WO2014155262A1 - Procédé de formation d'image d'un échantillon fluorescent - Google Patents

Procédé de formation d'image d'un échantillon fluorescent Download PDF

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Publication number
WO2014155262A1
WO2014155262A1 PCT/IB2014/060057 IB2014060057W WO2014155262A1 WO 2014155262 A1 WO2014155262 A1 WO 2014155262A1 IB 2014060057 W IB2014060057 W IB 2014060057W WO 2014155262 A1 WO2014155262 A1 WO 2014155262A1
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WO
WIPO (PCT)
Prior art keywords
points
sub
sample
fluorescent
detecting
Prior art date
Application number
PCT/IB2014/060057
Other languages
English (en)
Inventor
Daniela GANDOLFI
Jonathan MAPELLI
Egidio D'ANGELO
Paolo Pozzi
Original Assignee
Gandolfi Daniela
Mapelli Jonathan
D Angelo Egidio
Paolo Pozzi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gandolfi Daniela, Mapelli Jonathan, D Angelo Egidio, Paolo Pozzi filed Critical Gandolfi Daniela
Priority to EP14723476.9A priority Critical patent/EP2979122A1/fr
Priority to US14/778,351 priority patent/US20160054226A1/en
Priority to JP2016504800A priority patent/JP2016514837A/ja
Publication of WO2014155262A1 publication Critical patent/WO2014155262A1/fr

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/067—Electro-optic, magneto-optic, acousto-optic elements
    • G01N2201/0675—SLM

