WO2018182526A1 - Appareil d'analyse d'un échantillon - Google Patents

Appareil d'analyse d'un échantillon Download PDF

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Publication number
WO2018182526A1
WO2018182526A1 PCT/SG2018/050155 SG2018050155W WO2018182526A1 WO 2018182526 A1 WO2018182526 A1 WO 2018182526A1 SG 2018050155 W SG2018050155 W SG 2018050155W WO 2018182526 A1 WO2018182526 A1 WO 2018182526A1
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WO
WIPO (PCT)
Prior art keywords
optical
specimen
axis
lens
optical device
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Application number
PCT/SG2018/050155
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English (en)
Inventor
Quan Liu
Yao TIAN
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Nanyang Technological University
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Publication of WO2018182526A1 publication Critical patent/WO2018182526A1/fr

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Classifications

    • 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
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0208—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/021—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using plane or convex mirrors, parallel phase plates, or particular reflectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0216—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using light concentrators or collectors or condensers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0229—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using masks, aperture plates, spatial light modulators or spatial filters, e.g. reflective filters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2803—Investigating the spectrum using photoelectric array detector
    • 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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • 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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • 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
    • G01N2021/6463—Optics
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/001—Axicons, waxicons, reflaxicons

Definitions

  • the present invention relates to an apparatus for analysing a specimen.
  • Optical sectioning refers to an optical technique capable of acquiring images from multiple thin slices of a sample specimen, without need to physically sectioning the sample.
  • Optical sectioning microscopy has become an important tool in biological science research in the past decades due to its superb capability of rejecting out-of-focus light and preserving fine details at the focal spot.
  • Optical sectioning may be achieved experimentally or computationally. While computational optical sectioning techniques (e.g . deconvolution microscopy) are cost effective since they only require normal wide field illumination, they tend to suffer from limited performance and tradeoff between the speed and resolution of the optical sectioning achieved.
  • the experimental approaches for optical sectioning may be realized using one- photon or two-photon techniques.
  • Using a micro-lens array is able to help partially alleviate the problem , but however can induce additional chromatic aberration and require high precision in the fabrication of the pinhole array and micro-lens array.
  • Line scanning enables the acquisition of a line image from a line focus, but the requirement of a slit confocal aperture can yet degrade spatial resolution.
  • LSM Light sheet microscopy
  • SPIM selective plane illumination microscopy
  • Structured illumination microscopy illuminates a sample specimen with a patterned light beam and reconstructs an optically sectioned image from a minimum of three raw images obtained by laterally shifting the pattern on the sample specimen. This technique is able to image an entire field of view without scanning .
  • the theoretical resolution achievable with structured illumination microscopy is similar to confocal microscopy. But the need for several raw images requires a high signal-to-noise ratio and minimum spatial drift during raw image acquisition. This can limit its actual axial resolution and usage in dynamic image acquisition.
  • One object of the present invention is therefore to address at least one of the problems of the prior art and/or to provide a choice that is useful in the art.
  • an apparatus for analysing a specimen comprising: an optical device having reflective or refractive surfaces arranged at a first angle relative to a first axis of the optical device to reflect or refract optical beams emitted from the specimen , the optical beams optically reflected or refracted by the reflective or refractive surfaces to be projected as a distribution of optical points on a plane orthogonal or arranged at a second angle relative to a second axis of the optical device; and an imaging device to process the projected distribution of optical points into at least one image corresponding to a cross-sectional view of a portion of the specimen.
  • the apparatus enables parallel optical sectioning of specimen samples in the axial dimension, which thus eliminates need for scanning in the axial dimension. This has the effect of significantly speeding up 3D imaging of the samples.
  • the first angle may be configured to be a value between 40° and 50°.
  • the first angle may be selected to be 45°.
  • the apparatus may be an optical sectioning microscope adapted for three-dimensional spectroscopic imaging.
  • the reflective or refractive surfaces may be coated with at least a layer of metal to enable reflection or refraction of the optical beams.
  • the metal may include silver, aluminium or gold.
  • the optical device may be a conically-shaped mirror, a reflective device configured to perform total internal reflection, a lens-axicon doublet/triplet or an axicon pair/triplet.
  • the imaging device may be a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS) camera.
  • CCD charge-coupled device
  • CMOS complementary metal-oxide-semiconductor
  • the apparatus may further include an annular lens or an annular mirror configured to focus the reflected or refracted optical beams on the imaging device.
  • the annular lens or the annular mirror may be arranged intermediate the optical device and the imaging device.
  • the apparatus may further comprise at least one lens arranged along the second axis of the optical device to enable further focusing of the reflected or refracted optical beams on the imaging device.
