WO2016020684A1 - Tomographie optique multiplexée - Google Patents

Tomographie optique multiplexée Download PDF

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Publication number
WO2016020684A1
WO2016020684A1 PCT/GB2015/052272 GB2015052272W WO2016020684A1 WO 2016020684 A1 WO2016020684 A1 WO 2016020684A1 GB 2015052272 W GB2015052272 W GB 2015052272W WO 2016020684 A1 WO2016020684 A1 WO 2016020684A1
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WIPO (PCT)
Prior art keywords
imaging
lens
sample
image
optical
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PCT/GB2015/052272
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English (en)
Inventor
Paul French
James Mcginty
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Imperial Innovations Limited
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Publication of WO2016020684A1 publication Critical patent/WO2016020684A1/fr

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    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4795—Scattering, i.e. diffuse reflection spatially resolved investigating of object in scattering medium
    • 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
    • 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
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G02B21/0044—Scanning details, e.g. scanning stages moving apertures, e.g. Nipkow disks, rotating lens arrays
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • 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
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • 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

Definitions

  • the present invention relates to three-dimensional imaging systems, and in particular to optical tomography systems, for example for imaging mesoscopic biological samples .
  • OPT is the optical equivalent of X-ray computed tomography (CT), in which the 3 -D structure (a stack of X-Z slices) of a rotating sample is reconstructed from a memorized images (X-Y images) obtained at different proj ection angles .
  • CT X-ray computed tomography
  • OPT has been widely applied to anatomical studies of fixed, cleared sample s such as mouse embryos for research into developmental biology . However it would potentially be beneficial to apply it to histopathology and the study of disease mechanisms and potential therapies in disease models .
  • OPT image s can be formed using transmitted light, e . g . to map absorption coefficients, or using fluorescence radiation .
  • Figure 1 represents a transmission OPT system in which an optical light source 10 is located on one side of a sample chamber 12 and arranged to direct light towards the chamber 12, and a detector array 14, such as a CCD detector array, is located on the opposite side of the chamber to the source 10 and arranged to detect light from the source that is transmitted through the sample imaging chamber 12 and through the sample 13 located in the chamber.
  • Figure 2 represents a fluorescence OPT system in which the source 20 is located on one side of the sample chamber 22 and a detector array such as a CCD array 24 is located away from the axi s along which light is transmitted through the source, and arranged to detect light emitted as fluorescence from the sample chamber 22.
  • the system include s a means, such as a rotating sample holder, to rotate the sample inside the imaging chamber 12, 22, so as to rotate the sample 13 , 23 between a number of orientations to allow images to be acquired by the detector array 14, 24, for each of a number of proj ections .
  • the transmitted light or fluorescence radiation can be characterised to provide spectroscopic information, e . g .
  • fluorescence lifetime imaging FLIM
  • OPT offers the opportunity to directly obtain 3-D images of intact "volumetric" samples rather than the standard approach of mechanically slicing the samples and combining digital images of each section to reconstruct 3-D images. This is important because mechanical "sectioning" can damage fragile samples.
  • Absorption contrast can arise from endogenous chromophores, including blood, and from exogenous labels or stains, e.g. the standard H&E stains.
  • Fluorescence contrast can arise from endogenous fluorophores, such as elastin, collagen, NADH, flavoproteins etc., or from exogenous labels including dyes or genetically expressed fluorescent proteins - although the fluorescence properties of the latter can be degraded when certain chemical clearing processes are employed.
  • the autofluorescence can sometimes be used, e.g. by using spectroscopic parameters such as fluorescence lifetime, to provide a label-free readout of the state of biological tissue, e.g. to indicate disease or damage, or to contrast different types of tissue.
  • the image acquisition time can be particularly extended for FLIM OPT where a series of time-gated fluorescence intensity images may be acquired at each angular proj ection as shown in Figure 3 .
  • a light source in the form of an ultrashort pulsed laser 30 is arranged to direct pulses of light towards a sample chamber 22, in this case via a mirror 34.
  • the image acquisition system comprises a gated optical intensifier (GOI) 38 located in the imaging path in front of the detector array 36 with a filter 32 between the GOI and the chamber 22. This arrangement forms time-gated fluore scence images from a sample supported on a rotatable holder within the sample chamber 22.
  • GOI gated optical intensifier
  • the GOI is arranged to relay the fluorescence image to the detector array 36 only during short imaging periods .
