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Article Dans Une Revue Spectroscopy Europe Année : 2016

The CAL(AI)2DOSCOPE: a microspectrophotometer for accurate recording of correlated absorbance and fluorescence emission spectra

Martin Byrdin

Résumé

Microspectrometers are (sometimes bulky) spectrometers specifically designed for the study of microscopic samples. A variety of instruments have been developed that combine spot sizes down to sub-microns with spec-tral ranges from ultraviolet (UV) to infra-red (IR) for various optical spectroscopy modalities such as absorbance, transmit-tance, fluorescence or vibrational spec-troscopies. If, on top of being tiny, the samples are delicate objects such as protein microcrystals, other challenges than just tight and achromatic focusing arise: sample optical anisotropy, large refractive index and demanding envi-ronmental requirements make most commercially available devices unsuited. Therefore, at many places where micro-spectroscopy is required (for example at synchrotron sources to investigate protein crystals), home-made microspectrome-ters have been developed in recent years that strive to meet the above-mentioned challenges.1At the European Synchrotron Radiation Facility in Grenoble, France (ESRF), a dedicated system based on three mutu-ally aligned mirror objectives has been installed (“Cryobench”)2,3 that combines the absorption (A), fluorescence (F) and Raman modalities with the possibility of rotational adjustment of the sample (via a goniometric support), and the option to cool the sample down to 100 K due to a stream of gaseous nitrogen. Our experience with the powerful possibili-ties of this system, and the realisation of its limitations prompted us to design a next generation microspectrometer dubbed “CAL(AI)2DOSCOPE” which is presented in this article. The biological samples we study (fluorescent proteins used as genetically encoded markers for advanced fluorescence microscopy) exhibit an intricate behaviour highly sensitive to their micro-environment and notably to their illumination history. As a consequence, their absorption and fluo-rescence (giving access to their photo-physical properties) cannot be studied independently, but rather need to be followed (quasi-) simultaneously, requir-ing coordination in space and time. Existing solutions such as crossed beams (e.g. ESRF Cryobench)4 or optics rear-rangement (e.g. SwissLightSource)5 are not fully compatible with these require-ments. Therefore, we developed the alternative solution of a common optical path for both A and F, coupled to rapid switching of light sources and detectors by upstream mechanical shutters.
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Dates et versions

hal-03184018 , version 1 (29-03-2021)

Identifiants

  • HAL Id : hal-03184018 , version 1

Citer

Martin Byrdin, Dominique Bourgeois. The CAL(AI)2DOSCOPE: a microspectrophotometer for accurate recording of correlated absorbance and fluorescence emission spectra. Spectroscopy Europe, 2016, 28 (6), pp.14-17. ⟨hal-03184018⟩
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