  • the at least one lens may include an achromatic beam expander, at least one relay lens, and a plurality of objective lenses arranged along the second axis of the optical device to enable further focusing of the reflected or refracted optical beams on the imaging device.
  • the achromatic beam expander is arranged intermediate the annular lens and the relay lens
  • the relay lens is arranged intermediate the achromatic beam expander and the objective lenses
  • the objective lenses are arranged intermediate the relay lens and the imaging device.
  • the second axis of the optical device may lie in the z-axis and the plane orthogonal to the second axis of the optical device may lie in the x-y plane.
  • the projected distribution of optical points may collectively form an optical annular image on the plane orthogonal to the second axis of the optical device.
  • the imaging device may be arranged to be positioned on the plane orthogonal to the second axis of the optical device.
  • the apparatus may further include an illuminating source configured to illuminate the specimen using a collimated optical beam to cause the optical beams to be emitted from the specimen, the specimen being illuminated at at least one focal point lying along the second axis of the optical device, the second axis being orthogonal to a surface of the specimen.
  • the at least one focal point may include a plurality of focal points or a focal line lying along the axis orthogonal to the specimen.
  • the illuminating source may include a laser.
  • the apparatus may yet further comprise an achromatic beam expander, and a plurality of axicon lenses arranged along the axis orthogonal to the specimen to enable focused illumination of the collimated optical beam on the specimen.
  • the axicon lenses are arranged intermediate the illuminating source and the achromatic beam expander; and the achromatic beam expander is arranged intermediate the axicon lenses and the specimen.
  • the distribution of the optical points may be continuous or discrete.
  • the distribution of optical points may collectively form at least one circular or elliptical ring on the plane orthogonal to the second axis of the optical device.
  • the at least one circular or elliptical ring may also include a plurality of circular or elliptical rings.
  • a method for analysing a specimen using an apparatus which includes an imaging device and an optical device having reflective or refractive surfaces arranged at a first angle relative to a first axis of the optical device.
  • the method comprises: (i) optically reflecting or refracting optical beams emitted from the specimen by the reflective or refractive surfaces of the optical device, the reflected or refracted optical beams projected as a distribution of optical points on a plane orthogonal or arranged at a second angle relative to a second axis of the optical device; and (ii) processing the projected distribution of optical points by the imaging device into at least one image corresponding to a cross-sectional view of a portion of the specimen.
  • the apparatus may further include an illuminating source, and the method may further comprise: (iii) illuminating the specimen using a collimated optical beam generated by the illuminating source to cause the optical beams to be emitted from the specimen, the specimen being illuminated at at least one focal point lying along the second axis of the optical device, the second axis being orthogonal to a surface of the specimen.
  • FIG. 1 a is a simplified schematic diagram of an apparatus for analysing a specimen, according to a first embodiment.
  • FIG. 1 b is a detailed schematic diagram of the apparatus of FIG. 1 a.
  • FIG. 2 shows an illumination portion of the apparatus of FIG. b.
  • FIG. 3 shows an imaging portion of the apparatus of FIG. 1 b.
  • FIG. 4 is a flow diagram of a method for analysing a specimen using the apparatus.
  • FIGs. 5a-5d show respective views of a cone mirror used in the apparatus.
  • FIGs. 6a and 6b show respective perspective views of the cone mirror.
  • FIGs. 7a-7c show respective views of an annular lens used in the apparatus.
  • FIGs. 8a and 8b show respective perspective views of the annular lens.
  • FIG. 9 shows spot diagrams of simulated depth dependent fluorescence image pattern projected on an image plane of the apparatus.
  • FIG. 1 0a shows virtual emission points formed by the cone mirror.
  • FIG. 10b shows simplified imaging schematics of the annular lens.
  • FIG. 1 1 shows the cone mirror and the annular lens used in combination in the apparatus to enable parallel optical sectioning with high spatial resolution.
  • FIGs. 12a-12d show respective fluorescence images of a 1 1 ⁇ microbead imaged by the apparatus by progressively moving a sample of a specimen downwards to respective depths of 0 mm, 0.025 mm, 0.05 mm, and 0.075 mm.
  • FIGs. 1 3a-13d show respective reflectance images of a piece of silicon sample imaged by the apparatus by progressively moving the sample downwards to respective depths of 0 mm, 0.025 mm , 0.05 mm, and 0.075 mm .
  • FIG. 14a and 14b show respective setups of a parallel optical sectioning fluorescence microscope for 3D spectroscopic imaging, according to a second embodiment.
  • FIG. 15 shows collimated light propagation through an axicon lens to form a focal line.
  • FIG. 16 shows a setup of the parallel optical sectioning fluorescence microscope of FIG. 14 configured for parallel axial imaging.