  • a delay generator 40 controlled by a computer 42, is arranged to switch the GOI 38 in synchronism with the light source 30 to generate a memorize s time-gated images acquired at specific delays after the excitation pulse s reach the sample .
  • the computer 42 which is connected to the output of the CCD array 36, is arranged to store a s et of image data. Therefore a series of image data sets is built up corre sponding to the fluore scent images recorded at different delay times after the laser excitation pulse . This data can be used to generate FLIM OPT images as is well known .
  • spectral OPT For spectrally resolved OPT, it is usually necessary to acquire multiple spectrally-resolved images for each angular proj ection, e . g . by imaging through different spectral filters . Thus spectral OPT can increase the total image acquisition time by the number of spectral channels de sired .
  • Image quality can also be degraded by deviations from the parallel ray assumption that underlies the standard FBP algorithm .
  • NA numerical aperture
  • Figure 4 shows the approximate relationship between the depth of field (D OF) and the numerical aperture NA; in particular a higher NA results in a lower D OF .
  • D OF depth of field
  • Relatively high NA optics are necessary for producing high resolution images of small sample s and are generally desirable for fluore scence imaging because the light collection efficiency increases with numerical aperture .
  • Figure 4 shows the limiting case (sketched for a single resolution element) when the depth of field of the imaging system is comparable to the diameter of the sample . In this case the tomographic image of the whole sample is reconstructed from approximately plane wavefronts as expected for back proj ection.
  • image s acquired simultaneously at different proj ections could be used to locate specific feature s in 3 -D space and therefore the position of such features could be tracked with a time resolution limited by the frame rate of the cameras rather than the acquisition time for the OPT data set.
  • image data from different angular proj ections could be simultaneously acquired on the same camera using mirror assemblie s or optical fibre bundle s to convey the image information from different imaging channels, defined by different imaging obj ective lense s, to different regions of the imaging sensor.
  • the present invention provides an optical tomography system, which may b e an optical proj ection tomography system, comprising a support arranged to support an obj ect, and an imaging system comprising an obj ective lens arranged to receive light emitted from the obj ect over a range of directions, detection means arranged to detect the light, and means defining at least one aperture arranged to define a plurality of optical paths between the obj ective lens and the detection means whereby the detection means can detect image s of the obj ect from a plurality of different viewing directions .
  • the at least one aperture may be located between the obj ective lens and the detection means, and may be in the back focal plane of the obj ective lens .
  • the system may comprise a condenser lens and the at least one aperture may be located on the back focal plane of the condenser lens .
  • the imaging system may further comprise at least one optical component, for example a tube lens or lenses, arranged to focus light from the obj ective lens, for example onto the detection means .
  • the obj ective lens may be arranged to focus the light from the obj ect at infinity .
  • the detection means may comprise a single imaging detector or an array of detectors .
  • the detection means may have a plurality of areas, which in an array of detectors may comprise a plurality of sub-arrays, each arranged to receive light transmitted along a respective one of the optical paths .
  • the detected images may be formed by a single tube lens or by a number of lense s or other optical components .
  • the imaging system may comprise a plurality of imaging or 'tube ' lenses one in each optical path forming a 'multiplexed imaging channel ' .
  • the image s recorded may corre spond to different viewing directions .
  • Each of the tube lenses may be arranged to focus an image onto the detection means, for example onto a respective one of the sub -arrays .
  • the system may further comprise an image relay lens or lenses arranged to relay the images formed by the tube lense s on to the detection means .
  • Each of the optical paths and the lenses through which the optical paths pass, can b e considered as forming respective imaging sub-channels, each arranged to image the obj ect from a re spective viewing direction .
  • the imaging system may comprise a first imaging system or lens or set of lenses arranged to image the obj ect from a plurality of viewing directions to an intermediate image plane , and a second imaging system or lens or set of lenses arranged to magnify or de-magnify this plurality of images from different viewing directions onto the imaging detector.
  • the support may be arranged to rotate the obj ect about an axis, or it may b e stationary .
  • the viewing directions may be angularly spaced around the axis .
  • the realisation of angularly resolved imaging channels using a common obj ective lens is reminiscent of common-main-obj ective typ e stereomicroscopes although that application is quite distinct from what is proposed here .
