Method for correcting a signal backscattered by a sample and associated device

• US 9,594,024 B2
• Filed: 02/04/2016
• Issued: 03/14/2017
• Est. Priority Date: 02/06/2015
• Status: Active Grant

• Current Assignee: Commissariat a L'Energie Atomique (AES Corporation)
• Original Assignee: Commissariat a L'Energie Atomique (AES Corporation)
• Inventors: Roig, Blandine, Koenig, Anne, Dinten, Jean-Marc, Perraut, Francois
• Primary Examiner(s): Nur, Abdullahi
• Application Number:
  US15/015,325
• Publication Number:
  US 20160231249A1
• Time in Patent Office: 404 Days
• US Class Current: 1/1
• CPC Class Codes: G01J 3/0229 using masks, aperture plates, spatial light modulators or spatial filters, e.g. reflective filters
• CPC Class Codes: G01J 3/027 Control of working procedures of a spectrometer; Failure detection; Bandwidth calculation
• CPC Class Codes: G01J 3/44 Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/49 within a body or fluid
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 21/65 Raman scattering
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 2201/0833 Fibre array at detector, resolving
• CPC Class Codes: G01N 2201/121 Correction signals
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence

Abstract

The invention relates to a method for correcting an optical signal produced by a sample comprising the following steps:

• • illuminating a surface of the sample by a first light beam, produced by a first light source, the said first light source being coupled to a first optical system, focusing the said first light beam in an object focal plane of the first optical system, the said object focal plane being situated, in the sample, at a measuring depth z from the surface of the sample;
  • measuring, with a first photodetector, of a first optical signal backscattered by the sample in response to the first light beam, the first photodetector producing a first measured signal representative of the said first optical signal, a spatial filter being interposed between the first optical system and the first photodetector, the spatial filter comprising a window which transmits the said first optical signal towards the said first photodetector, the window being disposed in a conjugate focal plane of the object focal plane of the first optical system;
    wherein the method also comprises the following steps:
  • determining an optical scattering property of the sample;
  • applying a correction function to the first measured signal so as to generate a first corrected signal, the said correction function taking into account the said optical scattering property.

Claims

1. A method for correcting an optical signal produced by a sample comprising the following steps:

illuminating a surface of the sample by a first light beam, produced by a first light source, said first light source being coupled to a first optical system, focusing said first light beam in an object focal plane of the first optical system, said object focal plane being situated, in the sample, at a measuring depth z from the surface of the sample; and

measuring, with a first photodetector, of a first optical signal backscattered by the sample in response to the first light beam, the first photodetector producing a first measured signal representative of said first optical signal, a spatial filter being interposed between the first optical system and the first photodetector, the spatial filter comprising a window which transmits said first optical signal towards said first photodetector, the window being disposed in a conjugate focal plane of the object focal plane of the first optical system,

wherein the method also comprises the following steps;

determining an optical scattering property of the sample;

applying a correction function to the first measured signal so as to generate a first corrected signal, said correction function taking into account said optical scattering property, and

wherein the determination of the optical scattering property of the sample further comprises;

performing a second illumination of a surface of the sample with a second light beam, so as to form, on said surface, an elementary illumination zone, corresponding to the part of said surface illuminated by said second light beam;

detecting N second optical signals, backscattered by the sample, each second optical signal emanating from the surface of the sample at a backscattering distance from said elementary illumination zone, N being an integer greater than or equal to 1, so as to form as many second detected signals; and

determining at least one optical scattering property of the sample, by comparison between;

a function of each second detected signals, and

a plurality of estimations of said function of each second detected signals, each estimation being carried out by considering a predetermined value of said optical scattering property.

2. The method according to claim 1, further comprising determining said measuring depth between the first object focal plane of the first optical system and the surface of the sample, wherein the correction function takes the measuring depth into account.

3. The method according to claim 1, wherein the first light source is a laser source, the photodetector being a Raman spectrophotometer, such that the first measured signal is a Raman spectrum at the measuring depth, the method further comprising the determination of a correction term, as a function of the determined optical scattering property, the correction function comprising an exponential function, the exponent of the exponential function depending on said correction term and on said measuring depth.

4. The method according to claim 1, wherein the first light source is a laser source, the photodetector being a Raman spectrophotometer, such that the first measured signal is a Raman spectrum at the measuring depth, the method further comprising:

measuring, with said Raman spectrophotometer, a depth-wise Raman spectrum, representative of an optical signal backscattered by the sample in response to the first light beam, when said first object focal plane is situated, in the sample, at a depth from the surface, said depth being greater than a previously determined threshold depth;

determining a correction term, as a function of the determined optical scattering property; and

the correction function taking into account said depth-wise Raman spectrum and said correction term.

5. The method according to claim 4, wherein the correction function comprises an exponential function, the exponent of the exponential function depending on the correction term and on the measuring depth.

6. The method according to claim 5, wherein the correction function comprises a product of the Raman spectrum at the measuring depth by said exponential function.

7. The method according to claim 5, wherein the correction function comprises an addition of the depth-wise Raman spectrum to the product of said Raman spectrum at the measuring depth by the exponential function.

