DISCO

DISCO Beamline SRCD endstation provides users with a fully flexible circular dichroism spectrometer in the UV. Reference publication of the endstation :
 M. Réfrégiers, F. Wien, H.-P. Ta, L. Premvardhan, S. Bac, F. Jamme, V. Rouam, B. Lagarde, F. Polack, J.-L. Giorgetta, J.-P. Ricaud, M. Bordessoule and A. Giuliani DISCO synchrotron-radiation circular-dichroism endstation at SOLEIL J. Synchrotron Rad. (2012). 19, 831-835

SRCD advantages

Synchrotron radiation extends toward higher energies the spectral domain of circular dichroism. Moreover the exterme stability of the beam and the wide accessible spectral range are real asset for difficult solvants.

Data analysis

We strongly recommand you to analyse your data with BestSel a novel method for the secondary structure determination and fold recognition from protein circular dichroism spectra. More about the algorithm at Micsonai et al., Proc. Natl. Acad. Sci. USA (2015) doi:10.1073/pnas.1500851112.

Figure 1 d'après Wallace, B.A. and Janes, R.W. (2001) SRCD Spectroscopy of Proteins: Secondary Structure, Fold Recognition and Structural Genomics. Curr. Opin. in Chemical Biology, 5: 567-571

The high synchrotron brilliance allows to work with turbides and very absorbing samples and buffers.

Useful infos
Samples are sandwiched between CaF2 coverslips, a transparent material in the VUV.  Optical pathways may vary from 2,5 μm to 50 μm.

- Typical concentrations are between 0,1 and 10 mg/ml.
- Only few microliters are necessary: from 0.5 μl to 15 μl for optical pathways comprised between 2.5 μm and 50 μm.
- Full thermal scans from 0 to 100°C.

Some buffers and salts absorb a lot in DUV and shall be replaced.  This table adapted from Franz X. Schmid, p. 251 « Protein structure – a practical approach », IRL Press, Oxford 1989, gives absorbances of salts and buffers at 0.01 M in 0.1 cm pathlength.

Bientôt, ici, un logiciel avancé pour l'analyse des spectres de dichroïsme circulaire: CiDiPy

Waiting for a detailed manual (one day ?), you will find here, some useful infos on the two microscopes available at DISCO beamline.

Both microscopes receive   monochromatic light quickly tuneable between 200 and 600 nm. The first, POLYPHEME, is dedicated to DUV fluorescence microspectroscopy  fluorescence, the second, TELEMOS, is a DUV fast full field microscope with Z scanning for 3D reconstruction.

Référence: Jamme, F., Villette, S., Giuliani, A., Rouam, V., Wien, F., Lagarde, B., & Refregiers, M.Synchrotron UV Fluorescence Microscopy Uncovers New Probes in Cells and Tissues. Microscopy and Microanalysis, 2010, 16(5): 507-514

Samples preparation:

Both microscopes are inverted, POLYPHEME is a IX71, TELEMOS is an axioobserver Z1. Most samples will therefore be positioned upside downover a quartz coverslip. Our two main objective use immersion glycerin, it is also possible to observe samples with a air long working distance objective.

• Cells
  Nous possédons une cellule attofluor (Invitrogen). Elle permet de recevoir des cellules qui ont poussé sur une lamelle ronde en suprasyl (nous contacter si vous désirez recevoir de telles lamelles). Si vous possédez vos propres chambres d'incubation, n'hésitez pas à nous contacter.
• Tissues
  Nous conseillons de déposer les biopsies ou coupes sur des lames sans monter de lamelles sauf si vous avez des lamelles UV. Les épaisseurs peuvent varier entre 1 et 10 µm environ, pour des demandes spécifiques n'hésitez pas à nous contacter. Certains milieux de montage transparents aux UVs peuvent être utilisés.
• Fixation protocole (from Slavka Kascakova) :

1. Before seeding the cells on quartz slides, the slides need to be sterile. It's sufficient to sterilize them with 70% of EtOH (let slides from 30 min - 1 hour in 70% of EtOH and then let them dry under the flow). You can eventually, once the slides are dried, let them under the UV light for 30 min.
2. Afterthat the quartz slides are placed into the well plates or Petri dish and cells are deposit on it with its culture medium. The plate is then placed into the incubator with 5% CO2 and 37°C. We are making sure that cells nicely adapt on slide and proliferate.
3. Individual cell treatment.....Incubation with nanoparticles, drug or whatever product for different periods. After incubation, cells are washed with PBS or medium without FCS.
4. Cell treatment is followed by fixation for microscopic observation: Slide is gently removed from well and placed into the Petri dish with 4 % formalin in PBS for 20 min at room temperature. After this period, cells are then washed for 5 seconds with distilled water, to remove the residual PBS from the surface of the cells. The cells are then dried under ambient conditions and stored in fridge at 4°C. (For drying process, one can eventually deposit the slides on Whatman lens cleaning tissue (http://www.whatman.com/PRODLensCleaningTissue.aspx) (Whatman paper leaves no fibers and it's not damaging optical surfaces). It's also important to mention, that manipulation with slide should be very gentle - very easily one can do scratches on the surface of slide or break the slide.

