US 9,541,751 B2Granted

Method of observation of the emission of light from a sample by dynamic optical microscopy

AssigneeCentre National De La Recherche Scientifique

InventorsEmmanuel Fort, Sandrine Lévêque-Fort, Karla Balaa, Thomas Barroca

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Abstract

Method for observing an emission of light from a sample in a medium of refractive index nL disposed against a surface of a transparent support of refractive index nS, greater than nL, the emission of light comprising luminous components oriented toward the support and forming an angle θ and including supercritical luminous components and critical or subcritical luminous components, implementing an observation device applying filters to the signal; and transforming the signal into an image zone of the sample Modulation of the signal is carried out, in which components of the emission of light are allowed to pass through so as to obtain image zones, the modulation pertaining to all or some of the collected signals; and at least one useful image zone of the sample is produced by combining image zones, the combination evidencing differences between the image zones related to the modulation.

Claims14

Dependent claims

A method for observing a light emission of a sample in a medium with a refractive index nL, the sample being arranged on a surface of a transparent support of refractive index ns, which is greater than nL, the light emission comprising luminous components of a given amplitude and phase, oriented toward the support and forming an angle θ with a direction perpendicular to the surface, said luminous components including supercritical luminous components, for which the angle θ is strictly greater than a critical angle θc=arcsin(nL/ns), and also including critical or subcritical luminous components, for which the angle θ is less than or equal to the critical angle θc, the method comprising:

capturing, using an observation device, at least part of the light emission from a region of interest of the sample and obtaining a captured luminous signal comprising luminous components arising from the supercritical luminous components of the light emission;
applying filters, using the observation device, to the captured luminous signal in order to selectively decrease the amplitude and/or change the phase of certain luminous components of the captured luminous signal to obtain a filtered luminous signal;
and
transforming, using the observation device, the filtered luminous signal into an image zone of the region of interest of the sample;
wherein:
the method realizes a modulation of the filtered luminous signal, in which luminous components arising from critical or subcritical luminous components of the light emission are allowed to pass through in order to obtain a plurality of image zones of one and the same region of interest of the sample, the modulation pertaining to all or some of the luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission;
and
at least one useful image zone of the sample is produced by combining the plurality of image zones to produce at least one enhanced image zone for the region of interest, the combination evidencing differences between the image zones associated with the modulation.

5 more elements

A method for observing a light emission of a sample in a medium having a refractive index nL, the sample being arranged on a surface of a transparent support having a refractive index nS, the refractive index nS being greater than the refractive index nL, the light emission being oriented toward the transparent support and forming an angle θ with a direction perpendicular to the surface, the light emission comprising supercritical luminous components for which the angle θ is greater than a critical angle θc=sin−1nL/nS, critical luminous components for which the angle θ is equal to the critical angle θc, and subcritical luminous components for which the angle θ is less than the critical angle θc, the method comprising:

capturing at least a portion of the light emission from a region of interest of the sample to obtain a captured luminous signal, the captured luminous signal comprising the supercritical luminous components, the critical luminous components, and the subcritical luminous components;
applying filters to the captured luminous signal to selectively decrease an amplitude and/or change a phase of certain luminous components of the captured luminous signal to obtain a filtered luminous signal;
modulating the filtered luminous signal to obtain a first image zone of the region of interest of the sample by allowing luminous components arising from at least the supercritical luminous components and the subcritical luminous components to pass through an aperture of a diaphragm;
modulating the filtered luminous signal to obtain a second image zone of the region of interest of the sample by obscuring luminous components arising from the supercritical luminous components with the diaphragm and allowing luminous components arising from at least the subcritical luminous components to pass through the aperture of the diaphragm;
and
combining at least the first image zone and the second image zone to obtain at least one useful image zone of the region of interest of the sample, the combination evidencing differences between the first image zone and the second image zone associated with the modulation.

3 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/881,707Filed 25 Oct 2011
Priority: FR 10 5891328 Oct 2010
Published: US 2013/0278742 A124 Oct 2013
Granted: 10 Jan 2017
Term ends: Not estimatedThe source records don’t support an estimate.

Classification15 codes

G01N 21/6458
Spatial resolved fluorescence measurements; Imaging: fluorescence microscopy
G01N 21/648
Specially adapted constructive features of fluorimeters: using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
G02B 5/005
Optical elements other than lenses: diaphragms
G02B 21/0088
Specially adapted for specific applications: inverse microscopes