8. The method according to claim 1, wherein the first optical signal is a fluorescence signal.

9. The method according to claim 1, wherein the optical scattering property is a coefficient describing the scattering of the light in said sample, including a reduced scattering coefficient, a scattering coefficient or a scattering anisotropy factor.

10. The method according to claim 1, in which at least one backscattering distance is less than 200 μ

m.

11. A device for characterizing a sample, comprising:

a first light source, configured to emit a first light beam towards a surface of said sample;

a first optical system, configured to focus said first light beam in an object focal plane;

a first photodetector;

a spatial filter, interposed between said first optical system and said photodetector, said spatial filter comprising a window for transmitting a first optical signal, backscattered by said sample when it is exposed to said first light beam towards the first photodetector, said window being disposed in the conjugate focal plane of said object focal plane;

the first photodetector being configured to output a first measured signal from said first optical signal;

a microprocessor, being configured to apply a correction function to said first measured signal, said correction function depending on an optical scattering property of said sample;

a second light source, able to emit a second light beam towards a surface of said sample, so as to form, on said surface, an elementary illumination zone;

at least one detection optical fibre, extending between a proximal end, coupled to a second photodetector, and a distal end, configured to collect a second optical signal backscattered by the sample when it is exposed to said second light beam; and

a second optical system comprising all or part of the first optical system, said second optical system exhibiting a magnification factor and an optical axis,

wherein said second optical system conjugates the distal end of each detection optical fibre with an elementary detection zone located on the surface of the sample so that the distance between the elementary illumination zone and each elementary detection zone, perpendicularly to said optical axis, is dependent on said magnification factor.

12. The device according to claim 11, wherein the second optical system is also able to conjugate the second light source with an elementary illumination zone, located on the surface of the sample.

13. The device according to claim 11, also comprising a reflecting element, able to direct:

the first light beam towards the sample;

or the first optical signal backscattered by the sample towards the first photodetector;

or the second light beam towards the sample;

or the second optical signal towards the second photodetector.

14. The device according to claim 11, also comprising

a first detection optical fibre, whose distal end is situated at a first distance from the second light beam;

and

a second detection optical fibre, whose distal end is situated at a second distance from the second light beam, the second distance being greater than said first distance.

15. The device according to claim 11, wherein the second light source comprises an illumination optical fibre, extending between

a proximal end, configured to receive the second light beam;

and a distal end, configured to emit the second light beam towards the surface of the sample.

16. The device according to claim 11, wherein the first optical system and second optical system are merged and form a common optical system.

17. The device according to claim 16, wherein a detection optical fibre is disposed in said conjugate focal plane of said object focal plane of said common optical system, said detection optical fibre being configured to transmit the said first optical signal towards the first photodetector or towards the second photodetector.

18. The device according to claim 16, wherein the first photodetector and the second photodetector form one and the same photodetector.

19. The device according to claim 16, wherein the first light source and the second light source form one and the same light source.

20. A method for correcting an optical signal produced by a sample comprising the following steps:

determining a threshold depth of the sample;

measuring, with a Raman spectrophotometer, a depth-wise Raman spectrum, representative of an optical signal backscattered by the sample in response to a first light beam, when a first object focal plane is situated, in the sample, at a depth from the surface, the depth being greater than the previously determined threshold depth of the sample;

determining an optical scattering property of the sample;

determining a correction term, as a function of the determined optical scattering property;

determining a resulting correction function taking into account the said depth-wise Raman spectrum and the correction term;

illuminating a surface of the sample by a second light beam, produced by a laser light source, the laser light source being coupled to a first optical system, focusing the second light beam in the first object focal plane of the first optical system, the first object focal plane being situated, in the sample, at a measuring depth z from the surface of the sample, the measuring depth being lower than threshold depth;

measuring, with the Raman spectrophotometer, a second optical signal backscattered by the sample in response to the second light beam, the Raman spectrophotometer producing a second measured signal representative of the second optical signal at the measuring depth, thereby forming a Raman spectrum at the measuring depth, a spatial filter being interposed between the first optical system and the Raman spectrophotometer, the spatial filter comprising a window which transmits the said second optical signal towards the Raman spectrophotometer, the window being disposed in a conjugate focal plane of the object focal plane of the first optical system; and

applying the correction function to the Raman spectrum at the measuring depth to generate a corrected Raman spectrum at the measuring depth.

21. The method according to claim 20, wherein the optical scattering property is a reduced scattering coefficient.

22. The method according to claim 20, wherein the correction function comprises:

multiplying the Raman spectrum at the measuring depth by an exponential function, the exponential function depending on the correction term and on the measuring depth; and

adding the depth-wise Raman spectrum to the product of the Raman spectrum at the measuring depth by the exponential function.

23. The method for correcting an optical signal produced according to claim 20, wherein the Raman spectrum at the measuring depth is corrected such that:

SP_corr-z=F_corr(SP_z)=[SP_z−

SP_depth]e<sup>β

(μ

s′

)×

z</sup>+SP_depth

wherein;

SP_z is the spectrum measured at the measuring depth z;

SP_depth is the depth-wise spectrum;

β

(μ

_s′

) is the correction term as a function on the optical scattering property; and

F_corr is the correction function.