are made in close collaboration with the Mosaic and PhyTi teams from the Institut Fresnel in Marseille (Serge Monneret & Nicolas Bertaux).

Microscope available :
Nikon Ti Eclipse microscope with Andor Ixon 897 camera, Coherent Obis 405 and Innova-70C lasers

Example of results :

Actin cortex on COS-7 cells, work of Yannick Hamon and Roxane Fabre (He & Marguet’s lab, CIML)
Scale bars: 10 µm and 1 µm
Analysis software: Sébastien Mailfert – Nicolas Bertaux (He & Marguet’s lab, CIML and PhyTI’s lab, Institut Fresnel)

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Fluorescence Correlation Spectroscopy / Fluorescence Recovery After Photobleaching

Fluorescence Correlation Spectroscopy is a powerful technique which allows the measurement of diffusion processes. The specificity of our system is that we can perform FCS at different observation scales (spot variation FCS or svFCS) and thus explore confinement constraints within cell membranes. Spot FRAP experiments are also available on the same homemade setup. A second system is designed to perform Fluorescence Cross-Correlation Spectroscopy (FCCS) in order to study molecular interactions on living cells.
svFCS / spot-FRAP and FCCS systems are designed on Axiovert 200M microscopes with 488 and 633nm lasers, 40X water immersion objectives and SPCMs.
These setups are developed in close collaboration with the Mosaic team from the Fresnel Institute in Marseille.

Microscopes available :
Zeiss Axiovert 200M x2, Coherent Innova-70 and Sapphire lasers, Perkin Elmer SPCMs and incubation boxes

Example of results :

Detecting nanodomains in living cell membrane by fluorescence correlation spectroscopy. He HT, Marguet D. Annu Rev Phys Chem. 2011 May;62:417-36

Developments in progress:
We are developing new FCS approaches named FCCS (Fluorescence Cross-Correlation Spectroscopy) in order to study interactions between two proteins or 2PE-FCS (Two Photon Excitation Fluorescence Correlation Spectroscopy).

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Raman Spectroscopy

Spontaneous Raman scattering:
- provides fingerprint spectra (molecular identity) and informations about 3D structural changes (orientation or conformation) or intermolecular interactions
- Advantages: non-destructive, non-invasive; works in-situ & in-vitro for biological samples
- Limitations: weak signal (long integration times => Imaging can take several hours)


Microscope available :
Renishaw inVia microscope with 633 and 785nm lasers

The investigation of melanoma metastasis sections by Raman micro-spectroscopy reveals chemical modifications between tumor and non tumoral samples. Two specicic biochemical markers associated to the DNA and the collagen contents allow to dene a tumor index corresponding to the tumoral nature of the sample. The resulting tumor index heatmaps are then in complete agreement with histological analysis with staining.
Cyrille Billaudeau, Didier Marguet’s lab, CIML

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Cellular imaging

Widefield microscopy

Widefield microscopy is a technique using a fluorescence lamp in order to observe or acquire generally from one to four fluorescence colors on tissues, cells or organisms with a very good sensitivity and low photobleaching.
The inverted microscope Axiovert 200 is designed to observe transfected cells (expressing CFP, GFP, RFP) or cell colonies on plastic box or LabTeck with dry objectives (4X, 10X, 20X, 40X) or oil objectives (40x, 63X). The Axiovert 200 is equipped with a color camera.
The microscope Axioplan 2 is an upright microscope designed to observe and acquire fluorescence on slide only with oil objectives (16X, 40X, 63X and 100X). There are a wide choice for fluorescence filters (DAPI, DIC, GFP BP, GFP LP, Cy3, TxRed and Cy5). It is also well adapted to chromosomes counting or for FISH experiments.

Microscopes available :
Zeiss Axiovert 200 with Qimaging Qicam color cameraZeiss Axioplan 2 with Axiovision, CoolSnapFX Photometrics B&W CCD camera

Example of results :

Mouse Pancreas (Islet in red) injected with Macrophages (green), nuclei (cyan). X16
Carole Berruyer, Michael Sieweke’s lab, CIML

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Video-microscopy

The video-microscope is a widefield microscope dedicated to long term experiment and HCS (High Contents Screening) studies, for shorter time experiments like calcium imaging and visualization of fast events.
The Cell Observer microscope is an inverted microscope with an incubation box (temperature and C02) and an automatic focus (Definite Focus) to keep the focus stable. The Axiovision software allow to calibrate multi-position acquisition on multiple wells or acquire mosaic images. The Assay Builder software process very large amount of images. The algorithm is based on nucleus labeling. It segments individual cells in order to quantify fluorescence inside the nucleus or around each cell. Dotplot histograms are used for visualization and quantification of events (Number, Mean, Area, etc.).

Microscope available :
Zeiss Cell Observer DF, Photometrics HQ2 B&W CCD camera

Example of results :

Process of hundred of images and statistics representation are very powerful to obtain robust results. In the dot plot, each dot is linked to the corresponding cell in the image.