  • the goal is to acquire two angularly resolved images to provide depth cues when viewing a 3 -D scene, mimicking human vision .
  • the present invention concerns optical proj ection tomography which requires the acquisition of many angularly resolved 2-D image s sampling throughout 1 80 or 360 rotation in a plane around a sample to provide the set of angular proj ections to required reconstruct a complete 3 -D map of optical properties of a sample using filtered back proj ection or another tomographic reconstruction algorithm .
  • the system may further comprise data acquisition means arranged to acquire a plurality of sets of angularly-resolved image data from each of the imaging systems .
  • the data acquisition means may be arranged to acquire image s from each of the viewing directions of each of the multiplexed imaging channels simultaneously .
  • the support means may be arranged to rotate the obj ect between a plurality of orientations and the data acquisition means may be arranged to acquire at least one data set, or one data set from each multiplexed imaging channel, for each of the orientations .
  • the data acquisition means may be arranged to acquire a data set from each of the viewing directions simultaneously, or in successive sion, for each of the orientations .
  • the angular offset or spacing between the viewing directions about the axis of rotation may be an integer multiple of the angular spacing between the orientations, so that as the obj ect is rotated each of the optical paths can be used to generate image data sets from the same direction relative to the obj ect.
  • the angular offset or spacing between the viewing directions about the axis may be an integer multiple of the angular spacing between the orientations plus a fraction, such as a half, of that angular spacing , so that as the obj ect is rotated the different optical paths can be used to generate image data sets from directions which are angularly spaced relative to the obj ect more closely than, for example at half of, the angular spacing between the viewing directions .
  • the plurality of imaging channels may be focussed at respective focal points or planes which are equidistant from the axis of rotation of the obj ect.
  • the focal points or plane s may be at different distances from the axis of rotation . This means that as the obj ect is rotated, different parts of it will be imaged in focus by the two (or more) imaging channels or subchannels .
  • the system may further comprise proce ssing means, such as a processor, arranged to receive the image data sets and process them to generate a further image data set, which may be a tomographic or three-dimensional image data set.
  • proce ssing means such as a processor, arranged to receive the image data sets and process them to generate a further image data set, which may be a tomographic or three-dimensional image data set.
  • the system may be an optical proj ection tomography system, and for example may be a fluore scent imaging system . However it may be transmission imaging system, or even a scattering imaging system. In each case the system may further comprise a source of radiation which may be detected after transmission through, or scattering in, the obj ect, or which may cause the fluorescence which is then detected .
  • the system may further comprise a sample chamber.
  • the support means may be arranged to support the sample within the chamber.
  • the chamber may have a wall, part of which may be formed by the obj ective lens which also forms part of a plurality of the optical multiplexed imaging channel . Indeed each of the optical multiplexed imaging channels may include a lens which forms part of the wall of the chamber.
  • the chamber may be filled with an index matching fluid having a refractive index similar to that of the sample .
  • the invention further provides an optical tomography system comprising a support arranged to support an obj ect, and an imaging system comprising an obj ective lens arranged to receive light travelling from the obj ect over a range of directions, detection means arranged to detect the light and means defining at least one aperture or localised light source in the back focal plane of the condenser lens arranged to define a plurality of optical paths between the condenser lens and the sample whereby the sample can be illuminated from a plurality of different viewing directions .
  • Some embodiments of the invention may permit the use of multiple simultaneous imaging directions using a single obj ective lens, so that the directions can be more closely spaced than would be possible using multiple conventional obj ective lenses .
  • the system may further comprise any one or more features, in any combination, of the embodiments of the invention that will now be de scribed by way of example only with reference to the accompanying drawings .
  • Figure 1 is a schematic view of a transmission optical tomographyimaging system forming part of an embodiment of the invention ;
  • Figure 2 is a schematic view of a fluorescence optical tomography system forming part of an embodiment of the invention
  • Figu re 3 is a schematic view of a fluorescent lifetime imaging system forming part of an embodiment of the invention.
  • Figu re 4 is a diagram showing the depth of field and numerical aperture in an optical tomography system ;
  • Figures 5a and 5b are diagrams of different sized sample s in an optical tomography system ;
  • Figure 6 is a diagram of an optical tomography system according to an embodiment of the invention.