Platine PI de POLYPHEME

PI 542.2CD – 80x80 mm aperture – 100x100 μm travel range

Liste des objectifs

Liste des filtres accessibles sur TELEMOS :

• Dichroïques
  DM300, DM400, DM505
• Bandpass and longpass filtres
  Un panel de filtres passes bandes à l'émission sont utilisables. Ce sont des filtres 1" provenant de chez Omega Filters. Vous pouvez aussi amener vos propres filtres ou discuter avec l'équipe de ligne pour des achats spécifiques pouvant profiter au plus grand nombre.

Liste of  excitables fluorochromes in DUV  :

• Pour une liste détaillée de fluorochromes endogènes, nous vous conseillons de consulter :
Wagnieres G.A., Star W.M. and Wilson B.C. (1998) ln Vivo Fluorescence Spectroscopy and Imaging for Oncological Applications. Photochem. Photobiol. 68, 603-632

Les logiciels utilisés sur les deux microscopes :

1. Pour l'acquisition de données
   - En microspectrofluorimétrie : Labspec
   - En imagerie plein champ et 3D : µManager
2. Pour l'analyse 
   Pour le traitement d'images spectrales :
   - Labspec
   - Matlab
   Pour le traitement d'images 2D : ImageJ
   Pour la déconvolution d'images et la reconstruction d'images 3D: Huygens

Recording mosaïcs

1- In micromanager, check that the pixel size is calibrated

2- perform slide explorer until you obtain the area of interest in transmission mode, save it

3- define a rectangle (ROI) with imageJ tools and click on ROI -> POS

4- launch a multidimensionnal acquisition with multiposition on but only one filter.

How to perform mosaïcs of images for large area mapping on TELEMOS microscope at DISCO Beamline:
Recording mosaïcs
1- In micromanager, check that the pixel size is  calibrated (! for a binning of 1)

2- perform slide explorer until you obtain the area of interest in transmission mode, save it
3- define a rectangle (ROI) with imageJ tools and click on ROI -> POS
4- launch a multidimensional acquisition with multiposition on but only one filter.

Stitching mosaic

1- you will first need to reorder your data, this can easily be made by opening the saved folder in micromanager and clicking on save stack

2- in FIJI, use the plug-in:Stitchin:Deprecated:Stitch sequence of grid of images

3- use the following options : overlap 12% for the 10x.

That's it !

Do not forget to save the result.

La branche APEX de la ligne DISCO délivre des photons VUV entre 350 et 60 nm à pression atmosphérique sous une atmosphère de gaz rare (argon ou néon).
La branche peut être couplée à une source de photoionisation à pression atmosphérique (APPI pour atmospheric pressure photoionisation) pour des analyses par spectrométrie de masse.  Le spectromètre de masse utilisé dans ce cas est un QStar pulsar i de la société ABSciex.  La gamme de masse accessible va jusqu’à m/z 10000 avec un pouvoir de résolution de 9000 dans les deux modes d’ionisation.

Le pompage différentiel est décrit dans :
Giuliani, A., Yao, I., Lagarde, B., Rey, S., Duval, J. F. L., Rommeluere, P., Jamme, F., Rouam, V., Wien, F., de Oliveira, C., Ros, M., Lestrade, A., Desjardins, K., Giorgetta, J. L., Laprévote, O., Herbeaux, C., & Refregiers, M. A differential pumping system to deliver windowless VUV photons at atmospheric pressure. Journal of Synchrotron Radiation, 2011, 18, on-line first.
Les possibilités sont décrites dans :
Giuliani, A., Giorgetta, J. L., Ricaud, J. P., Jamme, F., Rouam, V., Wien, F., & Refregiers, M.Atmospheric pressure photoionization using tunable VUV synchrotron radiation. Nuclear Instruments and Methods B, 2012, on-line first.

Quelques applications possibles des photons VUV...