Microscopy Facility

This page describes the microscopy facility designed and maintained by the author at the Gly-CRRET laboratory, Science and Technology Faculty, UPEC University [1].
Several acquisition devices are available at the laboratory for image acquisition whether for macroscopic or microscopic specimens.

Histological preparations, stained by histochemistry or by immunolabeling are observable with microscopes from the standard color imagery with upright microscope to the epifluorescence or confocal fluorescence with inverted microscope. This can be done on glass slides or on fine bottom petri dishes.

 Confocal Spinning Disc Microscopy

The facility offers an IX 81 Olympus inverted microscope for epifluorescence equipped with a spinning disc confocal device working with non coherent lightning (metal halide arc). The acquisition is performed through a cooled Hamamatsu ORCA R2 CCD camera. This easy to use system allows confocal or not observation of the same field, at the same resolution, and this with 5 channels (phase contrast, bleu, green, red and near infra-red fluorescences).

• Bandwidth are given with precision on the following links:
  
   UV excitation, blue fluorescence (U-MNU2).
   Blue excitation, green fluorescence (U-MWIBA3).
   Green excitation, red fluorescence (U-MNG2).
   Red excitation, near IR fluorescence (CY5).

• This system can be used with the following objectives :
  
   4x phL NA 0.13 UplanFLU
   10 x ph1 NA 0.3 UPlanFLN
   20 x ph1 NA 0.45 LUCPlanFLN
   40 x ph2 NA 0.6 LUCPlanFLN
   60 x ph3 NA 1.25 Oil UplanFLN
   40 x NA 1.3 Oil UplanFLN

This apparatus provides greatly deblurred images in a single plan and authorises with axial stacks after deconvolution, 3D rendering and analysis of structures whose size is not lower than the order of the micrometer.

 Example of application : Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2. van Zoggel H, Carpentier G, Dos Santos C, Hamma-Kourbali Y, Courty J, Amiche M, Delbé J. PLoS One. 2012. (Article).

 Living Cells Observation

The device described above can be used for living cells observation by using a thermo-regulated incubation chamber placed onto the microscope stage. Cells
are maintained at constant hygrometry at the temperature of 37°C with an atmosphere containing 5% of CO2.

 Laser Scanning Confocal Microscopy

The laboratory is also equiped with a laser scanning confocal device (point to point acquisition) Olympus FV 1000.

This system allows five channels for observation of blue, green, red, near IR fluorescence and transmission.

• The excitation is provided by four lasers :
  
   Near UV laser diode (405 nm) for blue fluorescence (430-455 nm).
   Blue laser diode (473 nm) for green fluorescence (490-525 nm).
   Green HeNe gas laser (543 nm) for red fluorescence (560-620 nm).
   Red laser diode (635 nm) for near IR fluorescence (655-755 nm).

• This system can be used with the following objectives:
  
   60 x NA 1.35 Oil UplanSApo
   40 x NA 1.3 Oil UplanFLN

This device allows an effective resolution within the order of 50 nm per point by using an oil immersion 40x (NA 1.3) objective and about 25 nm per point with the 60 x objective.

 Exemple of application : Cossutta M, Carpentier G, Darche M, Houppe C, Ponzo M, Raineri F, Vallée B, Gilles ME, Villain D, Picard E, Casari C, Denis C, Paques M, Courty J, Cascone I. Weibel-Palade Bodies Orchestrate Pericytes During Angiogenesis. Arterioscler Thromb Vasc Biol (ATVBAHA). 2019 Sep;39(9):1843-1858. Article.

 Routinely Microscopy

Two other microscopes are available for routinely control or observation applications:

 Evos automatic microscope. This eyepiece less microscope allows color and fluorescence observations, as well as slide scanning by image mosaic acquisitions. Available objectives are 4x, 10x, 20x and 40x. Fluorescences in red, green and blue are typically useful for DAPI, GFP and RFP observations.

 Upright Olympus BH2 microscope for epifluorescence equipped with a Scion monochrome CCD camera, and 10 x, 20 x 40x and 100x objectives (the two last ones are oil immersion). Observations are possible for the classical red, green and blue fluorescences (typically DAPI, FITC and Rhodamine etc...) and phase contrast.

 Semi-microscopic and Macroscopic Image Acquisitions

 A Dino Lite mini microscope provided with a polarized filter allows image acquisition at the semi-microscopic scale (large histological slices, biopsies etc…).
 A monochrome BL-200 Panasonic video CCD camera coupled to a Scion LG3 grabber card can be used for macroscopic object images acquisition like gels, or bacteria colonies for example. The quality of the images is compatible with densitometric analysis.

 Computer Tools Developments Using the ImageJ Software

For several years now, original image analysis programs have been developed at the laboratory by Gilles Carpentier.

These tools were designed to answer to particular problems encountered by researchers in the laboratory. Some of these tools are available for the community on the ImageJ’s NIH website, or on the author’s website.

Among the treated subjects, we can cite dot blot and protein array analysis, quantitative in vitro and in vivo
angiogenesis analysis, Boyden chamber analysis, spacial calibration of images, helper tools for multidimensional image processing etc…

 Associated links: image.bio.methods.free.fr, ImageJ on Wikipedia, ImageJ software.

Manager, Gilles Carpentier. Contact : carpentier@u-pec.fr