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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

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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