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Phase Imaging

The SID4bio from Phasics is an innovative technique for quantitative phase imaging in light microscopy. Based on a patented technology (the 4-wave lateral shearing interferometry), SID4bio enables observing living cells with no marker, and running statistical and quantitative analysis on them (migration, growth, intracellular processes, etc.).
The SID4Bio provides an unrivalled contrast enhancement of biological specimen without the use of any marker. It enables: studying cell morphology and organelles, time lapse observation over long period of time, reliable post processing such as segmentation and Automatic counting.
By measuring the true phase shift introduced by the specimen, the SID4bio also gives quantitative information about the specimen. It enables: cell dry mass investigation, comparison between cells population, understanding organelles properties based on the optical refractive index assessment.
From http://www.phasicscorp.com

Microscope available :
Phasics SID4BIO on the Zeiss Cell Obsever (see the videomicroscopy part)

Example of results:

Left| COS7 cells division during 7h imaged by SID4bio (Phase and intensity). Pierre Bon, Hervé Rigneault’s lab, Fresnel Institute / Didier Marguet’s lab, CIML
Right| Mosaïc image reconstruction of a melanoma: phase (SID4bio), nuclei (DAPI), mitotic cells (pHH3).
Marie-Claire Blache, Didier Marguet’s lab, CIML

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Confocal Laser Scanning microscopy

Confocal microscopy is a powerful technique to avoid out of focus light in order to have more specific signal from a thin optical section. The modern confocal offers very interesting options like supercontinuum laser, spectral detectors, ultra high sensitivity detection and software options like FCS (Fluorescence Correlation Spectroscopy), FRAP (Fluorescence Recovery After Photobleaching), RICS (Raster Image Correlation Spectroscopy) techniques.

Microscopes available :
LSM 880 with spectral detection + Fast AiryScan and lasers at 405, 458, 488, 561, 633nm.
LSM 780 with spectral detection and lasers at 405, 440, 458, 488, 561, 633nm.
Leica SP5X with a supercontinuum laser and 5 PMTs

Example of results :

A LysoDC extends a dendrite through M cell into the gut lumen. Immunofluorescence staining of a section of a mouse Peyer’s patch showing a LysoDC (CD11c in cyan; lysozyme in yellow) sending a dendrite through an M cell (magenta). The dendrite stretches on a 7 µm length in the intestinal lumen. The basal lamina of the follicle-associated epithelium is highlighted with an integrin a6 labeling (green). T cells are in red (CD3 staining). Hugues Lelouard, Jean-Pierre Gorvel’s lab, CIML

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Intravital and large sample imaging

Multiphoton microscopy

Multiphoton microscopy use a pulsed laser to generate the biphoton effect. This phenomenon create a localized excitation in the focal volume of the objective by recombination from two infrared photons into one visible photon. It is in practice used to penetrate deeper in the tissue for in vivo microscopy with a confocal-like resolution.

The Leica MP5 is a biphoton using the Coherent Chameleon Ultra II laser (680-1080nm) combined with an OPO and an external precompensator. The OPO system generates a second beam (1000-1300nm) for excitation of Red proteins (td tomato, mCherry, etc.). It is also designed for explant organs or for in vivo experiments on mouse. This microscope is installed in a L2 laboratory.

Microscope available :
Leica SP5 MP with Coherent Ultra II and OPO Chameleon (140fs pulse width)

Example of results :


Two-photon excitation microscopy images illustrating the motility of thymocytes (blue) and dendritic cells (red) compared to the network of stromal cells (yellow) within the thymus of a mouse.
Marc Bajénoff, Marc Bajénoff’s lab, CIML

Development in progress : We are developing a 3 colors excitation 2 photon microscope based on the article « Multicolor two-photon tissue imaging by wavelength mixing “ by Pierre Mahou and Emmanuel Beaurepaire.The synchronization of the pump laser with the OPO will generate a third wavelength in the focal point .

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Light-sheet microscopy

Light-sheet microscopy allows multidimensional imaging with a low photodamage due to the use of a thin sheet of excitation vs. a confined scanning spot in classical confocal microscopy. 3D imaging is performed by scanning the sample with this light-sheet.
The light sheet microscope can image samples ranging in size from few µm up to more than 1 cm. In case of large samples no stitching is required due to a field of view diagonal from 1.7 mm to 17 mm.

Microscope available :
UltraMicroscope II from LaVisionBiotec

Example of results :

Hematopoietic cells (red), transcription factor Prox-1 (blue), blood vasculature (white) and neurons (green) within a mouse embryo at embryonic day 13.5.
Serge Van de Pavert

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Slide scanner

The slide scanner can acquire a tissu in coloration or fluorescence with a 20x/08 or 40x/0.95 magnification. A z stack acqusistion could be obtained to increase the contrast. using a maximum projection. The scanner can load up to 150 slides.

Microscope available :
Pannoramic  SCAN II with DAPI/A488/A555/A647 fluorescence cube

Example of results :

Reconstruction of a thymic lobe from an exhaustive set of serial sections processed by the homemade algorithm For3D.
Each image was acquired with the automated slide scanner. Medullary islets are color encoded according to their volume, from red (smallest) to yellow (largest).
The For3D method is described in Sergé et al. For3D: Full organ reconstruction in 3D, an automatized tool for deciphering the complexity of lymphoid organs. J Immunol Methods. 2015 Sep;424:32-42.
Magali Irla in Philippe Naquet’s lab

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