  • Figure 7 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figure 8 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figure 9 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 10 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 11 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 12 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 13 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 14 is a diagram of an optical tomography system according to a further embodiment of the invention
  • Figu re 15 is a diagram of an optical tomography system according to a further embodiment of the invention
  • Figure 16 is a diagram of an optical tomography system forming the basis of further embodiments of the invention.
  • Figu re 17 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 18 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 19 is a diagram of an optical tomography system according to a further embodiment of the invention.
  • Figu re 19a is a top view of part of the system of Figure 19 ;
  • Figu re 20 is a diagram of an optical tomography system according to a further embodiment of the invention . Description of Em bodiments of the Invention
  • an imaging system comprises a light source (not shown) and a sample chamber (not shown for clarity in this and subsequent figure s) having a rotatable sample holder for supporting a sample 62 and arranged to rotate the sample about an axis of rotation Z .
  • a detector array 66 in the form of a camera (which could be a CCD camera or another type of camera) is arranged to detect fluore scence emitted from the sample 62.
  • the array can be considered as made up of a number of sub-arrays 67, in this case three, each of which is arranged to detect emissions travelling to the array 66 from different angularly separated optical paths .
  • This multiplexed imaging channel forms part of an OPT system which is otherwise the same as that of Figures 1 to 3 and will not be described in detail here . It will be appreciated that the same imaging system could b e incorporated into other type s of OPT system .
  • Each of the detector sub-arrays 67 records an image from its own optical system defining a respective optical path or imaging sub -channel, in each case comprising a respective imaging lens or 'tube lens ' 63 and a respective aperture 68 in this case formed as an opening in an opaque aperture member.
  • a single obj ective lens 70 having a focal length fj forms part of all three imaging sub -channels and is located between the apertures and the sample chamber so that the aperture s 68 all lie in its back focal plane, which is als o known as the Fourier plane .
  • the tube lense s 63 which have a focal length f 2 are also all located in a common plane which is parallel to the Fourier plane of the obj ective lens 70 and to the plane of the detector array 66. They are located so that the apertures 68 also lie on the focal plane of the tube lenses, and so that the detector array lies on the back focal plane of the tube lense s .
  • each of the imaging sub-channels will transmit light emitted from the obj ect at a respective angle (or in fact range of angles) , centred on a respective viewing direction .
  • the three detector sub -arrays 67 will therefore receive light emitted from the obj ect in three respective different viewing directions .
  • Light travelling along each of the imaging sub-channels is focused on to a respective one of the detector sub-arrays 67 to form a respective image . Therefore the detector array 66 can detect images from three different viewing angle s simultaneously .
  • the tube lense s 63 in this embodiment can be separate lenses or formed as an integrated array of lenses parallel to the focal plane .
  • the detector array is then also divided into a corresponding array of sub-arrays . This could provide a set of imaging directions which are angularly spaced apart in two different directions, one parallel and one perpendicular to the axis of rotation of the sample Z . This could provide additional information that could be utilised using more sophisticated reconstruction algorithms than simple FBP, including iterative algorithms . This approach could be applied to aid tomographic reconstruction using scattered light.
  • the tube lense s 63 are located on the back focal plane of the obj ective lens 70.
  • the tube lenses 63 can then themselve s be integrated with the apertures that define the separate imaging channels .
  • the imaging system of Figure 6 can be de scribed as a multiplexed imaging channel and multiple such imaging channels, each with their own obj ective lens and detector array, can be combined in an OPT system to provide multiple sets of angularly resolved imaging sub-channels, as represented in Figure 7. If each imaging channel is focused at a different depth in the obj ect, this can be used to increase the numbers of imaging depths in one system . Alternatively the imaging channels or sub -channels can be arranged to image the same depth in the sample but could select light of different wavelengths (e . g . by using a filter) to provide spectrally resolved imaging channels . This can be used to decrease the required time to acquire spectrally resolved OPT data.
  • three multiplexed imaging channels 160 are spaced, in this case equally, around a sample chamber.
  • each multiplexed imaging channel 160 can image the obj ect from three different viewing directions, the whole system can image it from nine different directions simultaneously .
  • the exact number of imaging directions in each system and the number of imaging systems can be varied as appropriate .
  • This approach can be used to realise a One-shot' OPT imaging system in which the obj ect does not need to be rotated as it can be imaged from a large number of directions, preferably simultaneously, but optionally sequentially by moving the imaging channels .
  • This system can be used for sample s for which rotation is impractical, such as growing plants or live animals or intact tissues .