  • FIG. 1 7a depicts an image representative of a fluorescence bead with a diameter of 1 0 Mm .
  • FIG. 17b is a graph plot of fluorescence intensity integrated over all angles and plotted as a function of radial distance from the optical axis of the setup of FIG. 16.
  • FIG. 1 8 shows a cone mirror arranged in the form of a solid cone.
  • FIG. 1 a depicts a simplified schematic of an apparatus 1 00 for analysing a (sample) specimen 1 02, based on a first embodiment.
  • the specimen 102 is not part of the apparatus 100.
  • the apparatus 1 00 is a (parallel) optical sectioning microscope adapted for three-dimensional (3D) spectroscopic imaging but is not limited to as such. Broadly (with reference to FIG.
  • the apparatus 100 comprises: an optical device 104 having reflective or refractive surfaces arranged at a first angle 101 relative to a first axis 103 of the optical device 1 04 to reflect or refract optical beams emitted from the specimen 1 02, the optical beams optically reflected or refracted by the reflective or refractive surfaces to be projected as a distribution of optical points on a plane 1 05 orthogonal or arranged at a second angle 122 relative to a second axis 107 of the optical device 104; and an imaging device 1 06 to process the projected distribution of optical points into at least one image corresponding to a cross-sectional view of a portion of the specimen 102.
  • the second axis 107 of the optical device 1 04 lies in the z-axis and the first axis 103 is orthogonal to the second axis 107.
  • the first axis 103 may lie in the x-y plane.
  • the plane 1 05 orthogonal to the second axis 107 of the optical device 104 lies in the x-y plane.
  • the first angle 1 01 is preferably a value between 40° and 50°, but an optimal angle is determined to be about 45°.
  • the second angle 122 is to be about twice the value of the first angle 101 , and so correspondingly, if the first angle 1 01 is arranged to have a value between 40° and 50° , then the second angle 122 is to be between 80° and 1 00° for optimal imaging.
  • the imaging device 1 06 is a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS) camera.
  • CMOS complementary metal-oxide-semiconductor
  • the imaging device 106 is arranged to be positioned on the plane 1 05 orthogonal to the second axis 1 07 of the optical device 04.
  • the distribution of optical points collectively forms at least one circular or elliptical ring on the plane 1 05 orthogonal to the second axis 1 07 of the optical device 1 04.
  • the circular or elliptical ring is in the form of an optical annular image.
  • the definition of the at least one circular or elliptical ring also includes a plurality of circular or elliptical rings, as shown in FIG. 9 for example.
  • the distribution of the optical points is continuous or discrete, depending on both the illumination pattern and sample. For example, if the illumination pattern is discrete or the distribution of target molecules or particles in the sample is discrete, the distribution of the optical points is discrete. If both the illumination pattern and the distribution of target molecules or particles in the sample are continuous, the distribution of the optical points is continuous.
  • the reflective or refractive surfaces of the optical device 1 04 are coated with at least a layer of metal to enable reflection or refraction of the optical beams.
  • the metal includes silver, aluminium or gold, but other suitable materials may also be utilised.
  • the optical device 104 can be a conically-shaped mirror, a reflective device configured to perform total internal reflection, a lens-axicon doublet/triplet or an axicon pair/triplet.
  • a customized cone mirror 500 is used as the optical device 104, and FIGs. 5a-5d show respective views of the cone mirror 500.
  • FIGs. 6a and 6b show respective perspective views of the cone mirror 500.
  • the cone mirror 500 can be arranged in the form of a hollow cone (see FIGs.
  • a solid cone 1800 or a solid cone 1800 (see FIG. 1 8) with reflective or refractive interior surfaces (which optionally may have a layer of protective coating , such as silica, thereon).
  • a suitable material such as a gel, a liquid or a gas to enable configuration of a desired refractive index for the cone mirror 500.
  • the solid cone 1 800 itself is to be formed from a suitable lens material, depending on a refractive index desired for the cone mirror 500.
  • the reflective or refractive surfaces of the cone mirror 500 may also be arranged on exterior surfaces of the cone mirror 500, and in such an instance, the said reflective or refractive surfaces may be coated with silver or aluminium.
  • the cone mirror 500 has a generally cylindrical shape, and in the middle of the cylinder, an inverted conical depression 501 is then made in order to form the reflective/refractive surfaces 503 of the cone mirror 500, as shown in FIGs. 5a-5d.
  • the diameter 502 of the base 505 of the cone mirror 500 may be configured as around 20 mm wide, and the height 504 of the cone mirror 500 may be about 8 mm tall.
