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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / ADAM Virgile
Contact person(s) related to this article / BYRDIN Martin
Contact person(s) related to this article / BOURGEOIS Dominique

Super-resolution microscopy (PALM)

A key technique for our team is the well-known PhotoActivated Localization Microscopy (PALM). With this imaging method, invented in 2006 [1-3] we can use and characterize the phototransformable fluorescent proteins we develop for advanced fluorescence imaging applications. The principle of PALM is to circumvent the diffraction-limited optical resolution ( 200-300 nm) thanks to the observation of densely labeled samples, a few molecules at a time. The photoactivation of a very limited number of individual molecules per acquired frame (typically with a time exposure of a few ms), allow their precise localization because of their spatial separation. The accumulation of thousands of such frames, each containing a small number of single molecules that are observed until photobleached will permit the reconstruction of a subdiffraction image ( 20 nm resolution), also called super-resolution image.
Our home-made setup is equipped with five CW solid-state lasers whose wavelengths are ranging from violet to red and intensities between 50-400 mW. The output of these lasers is regulated by a computer-controlled acousto-optical tunable filter (AOTF) and a white lamp is also useable for specific wavelengths. Our motorized inverted microscope (Olympus IX81) is equipped with several objectives with magnification ranging from 5X to 100X, which can be coupled to an anti-drift “nose-piece” system (Olympus). The illumination of the sample is quickly switchable between direct wide-field and total internal reflection fluorescence (TIRF) modes. A fiber-coupling to a spectrophotometer is also possible for a better spectral characterization of a sample along with the image acquisition. A special home-made detection system allows recording images with two EMCCD cameras (Photometrics Evolve 512) on one to four color channels simultaneously.

(A) Example of PALM on a human cell. Actin fibers fused to a green-to-red photoconvertible fluorescent protein are observed in conventional wide field microscopy (left) and in PALM (right). The inset demonstrates the optical resolution improvement. (B) The PALM microscope room and a zoom on the optical table with our setup.

[1] Betzig, E., et al., Imaging intracellular fluorescent proteins at nanometer resolution. Science, 2006. 313(5793): 1642-5
[2] Rust, M.J., et al., Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods, 2006. 3(10): 793-5
[3] Hess, S.T., et al., Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J, 2006. 91(11): 4258-72

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Optical spectroscopy

Cryogenic Absorption/Luminescence Microspectrometer CAL(AI)2DOSCOPE

Our CAL(AI)2DOSCOPE is an apparatus that allows to quasi-synchronously record rapidly and continuously absorption and luminescence spectra of precisely identical portions of microscopic samples (nanoliters).

Optical spectroscopy is a versatile tool for characterization of newly developed/modified materials, both biological (e.g. proteins) and non-biological (e.g. dyes, nanomaterials). It allows easily and nondestructively assessing their purity, electronic properties, some physico-chemical parameters, solvent and temperature stability, etc.
A major limitation is that initially, often only tiny amounts of the substance to be characterized are available, calling for minimization of the sample volume - hence microspectrometry.
In such an instrument, in contrast to macroscopic samples, basically the whole sample volume is illuminated, excluding renovation by diffusion of the molecules under study. For intense illumination (such as laser excitation), this leads to rapid aging of the material under study and it is therefore highly desirable to study both absorption and fluorescence properties concomitantly or as close in time as possible.
In biological microspectroscopy, there exist worldwide a handful of absorption/fluorescence spectrometers that do work either with crossed beams or with manual optical rearrangements, hence they do record either not the same spot or not at the same time, or both.

We use a setup that features co-linear geometry and two prearranged optical paths that are alimented by a common source divided by dicroics/beamsplitters. By use of a single objective for absorption white light and fluorescence excitation and detection, separating/coupling the respective beams by mirrors/filters upstream of the objective, our design principally avoids both problems of mutual misalignment and temporal delay.

Mutual perturbation of absorption and fluorescence measurements is avoided by fast switching of the light sources and detectors by a system of software-controlled triggers and shutters. Thus, by construction, this instrument assures Alignment Independent Detection with Alternative Illumination, hence its name:

Cryogenic Absorption/Luminescence Alignment Independent Alternative Intermittent Detection Optical microSCOPE

Optical setup principle

Characteristic features/parameters

* Easily interchangeable beamsplitting mirrors with different reflection/transmission ratios allow for adaptable distribution of limited photons between the different channels.

* Fiber-coupled light sources and detectors guarantee modularity and easy evolution.

* A camera-coupled microscope-like mechanical design with the sample holder mounted on a motorized goniometer head assures maximal flexibility and convenience in sample handling and alignment, beam focusing, objective exchange.

* Optics (objectives, mirrors, beamsplitters, detectors) were optimized for maximal spectral flatness in the UV/VIS wavelength range (200-800 nm).

* A gaseous nitrogen cryostat allows maintaining the sample at controlled temperatures between 100 and 300K.

* Depending on fiber diameter (0.1 to 0.6 mm) and objective magnification (2x to 15x), spot sizes from 10 to 600 µm diameter can be realized, corresponding to sub-picoliter to sub-microliter sample volumes, respectively.

* Dichroic mirrors for Fluorescence excitation/emission separation available at 405,488 and 561 nm.

* Time resolution is limited by shutter operation and CCD-acquisition to >1 ms

Applications

The CAL(AI)2DOSCOPE allows to compare, e.g., the aging kinetics of a fluorescent protein by absorption and fluorescence and to distinguish transient (blinking) from definitive (bleaching) photodestruction.

Flyer

Clicking on the icon to the right, opens a two page pdf-summary of the instruments features

Press

In 2016, the instrument was featured in an article in "spectroscopy europe"
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Kinetic cristallography

Kinetic protein crystallography : a new approach to visualize reaction intermediates at the near-atomic scale

Kinetic crystallography consists in triggering biological function in the crystalline state of a protein, so as to capture functionally relevant conformational changes. The majority of protein structures presently solved by X-ray crystallography actually correspond to a static state, that represent only poorly the ensemble of conformations adopted in reality by a protein in action. “Kinetic protein crystallography” allows investigating structure in a dynamical fashion. See Bourgeois & Weik, Crystallography Rev. (2009) 15,87-118 and Bourgeois & Royant, Curr. Opin. Struc. Biol. (2005) 15, 1-10).
In a few favorable cases (for example the case of myoglobin), ultra-fast methods such as based on Laue diffraction allow to record real-time movies of proteins in action, whose time-resolution reach 100 ps. However, these methods are difficult to apply. Nevertheless, it is often possible to “trap” reaction intermediates within the crystal, and then to solve the structure of these intermediates with more standard data collection techniques (for an example in the team: see Katona et al, Science, (2007), 316, 449-52). To this aim, we have developed several instruments and methodological approaches based on photo-activation of endogenous or exogenous chromophores, precise temperature control of the samples, and monitoring of the reactions by “in crystallo” spectroscopy (UV-visible absorption and fluorescence, fluorescence lifetime, and Raman spectroscopy). We currently apply these techniques to the case of fluorescent proteins, which are particularly well adapted to kinetic crystallography approaches.

Enzymatic mechanism of superoxyde reductase fom Desulfoarculus baarsii. A lysine residue imports a water molecule essential for H2O2 production into the SOR active site. (Katona et al., 2007).

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