Definitions

  • the invention relates to a method of imaging a fluorescent sample using the technique known as two-photon fluorescence microscopy, which can be generally used to generate an image of the detected surface.
  • Fluorescence scanning is often performed using scan heads mounted to microscopes that can detect samples loaded with a fluorophore that allows detection of their edge and surface points, in other words, of their perimeter conformation in a detection plane.
  • this type of scans requires the use of a pulsating infrared laser beam which is focused into a very small volume of the sample to be scanned, of the order of a cubic micron.
  • the fluorescent dye If the fluorescent dye is present in this very small volume, it will emit a fluorescent light which is sensed by a camera or the like and transmitted to an apparatus that displays the fluorescence signal, e.g. on a display screen.
  • the whole surface of a sample can be only imaged by providing additional devices that form the scan head of the microscope and allow the laser beam to be moved in predetermined directions, typically along parallel overlapping rows, until the whole surface to be imaged is scanned, thereby obtaining the final image by capturing the individual fluorescence emissions of the fluorophore.
  • a larger scan of a sample surface may be also obtained using a device known as D.O.E. (Diffractive Optical Element), which provides appropriate phase modulation of the laser beam to divide it into a beam of parallel sub-beams which simultaneously illuminate multiple fluorescent points of a sample, thereby allowing a fixed distribution of points that may be used by an experimenter.
  • D.O.E. diffractive Optical Element
  • a first drawback is that microscope scan heads that can displace the laser beam for scanning all the points of the sample have a very high cost, due to their complex structure.
  • a second drawback concerning the use of the D.O.E. device is that, since the latter can only provide a fixed distribution of sub-beams, it must be associated with a beam displacing apparatus to obtain a final image having a sufficient resolution.
  • One object of the invention is to improve the prior art.
  • Another object of the invention is to provide a method of imaging a fluorescent sample that can provide images of the sample using a microscope that has no additional apparatus for displacing the scan beam over the sample surface that is required to be imaged for fluorescent point detection.
  • the invention provides a method of imaging a fluorescent sample as defined by the features of claim 1 .
  • the invention provides an apparatus for imaging a fluorescent sample as defined by the features of claim 4.
  • FIG. 1 is a first schematic example of a schematic view of a scan field, showing a first subset of points irradiated by a first distribution of scan beams;
  • FIG. 2 is a second schematic example of a schematic view of a scan field, showing a second subset of points, complementing the first subset, which are irradiated by a second distribution of scan beams;
  • FIG. 3 is a schematic example of a general view of a set of scannable fluorescent points
  • FIG. 4 is a third schematic example of a schematic view of a scan field, showing a third subset of points irradiated by a third distribution of scan beams;
  • FIG. 5 is a fourth schematic example of a schematic view of a scan field, showing a fourth subset of points irradiated by a fourth distribution of scan beams;
  • FIG. 6 is a fifth schematic example of a schematic view of a scan field, showing a fifth subset of points irradiated by a fifth distribution of scan beams;
  • FIG. 7 is a schematic example of a general view of an additional set of scannable fluorescent points
  • FIG. 8 is a general schematic view of a scanner apparatus for implementing the method of the invention.
  • FIG. 9 is a flow-chart of the steps of the method of the invention.
  • numeral 1 designates a sample element which, for example, is represented by a grid having rows “F1 -F7" and columns “C1 -C7" of cells that generally form a scan field 2, hereinafter briefly referred to as field 2, here a flat square geometric figure.
  • the illustrated grid may have any perimeter and composition and that, as mentioned above, it is merely a schematic, non-limiting example of a sample to be analyzed.
  • the scan field 2 contains a set of detectable points that have the common feature of being fluorescent and hence detectable when a scan beam, such as a laser beam 4, indicated with a broken line in Figure 8 and emitted by a source 5, impinges thereupon.
  • the laser beam 4 is modulated by a known S.L.M. (Spatial Light Modulator) device, referenced 10, which divides it into a first predetermined number of sub-beams 8A that form a first beam of scan laser sub-beams.
  • S.L.M. Spatial Light Modulator
  • first distribution of beams 8 When the first distribution of beams 8 irradiates the sample 1 in a first initial detection step, it generates a first subset of first detected points, conventionally indicated as small circle 9 in Figures 1 to 3, also referred to hereinafter as first points 9, on a first perpendicular scan plane, typically the plane "P1 " on which the field 2 lies.
  • This first subset of first scanned points provides a first part of the image (in pixels), which is transmitted to a display apparatus, schematically referenced 1 2 in Figure 8, which reproduces a first part of the overall image to be captured.
  • a second beam of detection laser sub-beams 8B is obtained, which is spatially different from the first beam 8A such that, when it irradiates the sample 1 in a later detection step, it will generate a second subset of second detected fluorescent points, conventionally indicated in the figures with cross symbols 1 1 , and hereinafter also referred to as second points 1 1 .
  • the number of second points 1 1 of the second subset complements the number of first points 9, to reach the total number of points that form the set of the detectable points of the field 2.
  • the second image part of this second subset is also sent to the display apparatus 1 2 which integrates it with the first previously captured part to form the whole image of the field 2 of the sample 1 .
  • three-dimensional images may be also captured and reproduced, by adjusting the focus of a lens 1 3 of a focusing apparatus 20 situated downstream from the S.L.M. device, through which the beams 8A and 8B pass.
  • the second subset of scanned points 1 1 is detected on a second plane "P2" other from the plane "P1 " and normally parallel thereto.
  • S.L.M. device is intended to mean that a phase distribution of the laser beam 8B is selected to perform a second detection, such that an illumination distribution selected by the experimenter and different from that of the beam
  • the step 1 00 indicates the start of the method of capturing/acquiring an image, followed by a step 1 01 of selecting the detection of a first subset of first detectable points 9 of a scan field 2.
  • the step 1 01 is followed by a further step 1 02, which is the starting step during which the first beam 8A of laser sub-beams is generated, using a hologram-template, by the S.L.M. device 1 0 which irradiates the sample in the first subset of the first selected points 9.
  • the first subset of detected points 9 is sent to the display means 1 2 along the flow line 1 04 that comes out of the selection step 1 1 0 and reaches the step 1 05, in which reconstruction of an image to be constructed and displayed starts.
  • a first part of an image to be constructed in the display means 1 2 is opened, and a pixel intensity is assigned to this first part, in the following step 107.
  • this first part of the detection method of the invention is repeated in a subsequent step for detection of at least one second subset of detectable points 1 1 , which defines a second part of the image to be constructed and which, like the previous one, is later sent to the step 1 05 and to those that follow, 1 06 and 1 07.
  • a selection step 1 1 is provided, in which the method is repeated from the step 1 06 for any further subset of detected points.
  • the method of the invention includes the image reconstruction step 1 1 3, which is followed by the step 1 14 of selecting the particular points of interest of the image reconstructed by joining the subsets of detected points 9 and 1 1 , the step 1 1 5 of generating a hologram template of the points of interest, the step 1 1 6 of programming the S.L.M. device 1 0 with the hologram template and the step 1 17 of capturing the image by a camera.
  • the invention has been found to fulfill the intended objects.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Health & Medical Sciences (AREA)
  • Multimedia (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé d'imagerie d'un échantillon fluorescent comprenant les étapes consistant : à balayer des points fluorescents (9, 11) dudit échantillon à l'aide d'un moyen de scanneur (10, 8A, 8B), ce qui permet d'obtenir ainsi des points fluorescents balayés; à imager lesdits points fluorescents balayés sur un moyen d'affichage (12), ledit balayage comprenant les étapes consistant : à prédéfinir un champ de balayage (2) dudit échantillon, qui comprend un ensemble de points fluorescents pouvant être balayés (9, 11); à irradier de façon séquentielle, à l'aide d'un moyen d'irradiation (4, 8A, 8B), au moins un premier sous-ensemble de points dudit ensemble de points et au moins un second sous-ensemble dudit ensemble de points, qui complète ledit premier sous-ensemble par rapport audit ensemble de points. Les premier et second sous-ensembles peuvent être irradiés à des distances focales d'irradiation différentes (P1, P2).
PCT/IB2014/060057 2013-03-25 2014-03-22 Procédé de formation d'image d'un échantillon fluorescent WO2014155262A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP14723476.9A EP2979122A1 (fr) 2013-03-25 2014-03-22 Procédé de formation d'image d'un échantillon fluorescent
US14/778,351 US20160054226A1 (en) 2013-03-25 2014-03-22 An image forming method of a fluorescent sample
JP2016504800A JP2016514837A (ja) 2013-03-25 2014-03-22 蛍光サンプルの画像形成方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMO20130478 2013-03-25
ITMO213A000078 2013-03-25

Publications (1)

Publication Number Publication Date
WO2014155262A1 true WO2014155262A1 (fr) 2014-10-02

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5923466A (en) * 1993-10-20 1999-07-13 Biophysica Technologies, Inc. Light modulated confocal optical instruments and method
US20030021016A1 (en) * 2001-07-27 2003-01-30 Grier David G. Parallel scanned laser confocal microscope
US20060209399A1 (en) * 2005-02-28 2006-09-21 Yokogawa Electric Corporation Confocal microscope
WO2011023593A1 (fr) * 2009-08-24 2011-03-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé et appareil pour l'imagerie d'un échantillon cellulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5923466A (en) * 1993-10-20 1999-07-13 Biophysica Technologies, Inc. Light modulated confocal optical instruments and method
US20030021016A1 (en) * 2001-07-27 2003-01-30 Grier David G. Parallel scanned laser confocal microscope
US20060209399A1 (en) * 2005-02-28 2006-09-21 Yokogawa Electric Corporation Confocal microscope
WO2011023593A1 (fr) * 2009-08-24 2011-03-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé et appareil pour l'imagerie d'un échantillon cellulaire

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