  • the cone mirror 500 has a hole 507 arranged at the apex portion 506 of the cone mirror 500 to allow light to pass through; in other words, the apex portion 506 is truncated.
  • the hole 507 has a diameter 509 configured to be around 4 mm wide.
  • the cone mirror 500 has the reflective/refractive surfaces 503 which are preferably arranged at 45° (with a tolerance of ⁇ 0.01 °) relative to the horizontal axis 508 of the cone mirror 500. It is to be appreciated that the angle of 45° is disclosed with reference to the first angle 1 01 of FIG. 1 a. It is however to be appreciated that the measurements depicted in FIGs. 5a-5d are not to be construed as limiting on dimensions possible for the cone mirror 500 in any way.
  • FIG. 1 b A detailed schematic diagram of the apparatus 100 is shown in FIG. 1 b. It is to be appreciated that in FIG. 1 b, the depicted sizes of different components of the apparatus 100 in relation to one another are not shown or drawn in scale, and so should not be construed as limiting in any sense.
  • the apparatus 100 has an illumination portion 200 and an imaging portion 202, both of which will be elaborated below.
  • the apparatus 100 may optionally further include an annular lens 108 (or alternatively, an annular mirror) configured to focus the reflected or refracted optical beams on the imaging device 1 06.
  • FIG. 7a shows a top view of the annular lens 108
  • FIG. 7b shows a side view of the annular lens 108
  • FIG. 7a shows a top view of the annular lens 108
  • FIG. 7b shows a side view of the annular lens 108
  • FIG. 7c shows a cross-sectional view of the annular lens 1 08.
  • FIGs. 8a and 8b show respective perspective views of the annular lens 1 08.
  • the annular lens 1 08 is generally arranged in the shape of a convex lens having a circular base 700, but in this case the annular lens 1 08 is particularly configured with aspherical surfaces 702 that contiguously form a generally convex structure.
  • the aspherical surfaces 702 are arranged to rise from the circular base 700 to collectively form the convex structure.
  • the annular lens 1 08 may have a diameter 706 of about 46.43 mm, while the central piano surface 704 may have a diameter 708 of about 3.63 mm.
  • the convex structure may have a height 71 0 of about 1 0 mm . It is also to be appreciated that these dimensions for the annular lens 108 are not limiting in any way; other suitable dimensions are possible too.
  • the illumination portion 200 of the apparatus 100 includes an illuminating source 1 1 0 (e.g. a laser) configured to illuminate the specimen 102 using a collimated optical beam to cause the optical beams to be emitted from the specimen 102.
  • the specimen 102 is illuminated at at least one focal point lying along the second axis 1 07 of the optical device 104, in which the second axis 1 07 is orthogonal to a surface of the specimen 102. More specifically, the at least one focal point includes a plurality of focal points or a focal line lying along the axis orthogonal to the specimen 102.
  • the illumination portion 200 further comprises an achromatic beam expander 1 12.
  • the achromatic beam expander 1 12 comprises a plurality of optical elements. For illustration purposes, three optical elements are shown in FIG. 2 - a first optical element 1 12a, a second optical element 1 12b, and a third optical element 1 12c.
  • the illumination portion 200 further comprises a plurality of axicon lenses 1 14a, 1 14b (e.g . a pair is shown in FIG. 2) arranged along the axis orthogonal to the specimen 102 to enable focused illumination of the collimated optical beam on the specimen 102.
  • the axicon lenses 1 14a, 1 14b may be identical in one embodiment. In other embodiments, the axicon lenses 1 14a, 1 14b may be different (e.g. having different optical characteristics, different sizes, etc), and moreover other suitable optical elements in place of the axicon lenses 1 14a, 1 14b may also be utilised (if necessary).
  • the axicon lenses 1 14a, 1 14b are arranged intermediate the illuminating source 1 1 0 and the achromatic beam expander 1 12, whereas the achromatic beam expander 1 12 is arranged intermediate the axicon lenses 1 14a, 1 14b and the specimen 102.
  • the first and second optical elements 1 12a, 1 12b, 1 12c are arranged adjacent to each other and are also collectively nearer to the annular lens 1 08 than the third optical element 1 12c. It is highlighted that the first, second and third optical elements 1 1 2a, 1 12b, 1 12c collectively form a beam expander 1 12, and their purpose is to optically expand an optical beam passing in the direction from the third optical element 112c to the second optical element 112b and the first optical element 112a.
  • the third optical element 112c has a smaller size than the first optical element 112a and the second optical element 112b to optically expand the optical beam.
  • the three optical elements 112a, 112b, 112c are optional for the disclosed apparatus 100 in relation to analysing the specimen 102.
