Method and device to achieve spatially confined photointeraction at the focal volume of a microscope

Patent metadata

Field	Value
Publication number	US-9971136-B2
Application number	US-201414778618-A
Country	US
Kind code	B2
Filing date	Mar 20, 2014
Priority date	Mar 21, 2013
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

The invention relates to a method and a system to achieve spatially (e.g. three-dimensionally) confined photomodulation at the focal volume ( 50 ) in a ample ( 55 ) mounted in a microscope system, comprising two or more laser light sources ( 41, 42 ) emitting light ( 32, 34 ) of different wavelengths adapted to excite a material in an identical number of independent excitation steps to a higher vibrational state from which the material relaxes, either emitting a conversion light to be detected (“photoexcitation”) or modulating the spectral properties of the material (“photomodulation”).

First claim

The invention claimed is:

1. A method for spatially confined photomodulation or photoexcitation at a focal volume ( 50 ) of a microscope system ( 90 ), comprising the steps:
providing means ( 41 , 42 , 85 , 185 ) to generate a priming laser light beam ( 32 , 302 ) comprising a priming wavelength band ( 32 , 302 ) and a converting laser light beam ( 34 , 304 ) comprising a conversion wavelength band ( 34 , 304 ) different to the priming wavelength band ( 32 , 302 ),
providing a sample, comprising a photoexcitable probe,
wherein said probe comprises a low energy level ( 31 ) and a primed energy level ( 33 ) having a greater energy than the low energy level ( 31 )
illuminating the probe with the priming laser light beam, such that the probe is transitioned by photointeraetion with the priming laser light beam from the low energy level ( 31 ) to the primed energy level ( 33 ) in an excitation step,
illuminating the probe with the converting laser light beam ( 34 , 304 )
such that the probe transitions ( 37 ) to a photomodulate state ( 38 ) altering the spectral properties of the probe, or
such that said probe transitions ( 305 ) under the emission of a photon to the low energy level ( 31 ).

2. Method according to claim 1 , wherein the probe further comprises a conversion energy level ( 35 ) having a higher energy than the low energy level ( 31 ) and the primed energy level ( 33 ), and wherein under illumination of the probe with the converting laser light beam ( 34 , 304 ), said probe transitions from the primed energy level ( 32 , 302 ) to the conversion energy level ( 35 )
from where the probe transitions ( 37 ) to said photomodulated state ( 38 ),
or
from where said probe transitions ( 305 ) under the emission of a photon from said conversion energy level ( 35 ) to the low energy level ( 31 ).

3. Method according to claim 1 , the photon emitted from the probe upon transition to the low energy level, stems from a spontaneous emission process.

4. Method according to claim 1 , wherein the photomodulated state ( 38 ) is a photoconverted, photoactivated or photoswitched state of the probe.

5. Method according to claim 1 , wherein the probe comprises a photoconvertible, photoswitchable or photoactivatable protein.

6. Method according to claim 1 , wherein the probe is illuminated simultaneously or sequentially with the priming laser light beam ( 32 , 302 ) and the converting laser light beam ( 34 , 304 ).

7. Method according to claim 1 , wherein the probe is illuminated with a third laser light beam comprising a third wavelength hand and wherein said third laser light beam is either a second priming laser light beam or a second convening laser light beam.

8. Method according to claim 1 , wherein the priming laser light beam ( 32 , 302 ) and the converting laser light beam ( 34 , 304 ) are focused within the focal volume ( 50 ) of the microscope system ( 90 ).

9. Method according to claim 1 , wherein the priming laser beam ( 32 , 302 ) comprises a priming wavelength band that comprises at least a portion of the blue or blue/green visible spectrum, and that the converting laser light beam ( 34 , 304 ) comprises a conversion wavelength hand that comprises at least a portion of the visible far red and/or near infrared spectrum.

10. Microscope system ( 90 ), comprising:
means ( 41 , 42 , 85 , 185 ) for generating a priming laser light beam ( 32 , 302 ) of a priming wavelength band and a converting laser light beam ( 34 , 304 ) of a conversion wavelength band different to the priming wavelength band,
wherein the microscope system ( 90 ) comprises a photoexcitation device ( 51 , 61 , 81 , 82 , 181 , 182 , 281 , 184 ) that is designed to superimpose the priming laser light beam ( 32 , 302 ) and the convening laser light beam ( 34 , 304 ) within the focal volume ( 50 ) of the microscope system ( 90 ), such that the priming, laser light beam ( 32 , 302 ) the convening laser light beam ( 34 , 304 ) are superimposed only within the focal volume ( 50 ),
wherein said photoexcitation device ( 81 , 82 ) comprises a first objective lens ( 81 ) and a first optical element ( 82 ) arranged at the first objective lens ( 81 ), wherein said first optical element ( 82 ) comprises a first and a second portion ( 121 , 122 , 111 , 112 ), designed to superimpose the priming and the converting laser light bean ( 32 , 34 , 302 , 304 ), only within the focal volume ( 50 ) are passed through said first optical element ( 82 ) and the first objective lens ( 81 ), or
wherein said photoexcitation comprises a first lens ( 181 ) and a first optical element ( 182 ) threaded on the first lens ( 181 ), and a second lens ( 281 ) and a second optical element ( 184 ) threaded on the second lens ( 281 ), and wherein the first and the second optical element ( 182 , 184 ) are designed such that the priming and the converting laser light beam ( 32 , 34 , 302 , 304 ) are superimposed only within the focal volume ( 50 ).

11. Microscope system according to claim 10 , wherein the first portion ( 111 , 121 ) of the optical element ( 82 ) is adapted to transmit only the priming laser light beam ( 32 , 302 ) and the second portion ( 112 , 122 ) of the optical element ( 82 ) is adapted to transmit only the converting laser light beam ( 34 , 304 ), wherein said portions ( 111 , 112 , 121 , 122 ) comprise dichroic mirrors.

12. Microscope system according to claim 10 , wherein the first and the second portion ( 121 , 122 ) of the first optical element ( 82 ) are arranged concentrically ( 121 , 122 ) with respect to a common center or that the first and the second portion ( 111 , 112 ) of the first optical element ( 82 ) are arranged such that they each form a halve of the first optical element ( 82 ).

13. Microscope system according to claim 10 , wherein said photoexcitation device ( 51 , 61 ) comprises a first and a second lens ( 51 , 61 ), wherein the first and the second lens ( 51 , 61 ) are arranged such that the priming and the converting laser light beam ( 32 , 34 , 302 , 304 ) are superimposed only within the focal volume ( 50 ), wherein the first and the second lens ( 51 , 61 ) are arranged such that the priming and the converting laser light beam ( 32 , 34 , 302 , 304 ) are propagated at an angle to each other.

14. Microscope system according to claim 10 , characterized in that either the priming or the convening laser light beam ( 32 , 34 , 302 , 304 ) is shaped as a light sheet after passing through the first or second lens ( 51 , 61 ).

15. Microscope system according to claim 10 , characterized in that the priming laser beam ( 32 , 302 ) comprises a priming wavelength band that comprises at least a portion of the blue or blue/green visible spectrum, and that the convening laser light beam ( 34 , 304 ) comprises a conversion wavelength band that comprises at least a portion of the visible far red and/or near infrared spectrum.

Assignees

• Eth Zuerich
• California Inst Of Techn

Inventors

• Pantazis Periklis
• Dempsey William P
• Truong Thai V
• Fraser Scott E
• Georgieva Lada

Classifications

• G02B5/201

  in the form of arrays · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G02B27/141

  using dichroic mirrors · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N2021/1785

  Three dimensional · CPC title