  • the system can also be used to capture 3 -D fluorescence distributions at the frame rate of the cameras and be applied to dynamic samples, including specimens passing through a channel in the (vertical) z axis perpendicular to the plane of the diagram in Figure 7.
  • This can be used to implement 3 -D imaging in flow cytometry or for high throughput 3 -D imaging of sample s such as tumour spheroids or rapid imaging of small organisms such as nematode worms or drosophila.
  • the entire OPT configuration can be scanned vertically to map the 3 -D distribution of a long obj ect such as a plant stem .
  • the optical configuration in Figure 6 is one possible configuration to provide multiple angularly separated imaging sub -channels using a single obj ective lens but it may not be the most convenient, for example in terms of magnification .
  • Figures 8 to 10 show three further embodiments, each having multiple angularly multiplexed imaging sub-channels, using a single obj ective lens, which can provide flexibility in terms of magnification and other imaging properties .
  • a single multiplexed imaging channel is shown, and the systems are the same as that described in Figure 6 unle ss otherwise described, and corre sponding components are indicated by the same reference numbers increased by 200, 300 and 400 respectively .
  • corre sponding components are indicated by the same reference numbers increased by 200, 300 and 400 respectively .
  • the obj ective lens 270 , aperture s 268 , and tube lenses 263 are arranged as in the embodiment of Figure 6.
  • the CCD camera including the detector array 266 is further away from the tube lense s 263 .
  • a further image relay lens 280 is located between the back focal plane 282 of the tube lense s and the detector array 266, and is arranged to focus images from all of the three imaging sub -channels onto the plane of the detector array 266.
  • the arrangement in Figure 8 can be adapted to provide different imaging channels focusing at different depths in the sample by translating the tube lenses along their optical axe s so that they are at different distances from the focal plane of the obj ective lens 270.
  • the tube lenses 263 can be adjustably mounted so that their positions can each b e adjusted independently along their optical axes .
  • the configuration of Figure 8 can be further extended by replacing the image relay lens 380 with a pair of lenses 380 , 384, one behind the other.
  • the lens 380 closer to the tube lense s is adj ustably mounted so that its position can be adj usted along its optical axi s .
  • the arrangement in Figure 9 can also be adapted to realise different imaging sub-channels focusing at different depths in the sample by translating the tube lenses , which are also adj ustably mounted, along their optical axe s as with the embodiment of Figure 8. By translating lens 380 along its optical axis, all the imaging channels can be adjusted together to focus to the same adj ustable depth in the sample .
  • FIG. 6-9 relay all the multiplexed imaging subchannels associated with a specific obj ective lens to the same detector array i . e . the same camera sensor. It is also possible to use multiple camera sensors in a common image plane, one for each imaging channel . However, referring to Figure 10, in a further embodiment a separate camera is used for each imaging sub -channel and the imaging plane s, i . e . the planes of the detector arrays 466 of the three cameras, are not coincident, but facing in different directions, in this case being at right angle s to each other.
  • a mirror 486 is placed in the optical path between the tube lens 463 and the two imaging lense s 480, 484 so that the optical path is turned through 90° .
  • This approach has the advantage that the OPT image s can be acquired with a large number of camera pixels, thereby potentially increasing the achievable spatial resolution of the OPT reconstructed 3 -D image .
  • these multiplexed OPT set-ups can also incorporate adaptive optics elements such as spatial light modulators (SLM) or segmented mirrors (SM) .
  • SLM spatial light modulators
  • SM segmented mirrors
  • the se adaptive optics elements can als o be used to realise focusing of different angularly multiplexed imaging sub - channels to different depths in the sample .
  • an adaptive optics element such as a segmented mirror to simulate the function of the array of lense s .
  • Such an element can also incorporate compensation of optical aberrations in the system and provide focusing of different imaging channels to different depths in the sample .
  • Figure 1 1 1 a further embodiment is shown .
  • Features corresponding to thos e in Figure 10 are indicated by the same reference numerals increased by 100.
  • This numbering convention is used for the further embodiments de scribed below with the numbers increasing by a further 100 for each embodiment. In each case the features are the same unle ss the de scription or drawings indicate otherwise .
  • the systems of Figure s 6 and 7, and indeed those of Figure s 8 , 9 and 10 can also be used for imaging with transmitted light.