  • the conical surfaces of the pair of axicon lenses 114a, 114b are arranged to be diametrically opposing to create a light ring.
  • the imaging portion 202 of the apparatus 100 may broadly include at least one lens arranged along the second axis 107 of the optical device 104 to enable further focusing of the reflected or refracted optical beams on the imaging device 106.
  • the at least one lens specifically comprises the same beam expander 112 (e.g. the same plurality of optical elements 112a, 112b, 112c) of the illumination portion 200, at least one relay lens 116, and a plurality of objective lenses 118a, 118b (e.g. a pair is shown in FIG, 3) arranged along the second axis 107 of the optical device 104 to enable further focusing of the reflected or refracted optical beams on the imaging device 106.
  • the achromatic beam expander 112 is arranged intermediate the annular lens 108 and the relay lens 116.
  • the relay lens 116 is arranged intermediate the achromatic beam expander 112 and the objective lenses 118a, 118b. It is to be appreciated that the relay lens 116 is optional for the disclosed apparatus 100 in relation to analysing the specimen 102.
  • the objective lenses 118a, 118b are arranged intermediate the relay lens 116 and the imaging device 106.
  • the annular lens 108 is arranged intermediate the optical device 104 and the imaging device 106. This also correspondingly means the achromatic beam expander 112, the relay lens 116, and the objective lenses 118a, 118b are arranged intermediate the annular lens 108 and the imaging device 06.
  • first, second and third optical elements 112a, 112b, 112c i.e. the beam expander 112 in the imaging portion 202 of the apparatus 100 (as shown in FIG. 3) can be used to optically reduce/narrow an optical beam passing in the direction from the first optical element 112a and the second optical element 112b to the third optical element 112c.
  • the working principles of the apparatus 100 are described in detail below. First, it is to be highlighted that direction of light propagation in FIG. 1 b is indicated by the arrows of beam paths, and shared beam paths between illuminating laser beam and the outgoing fluorescent lights are drawn with double arrows.
  • a light i.e. a laser is used as the illuminating source 1 10) is introduced into the apparatus 100 and passed through the pair of identical axicon lenses 1 14a, 1 14b.
  • a Gaussian laser beam is used as the light arranged to be introduced into the apparatus 100, but should not be construed as limiting.
  • the circular profile of the light is transformed into a collimated, hallow-ring pattern.
  • the beam diameter is now enlarged by about 2.5 times by the optical elements 1 12a, 1 12b, 1 12c (which are custom-built in this case) to accommodate the size of the annular lens 108 (which is also custom-built).
  • the beam diameter enlarged by the achromatic beam expander 1 12 comprising the optical elements 1 12a, 1 12b, 1 1 2c may vary in different situations, and is therefore not restricted to being enlarged by 2.5 times.
  • the Gaussian laser beam (with the enlarged diameter) is focused by the annular lens 1 08 and consequently bent inwards and towards the optical device 1 04 (i.e. the customized cone mirror 500), in which half of the apex angle (i.e. the first angle 101 ) is 45° with its tolerances precisely controlled under ⁇ 0.01 °, as afore disclosed.
  • the specially devised configuration disclosed in FIG. 1 b rather than a focal point like those projected by conventional microscopes, a Bessel beam-like laser focal line is generated by the apparatus 100.
  • a Bessel beam may also be created in a similar manner using other types of optics such as an objective lens, an axicon lens, or an annular apodization mask.
  • the laser focal line shines into the sample 102, and the points along the focal line shone into within the sample 102 are illuminated simultaneously. Consequently, fluorescent or Raman photons at the locations of different depths of the sample 102 are emitted at the same time, which is also illustrated in FIG. 1 b by the arrows 3500 of the beam paths.
  • These fluorescent photons reflected by the cone mirror 500 are refocused by the annular lens 1 08, shrunk through the optical elements 1 12a, 1 1 2b, 1 12c and diverted 90° by a beam-splitter 120 towards the imaging device 1 06.
  • an intermediate image formed by the relay lens 1 16 is magnified by the objective lenses 1 1 8a, 1 1 8b and projected onto an image plane, at which the imaging device 1 06 is positioned adjacently in order to digitally take an image of the projected distribution of optical points (corresponding to the fluorescent photons falling onto the image plane).
  • the lenses 1 1 8a, 1 1 8b may have a 10x magnifying power.
  • objective lenses with other suitable magnifying powers may be used, depending on requirements of intended applications for the apparatus 100.
  • the image plane lies in the x-y plane.
  • FIG. 9 shows simulated spot diagrams 900 of the photons emitted at different depths (i.e. increasing from 0 ⁇ to 40 pm at a 5 pm depth interval) of the sample 102, as projected on the image plane.
