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Pierre-François Lenne
pierre-francois.lenne@univ-amu.fr

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Imagerie optique

Microscopie optique pour l'imagerie biologique, de l'échelle subcellulaire à celle de l'organisme entier

Le département de microscopie optique du centre d’imagerie PiCsL-IBDM fournit à la communauté scientifique locale, nationale et internationale une expertise et des systèmes d’imagerie optique pour l’imagerie à multi-échelle, des cellules aux petits organismes entiers. Notre offre de services comprend des conseils techniques, des formations et des conceptions expérimentales

La tarification de la réservation des microscopes, de notre offre de services et de notre offre de formation est basée sur des tarifs validés par le CNRS.

Décision de prix du CNRS pour l’imagerie IBDM – 2020

Coût horaire prestation imagerie – 2020

FBI : France BioImaging

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Microscopie à fluorescence

Techniques standard de microscopie avec contraste de fluorescence limité par diffraction

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Nanoscopie par fluorescence

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Fluorescence microscopy

DWM Widefield-Microscopy

Deconvolution is a computational technique for improving the contrast and resolution of digital images. It includes a suite of methods that seek to remove or reverse the blurring present in microscope images caused by the limited aperture of the microscope objective lens. DWM typically causes less photoxicicity on the samples, so it is well suited for observation of cells in culture for long periods, and it easily allows the observation of unstained live-cells using DIC/Nomarski or Phase-contrast.

SDCM Spinning Disk

Spinning disc confocal microscopy has advanced significantly in the past decade and now represents one of the optimum solutions for both routine and high-performance fluorescence live-cell imaging applications. Furthermore, cameras (often EMCCDs or sCMOS), typically have quantum efficiencies 2-3x higher than PMTs, so much less laser light is deli Decreased excitation energy is necessary and that reduces photo-toxicity and photo-bleaching of a sample,  often making it the preferred system for imaging live cells or organisms when optical slicing is necessary

CLSM Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM; also known as laser scanning confocal [LSCM]), often colloquially referred to  or simply as “confocal” microscopy, is a technique for obtaining high resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer. This allows three-dimensional reconstructions of topologically complex objects. CLSM typically requires high-laser power excitation, which can lead to phototoxicity and limited observation of live cells

MPM/2PM/SHG/THG

Multiphoton microscopy (more commonly in the form of two-photon microscopy) is a fluorescence imaging technique that allows observation imaging of living tissue up to about one millimeter in depth. It uses pulsed red-shifted excitation laser light, which can also excite visible fluorescent dyes.

TPEM is often used to image intravitally, and it may facilitate imaging of unlabelled tissues, using autofluorescence or SHG (second harmonic generation).

Fluorescence Nanoscopy

SMLM Palm

Photo activated localization microscopy (PALM) is a widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. PALM is based on collecting a large number of images each containing just a few active isolated fluorophores. The imaging sequence allows for the many emission cycles necessary to stochastically activate each fluorophore from a non-emissive state to a bright state, and back to a bleached state. During each cycle, the density of activated molecules is kept low enough that the molecular images of individual fluorophores do not typically overlap.

STED Stimulated emission depletion microscopy

STimulated Emission Depletion microscopy (STED) is one of the techniques that allows that make up super-resolution microscopy. It is similar to confocal microscopy, in that is uses laser scanning imaging, however it creates super-resolved images by the selective deactivation/depletion of fluorophores in the peripheral area of the illumination PSF, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. STED can provide 3D datasets of samples, often with a resolution down to 50nm or less.

Functional imaging

FRET Ratiometric / Acceptor Bleaching

Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through non-radiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance and therefore an excellent reporter on molecule proximity and interaction.

FLIM/FLIM-FRET Fluorescence Lifetime Imaging Microscopy

Fluorescence-lifetime imaging microscopy (FLIM) is an imaging technique for producing an image based on differences in the fluorescence-lifetime rather than its intensity. By quantifying variations in the exponential decay rate of the fluorescence from a fluorescent sample (fluorescence-lifetime) it is possible to report on molecule proximity, pH changes and even polarity. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy and multiphoton tomography. Since the fluorescence-lifetime is insensitive to changes in fluorophore intensity or concentration, it is the most quantitatively precise technique to report on fluoresce resonance energy transfer (FRET).

FRAP Fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) denotes an optical technique capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes. This technique is very useful in biological studies of cell membrane diffusion and protein binding as it not only reports on the diffusion rates of mobile fractions of molecules but also provides information about the proportion of immobile molecules. In addition, surface deposition of a Photo-manipulation/ -ablation

FCS Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuations in the fluorescence intensity. The analysis provides information on physical parameters of the fluorescent particles (molecules) in solution, such as concentration, average fluorescence intensity and diffusion speed. By following changes on these parameters it is possible to study binding events of the molecules or even conformational changes on them.

High-speed imaging >100 frames / sec

Mesoscopie imaging

SPIM/dSLSM Light Sheet Microscopy

light- sheet microscopy, often referred to as single plane illumination microscopy (SPIM), is a rapidly emerging technology that combines optical sectioning with multiple-view imaging to observe tissues and living organisms with impressive resolution. Unlike the conventional techniques of widefield and confocal fluorescence microscopy, the light sheet technique illuminates the illuminates on the region surrounding the focal plane of the detection objective in a twin objective configuration.