  • the obj ective lens 570, apertures 568 , tube lenses 563 and detector array 566 are the same as in the embodiment of Figure 6 , but the light source 5 10 is located on the optical axis of the obj ective lens 570 on the oppo site side of the sample chamber 522, and a condenser lens 526 is provided between the light source and the sample to relay light from the source 5 10 onto the sample from a range of directions .
  • This form of transmitted light OPT can be implemented with incoherent light sources although it can also b e undertaken with laser illumination . In the latter case it may be necessary to addre ss the issue of speckle , in some embodiments using a rotating diffuser to time average the phase of the laser radiation across the field of view .
  • the required separation of the simultaneous imaging sub-channels could b e provided by using mirrors at different angles of incidence to "fold" the optical imaging arm and reflect the different imaging channels at different angle s such that they do not overlap on the camera sensor.
  • the use of prisms and other optical components to separate angularly resolved imaging channels to realise a plenoptic camera was recently described in an MSc thesis at Imperial College London [H. D. Young, MSc thesis : "Plenoptic Cameras " (201 3) Imperial College London] .
  • this prior work concerned plenoptic depth-resolved imaging of 3 -D obj ects, rather than OPT with reconstruction of the complete 3 -D map of optical propertie s throughout the sample using filtered back proj ection or an alternative tomographic image reconstruction technique .
  • the author does suggest applying the plenoptic camera to a rotating sample to increase the light detection efficiency by collecting light through multiple apertures but does not identify the different sub-image channels with different angular proj ections for OPT.
  • the camera 666 can be arranged to sequentially acquire different angular proj ections without sample rotation .
  • a spatial light modulator could be used to provide an electronically adj ustable aperture or multiple apertures that could be scanned to acquire different angular proj ections .
  • the sequential acquisition of the angular proj ections enable s each of the images to be captured with the full pixel count of the camera sensor, i . e . using the same full area of the detector array for each proj ection, and so this approach can lead to higher resolution of the reconstructed OPT image than configurations where multiple angular proj ections are recorded on different areas of the same camera sensor.
  • this arrangement can b e implemented with a number of obj ective lense s 770 angularly spaced around the sample, each with its own set of imaging channels and detector array, as illustrated in Figure 13 .
  • this can be applied to samples for which rotation is impractical, such as growing plants or live animals or intact tissues .
  • several imaging systems as shown in Figure 13 are angularly spaced around the axis of a tube , which extends along the z axis perpendicular to the plane of the page of Figure 13 , but also spaced axially along the tube .
  • the aperture could be implemented by one or more spiralling slits that effectively present an aperture or apertures corresponding to varying angular proj ections along the z axi s .
  • This arrangement can image a sample travelling through the tube through apertures in the tube wall at different angles as a function of distance along the tube .
  • a sequence of images corre sponding to different angular proj ections can be acquired with no moving parts .
  • the proce ssor which generates the image from the detector signals could be arranged to us e helical tomographic reconstruction techniques, such as those described in [Alicia Arranz, Di Dong, Shouping Zhu, Markus Rudin, Christos Tsatsanis,Jie Tianand Jorge Ripoll, Opt Exp 2 1 (20 13 ) 259 12] .
  • illumination from a source 8 10 can be used to image with scattered light.
  • imaging channels can collect the scattered light detected at different angle s with respect to the illumination direction and determine the structure of the sample by reconstructing the amplitude and phase of the scattered light, for example using phase retrieval technique s in a manner analogous to ptychography or to Fourier ptychography, which is an extension of ptychography but utilise s illumination of the sample at different angles .
  • Rotation of the sample in such a system can provide a wide range of illumination angles that can lead to a high resolution in the reconstructed image .
  • More strongly scattering sample s can also be imaged by collecting scattered radiation and/or fluore scence at a range of illumination angles for tomographic reconstruction without phase information .
  • imaging in scattering sample s including biological sample s such as live zebrafish, it is possible to use more sophisticated algorithms than simple filtered back proj ection to reconstruct tomographic image s .
  • Some of these algorithms can include models of light scattering and it may b e useful to acquire information about the scattering of the illumination radiation in order, for example, to use inverse scattering approache s to reconstruct tomographic images .
  • This approach can make use of measurements analysing the transmitted and/or fluore scence light with respect to spectroscopic and other parameters, which can include wavelength, polarisation, scattering angle and/or photon arrival time or path length in the sample and can be applied to obtain improved transmitted light tomographic image s and also to inform the reconstruction of fluorescence tomographic image s .