  • centre spots of the spot diagrams 900 are images of lights emitted from the focal point of the annular lens 108.
  • each consecutive optical ring is imaged from a depth of about 5 pm deeper from the preceding optical ring.
  • the spot diagrams 900 individually is fairly well separated spatially, which suggests that depth information along the focal line of the sample 102 can simultaneously be extracted by the proposed apparatus 1 00.
  • FIG. 1 1 depicts 1 1 00 the cone mirror 500 and the annular lens 1 08 used together in the apparatus 100 to enable parallel optical sectioning with high spatial resolution.
  • the axial symmetry of the apparatus 100 is herein utilized. Also, the discussions are limited to the y-z plane and the complete results are easily obtainable with a 360° rotation along the optical axis (i.e. the z-axis).
  • the role of the cone mirror 500 is first considered.
  • the light emitted from various points along the z-axis may be viewed as being emitted from virtual points ("virtual emission points") on the y-axis.
  • virtual emission points virtual points
  • FIG. 10a shows lines of the light propagation (as depicted) are intentionally extended and consequently all the lines are found to converge to the virtual emission points.
  • the lights emitted at several z locations are shown magnified in a dashed circled portion 1000 depicted in FIG. 10a. For simplicity, only three different depths are shown in FIG. 10a (and are not to be construed as limiting).
  • the cone mirror 500 is able to transform a line object (denoted by "O" in FIG. 1 1 ) along the second axis 1 07 of the cone mirror 500 (corresponding to the focal line or a plurality of focal points) to a ring image along the lateral dimension (i.e. the x-y plane) orthogonal to the second axis 1 07, which makes it suitable for image detection by a two-dimensional (2D) detector (e.g. a CCD).
  • 2D two-dimensional
  • periphery outline of the ring image is depicted by the word markings "l L " and "l R " .
  • the cone mirror 500 enables the apparatus 100 to carry out parallel optical sectioning at high spatial resolution. It is to be appreciated that the ring image has a finite radial thickness and its radial thickness is equal to the height of the focal line.
  • a problem with the ring image (as formed) is that there is a large hole in the middle of the ring image, which however does not comprise any useful information. As a result, this wastes a large portion of an effective area in the middle of the CCD, if the ring image is simply imaged via conventional convex/objective lenses because the ring at the periphery of the ring image (which has useful information), and the hole in the middle of the ring image (which has no useful information) are both magnified proportionally, resulting in a hollow image on the CCD. This undesirably limits the highest spatial resolution that may be attained by the apparatus 100.
  • the ring image is magnified by the annular lens 108, rather than using a conventional convex/objective lens, to form a magnified ring image.
  • periphery outline of the magnified ring image is depicted by the word markings " I'L" and ' V'- Because the optical axis of the annular lens 108 forms a ring that has the same size as the hole in the middle of the ring image, subsequently only the ring in the periphery (comprising the useful information) is magnified towards the central axis of the apparatus 100.
  • the magnified ring image (an example as illustrated in FIG. 9) indicated by "l' L “ and “l ' R " shows no hole in the middle, and thus the magnified ring image may then be further magnified using conventional convex lens assembly to fully utilize the sensor area of the CCD to attain high-resolution imaging .
  • FIG. 10b a simplified optical setup is shown in FIG. 10b, where the cone mirror 500 is omitted and the emitted optical points are fixed on the y- axis.
  • the object plane 1 002 is positioned at the front of the focal plane 1004 of the annular lens 108.
  • an ideal lens to collectively represent the relay lens 1 16 and the objective lenses 1 1 8a, 1 1 8b
  • the ideal lens is not shown in FIG. 1 0b.
  • the optical axis of the annular lens 108 i.e. depicted as dash lines in FIG. 1 0b
  • forms a ring i.e.
  • FIG. 1 0b depicted as a shaded region in FIG. 1 0b) in the middle of the image.
  • the central plane region of the annular lens 108 is decorated with shades.
  • there are two sets of focal points 1 006a, 1006b i.e. the first set of focal points 1 006a is shown in a first magnified image 1008 and the equivalent second set of focal points 1 006b lies directly opposing to and below the first set of focal points 1 006a, as shown in FIG. 10b
  • Photons emitted from the two focal points are collimated by the annular lens 108, and thereafter, the photons pass through the ideal lens to form the smallest spot in the centre of the image plane 1 01 0.
  • photons not emitted from the focal point cannot be collimated by the annular lens 1 08. Instead, those photons propagate with a finite convergence and form spots at other locations on the image plane 1 01 0 which can be seen from a second magnified image 1012 of the image plane 1 010 in FIG.10b. Referring to the second magnified image 1 012, it is seen that the spots do not overlap spatially, and thus if this pattern is rotated along the optical axis of the apparatus 100 by 360°, ring patterns can be generated as afore discussed (i.e. see FIG. 9).
  • the method 400 comprises: at step 402, optically reflecting or refracting optical beams emitted from the specimen 102 by the reflective or refractive surfaces of the optical device 104, the reflected or refracted optical beams projected as a distribution of optical points on a plane 1 05 orthogonal or arranged at the second angle 122 relative to the second axis 107 of the optical device 1 04; and at step 404, processing the projected distribution of optical points by the imaging device 1 06 into at least one image corresponding to a cross-sectional view of a portion of the specimen 102.
  • a transparent PDMS sample is first prepared with 1 1 ⁇ fluorescent microbeads embedded therein.
  • the PDMS sample is put on a sample stage and a 532 nm laser is focused on the top surface of the PDMS sample.
  • an area (of the PDMS sample) with isolated microbeads is searched for by looking at a fluorescence image of the PDMS sample.
  • a fluorescent image of one single microbead is taken.
  • the PDMS sample is moved downwards to a series of depth locations and a fluorescence image of the PDMS sample is taken at each depth location.
  • Four fluorescence images 1200, 1202, 1204, 1206 at different depth locations i.e.
  • FIGs. 12a-12d 0 mm , 0.025 mm, 0.05 mm, and 0.075 mm respectively. It is observed that the ring patterns can clearly be seen in all four images 1200, 1202, 1204, 1206, which validate the simulated results. It is also to be emphasized that the hollow centre observed in FIG. 12a is due to the microbeads not being located at the surface of the PDMS sample. To support this observation, a series of reflectance images of a small piece of the PDMS sample are taken. The small piece of sample is put to the focus and reflectance images are taken in the same manner as the fluorescence images of the original PDMS sample.
  • the proposed apparatus 1 00 beneficially enables a simple and yet powerful technique (i.e. parallel optical sectioning) to allow parallel optical sectioning in the axial dimension, which eliminates need for scanning in the axial dimension. This in turn significantly speeds up 3D imaging .
  • the proposed apparatus 100 has advantages over conventional optical sectioning techniques that include confocal microscopy (CM), light sheet microscopy (LSM) and structured illumination microscopy (SIM).
  • CM confocal microscopy
  • LSM light sheet microscopy
  • SIM structured illumination microscopy
  • parallel optical sectioning is substantially faster than structured illumination microscopy. Since parallel optical sectioning eliminates the need for axial scanning, it is able to speed up optical sectioning by one to two orders of magnitude, while retaining benefits relating to out-of- focus light rejection.
  • parallel optical sectioning provides high spatial resolution compared to light sheet microscopy and structured illumination microscopy. Then, parallel optical sectioning is cheaper to perform than structured illumination microscopy/light sheet microscopy/structured illumination microscopy, due to simplicity in the setup structure of the proposed apparatus 100.
  • a parallel optical sectioning fluorescence microscope 1400 for three-dimensional (3D) spectroscopic imaging as shown in first and second setups in FIGs. 14a and 14b respectively.
  • coliimated laser light passes through a dichroic mirror (DM), and then hits the top surface of an axicon lens (AL) after being reflected by two galvo mirrors (GM), which are arranged for x- axis and y-axis scanning.
  • GM galvo mirrors
  • I n contrast to a normal objective lens that forms a focal point, coliimated light passing the axicon lens instead forms an elongated focal line as shown in FIG. 15.
  • the focal line generated in this manner is relatively long and wide, which however is not suitable for obtaining high- resolution fluorescence images. Therefore an objective lens (OL) is used to de- magnify the focal line. Importantly, the shrunk focal line is able to excite fluorophores. Fluorescence light is emitted in all directions but only the portion returning in the same optical path as the excitation light will be collected by the objective lens and forwarded to the axicon lens, which is explained below. Also, it is highlighted that the laser source in FIGs.
  • 14a and 14b is in fact a combination of multiple lasers coupled together with multiple dichroic mirrors, in which an active laser to be operated may easily be switched from one laser source to another laser source by sending transistor-transistor logic (TTL) signals to switch on a desired laser source and switch off the remaining laser sources.
  • TTL transistor-transistor logic
  • the returning fluorescence light includes two portions - one portion in the same optical path as the excitation light beam but travelling in the opposite direction forms a coilimated beam, whereas the other portion forms the non-collimated light.
  • each focal depth is translated to a different radial distance on the top surface of the axicon lens as depicted in FIG. 1 5.
  • the coilimated beam is then passed through a long pass filter (LPF) and finally image captured by a camera.