  • Angularly resolved detection of transmitted and scattered light can b e implemented by incorporating an aperture 968 scanning in the BFP of the obj ective lens 970 , as illustrated in Figure 1 5 .
  • This scan is undertaken at each rotational position of the sample, so that the full CCD pixel count of the detector array 966 can be used to acquire the information from angular subsets of the scattered light and using this angularly resolved information can improve the precision with which the light scattering is characterised .
  • a spatial light modulator could be used to provide an electronically adj ustable aperture or multiple aperture s in the BFP of the obj ective lens that could be employed to provide different angular proj ections .
  • Figure 16 shows a further embodiment in which transmitted light and scattered light image s are acquired by respective imaging systems, each the same as that in Figure 6. It would be possible to acquire transmitted light and scattered light images in one multiplexed imaging channel and spectrally resolved fluore scence images at different angular proj ections in a different multiplexed imaging channel .
  • all the angularly resolved image channels can be used to record transmitted or scattered illumination light or the image channels could all acquire fluorescence images in the same spectral band .
  • Clearly other configurations are pos sible including the use of time- resolved and or polarisation-resolved detectors .
  • the Fourier multiplexing approach can also be extended to illumination of the sample where, in further embodiments, aperture s or point illumination sources can be deployed in the back focal plane of the condenser to realise angularly resolved illumination .
  • FIG. 17 can be employed where an aperture 1 168 is scanned in the BFP of the condenser lens 1 124 to provide illumination at different angles while the sample is stationary .
  • a spatial light modulator could be used to provide an electronically adj ustable aperture or multiple apertures in the BFP of the condenser lens 1 124 that could be employed to provide different angular proj ections .
  • a focussed laser beam waist could be translated in the back focal plane of the condenser lens 1 124 to provide illumination at different angular proj ections with greater efficiency .
  • the set-up depicted in Figure 17 could be modified to replace the single moving aperture 1 168 in the BFP of the condenser lens with multiple aperture s 1268 , as indicated in Figure 1 8.
  • the illumination systems of Figure s 17 and 1 8 can in some case s be combined with multiple or moving apertures between the obj ective lens and the CCD camera.
  • the interference of coherent laser emanating from two or more points in the back focal plane of the condenser, using moving or multiple apertures such as those in Figure s 17 and 1 8 , could be used to realis e structured illumination of the sample during the OPT acquisition as is shown in Figure 19.
  • the two illuminating beams 13 1 0a, 13 10b are displaced in the vertical plane as shown in Figure 19a so as to produce horizontal fringes in the sample such that the structured illumination would look similar for different angularly multiplexed imaging channels, i . e . channels angularly spaced around the vertical rotational axis of the sample .
  • the excitation beams may be periodically modulated in amplitude or phase such that fluorescence emitted from the focal volume where two or more excitation beams overlap will be temporally modulated . If the excitation beams are modulated at different frequencies, then the fluore scence from the focal volume will include a contribution that is modulated at the difference frequency . Alternatively, if the phase between the excitation beams is modulated at a single frequency, the fluorescence collected from the focal volume may be modulated at the same frequency . Using appropriate demodulation techniques, optically sectioned images of fluorescence from the focal volume can be acquired .
  • the set-ups for OPT with laser illumination could be modified to arrange that the spectral components of incident laser radiation are spatially dispersed in the BFP of the condenser lens . This could be used to implement 'temporal focusing ' as has been de scribed to provide wide-field optical section in combination with multiphoton excitation, e . g . [D. Oron, E. Tal, and Y. Silberberg, Optics Express vol 13 (2005), 1468-1476] .
  • the set-ups depicted in Figures 20-22 could be further modified to combine the approach of temporal focussing with the technique of structured illumination, as discussed in [H. Choi e t al, Biomedical Optics Express, vol 4 (2013) 995-100] , to enable clearer images of the sample within the depth of field of the imaging system to be acquired, particularly in the presence of scattered light.
  • OPT techniques of the various embodiments described above can all b e combined with spectroscopic contrast such as excitation or emission wavelength, polarisation or fluorescence lifetime .