  • LPF long pass filter
  • optical sectioning is herein achieved by first transforming pixels at a range of depths to those at a range of radial distances using the axicon lens, and directly imaging these radially separated pixels onto the camera using an imaging lens (I L). In this manner, parallel optical sectioning is achieved in a snapshot without using a pinhole (PH). This beneficially causes the signal to noise ratio of the resulting image to be increased.
  • the first setup of FIG. 14a works well for sparsely distributed fluorophore molecules such as in 3D particle tracking .
  • the resulting image will be influenced by fluorophore molecules on the lateral dimension in a sample with densely packed fluorophore molecules such as tissue samples.
  • a further focusing lens (FL) and an additional pinhole (PH) may be added to the first setup (which results in the second setup of FIG. 14b) to reduce the influence from other fluorophore molecules laterally close to the focal line.
  • the parallel optical sectioning fluorescence microscope 1400 may conveniently be switched to a Raman microscope or a reflectance microscope, and still be arranged with the capability for optical sectioning.
  • a Raman microscope working with a near infrared (NI R) laser for excitation the filter sets and lens are also to be changed to maximize transmission for N I R light.
  • a reflectance microscope two linear polarizers with perpendicular orientation in polarization are to be placed at the entrance and exit ports of the dichroic mirror (DM) cube to minimize specular reflectance and all long pass filters need to be removed.
  • DM dichroic mirror
  • the parallel optical sectioning fluorescence microscope 1400 is configured for parallel axial imaging using an axicon lens (i.e. see setup 1600 of FIG. 16).
  • the middle dash-dotted line is the optical axis of the setup 1 600.
  • a collimated laser beam (which is not shown in FIG. 16) passing through a beam expander forms a focal line behind the axicon lens.
  • each pair of dashed lines in the sample represents one cone shell corresponding to a different radial distance from the optical axis on the top surface of the axicon lens.
  • three pairs of dashed lines show that fluorescence emitting from each depth of the sample is mapped to a different radial position in the camera.
  • the dashed line shows that fluorescence from another depth can interfere with the fluorescence from the middle dashed line to blur the resulting image, because the two lines land on a same radial position on the camera.
  • FIG. 1 7a depicts an image 1 700 representative of a fluorescence bead with a diameter of 10 ⁇ ⁇ . In line with expectation, one bead yields a single ring and the ring thickness is proportional to the beam diameter.
  • 1 7b is a graph plot 1750 of fluorescence intensity integrated over all angles, and plotted as a function of radial distance from the optical axis of the setup 1600 of FIG. 16. It is to be appreciated that the FWHM of the peak corresponding to the fluorescence beam is about 3 pixels-wide in the camera. Consequently, this suggests that the (un-optimized) setup 1600 has an axial resolution of about 3.3 Mm.
  • the incorporation of a universal spectroscopic imaging module enables imaging of a single fluorophore with arbitrary fluorescence peaks or the concentrations of multiple fluorophores directly in one imaging session, without requiring use of a tunable filter or switching interference filters.
  • this technique does not need side illumination, it is usable to image a turbid sample just like confocal microscopy.
  • the proposed new technique is desirable in numerous applications that require high-speed optical sectioning and/or spectroscopic imaging of single/multiple fluorophores, such as in the study of ischemia reperfusion injury in a mouse model.

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Abstract

La présente invention concerne un appareil (100) destiné à analyser un échantillon (102). L'appareil comprend un dispositif optique (104) ayant des surfaces réfléchissantes ou réfractrices disposées selon un premier angle (101) par rapport à un premier axe (103) du dispositif optique pour réfléchir ou réfracter des faisceaux optiques émis à partir de l'échantillon, les faisceaux optiques réfléchis ou réfractés optiquement par les surfaces réfléchissantes ou réfractrices à projeter sous la forme d'une distribution de points optiques sur un plan (105) orthogonal ou agencé selon un second angle (122) par rapport à un second axe (107) du dispositif optique ; et un dispositif d'imagerie (106) pour traiter la distribution projetée de points optiques en au moins une image correspondant à une vue en coupe d'une partie de l'échantillon. L'invention concerne également un procédé correspondant d'analyse de l'échantillon à l'aide de l'appareil.
PCT/SG2018/050155 2017-03-31 2018-03-29 Appareil d'analyse d'un échantillon WO2018182526A1 (fr)

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TWI788231B (zh) * 2021-02-25 2022-12-21 國立臺灣大學 共軛光學模組、空間轉換光學模組以及其共軛光學之彩色共焦量測系統
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TWI788231B (zh) * 2021-02-25 2022-12-21 國立臺灣大學 共軛光學模組、空間轉換光學模組以及其共軛光學之彩色共焦量測系統
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