  • the pre sent invention can be applied to any current application of OPT including developmental biology of both animals and plants, volumetric histopathology of ex vivo sample s, in vivo imaging of live organisms including live disease models such as zebrafish for drug discovery and studie s of disease mechanisms .
  • OPT organic radical polymerase chain reaction
  • Some embodiments of the present invention can address this critical issue by reducing the image acquisition time to acquire high resolution images and increasing the light collection efficiency by enabling the use of higher NA imaging systems .

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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un système de tomographie optique comprenant un support conçu pour soutenir un objet, et un système d'imagerie comprenant un objectif (70) conçu pour recevoir la lumière se déplaçant depuis l'objet (62) sur une plage de directions, des moyens de détection (66) conçus pour détecter la lumière, et des moyens (68) définissant au moins une ouverture conçue pour définir une pluralité de chemins optiques entre l'objectif et les moyens de détection, les moyens de détection pouvant détecter des images de l'objet à partir d'une pluralité de directions de visualisation différentes.
PCT/GB2015/052272 2014-08-05 2015-08-05 Tomographie optique multiplexée WO2016020684A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017013054A1 (fr) * 2015-07-17 2017-01-26 Leica Microsystems Cms Gmbh Microscope à feuille de lumière pour la représentation simultanée de plusieurs plans d'objet
CN107037576A (zh) * 2017-05-03 2017-08-11 中国科学院苏州生物医学工程技术研究所 光学显微成像设备及其成像方法
WO2017191121A1 (fr) * 2016-05-03 2017-11-09 Leica Microsystems Cms Gmbh Microscope et procédé de localisation de molécules fluorescentes dans trois dimensions spatiales
CN108036989A (zh) * 2017-12-07 2018-05-15 金恩升 一种食品安全检测用制样装置
WO2018167786A1 (fr) * 2017-03-13 2018-09-20 Technion Research And Development Foundation Ltd. Système et procédé fondés sur la ptychographie
EP3413033A1 (fr) * 2017-06-09 2018-12-12 Roche Diagnostics GmbH Procédé et appareil de détermination des propriétés d'un échantillon de laboratoire contenu dans un récipient d'échantillons de laboratoire
US10365219B1 (en) 2018-02-06 2019-07-30 Xidian University Two-mode Raman optical projection tomography system
WO2019153530A1 (fr) * 2018-02-06 2019-08-15 西安电子科技大学 Système bimodal d'imagerie par tomographie à projection optique et par effet raman
CN110799829A (zh) * 2017-07-06 2020-02-14 特拉维夫大学拉莫特有限公司 用于在环境室中的生物细胞样品的三维无标记光学成像的系统和方法
GB2589327B (en) * 2019-11-26 2023-09-13 Andor Tech Limited Differential phase contrast microscope

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5889593A (en) * 1997-02-26 1999-03-30 Kla Instruments Corporation Optical system and method for angle-dependent reflection or transmission measurement
US20030085335A1 (en) * 2001-11-07 2003-05-08 Gilad Almogy Spot grid array imaging system
US20060061857A1 (en) * 2004-09-22 2006-03-23 Lee Suk-Won Confocal scanning microscope using two nipkow disks
US20080180792A1 (en) * 2007-01-25 2008-07-31 Georgiev Todor G Light Field Microscope With Lenslet Array
WO2013132257A1 (fr) * 2012-03-07 2013-09-12 Imperial Innovations Limited Tomographie à projection optique multiplexée angulaire
CN203323713U (zh) * 2013-06-28 2013-12-04 湖南长重机器股份有限公司 一种无执行机构的曲面形状精密测量装置
US20140209821A1 (en) * 2013-01-31 2014-07-31 Hewlett-Packard Development Company, L.P. Viewing-angle imaging

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5889593A (en) * 1997-02-26 1999-03-30 Kla Instruments Corporation Optical system and method for angle-dependent reflection or transmission measurement
US20030085335A1 (en) * 2001-11-07 2003-05-08 Gilad Almogy Spot grid array imaging system
US20060061857A1 (en) * 2004-09-22 2006-03-23 Lee Suk-Won Confocal scanning microscope using two nipkow disks
US20080180792A1 (en) * 2007-01-25 2008-07-31 Georgiev Todor G Light Field Microscope With Lenslet Array
WO2013132257A1 (fr) * 2012-03-07 2013-09-12 Imperial Innovations Limited Tomographie à projection optique multiplexée angulaire
US20140209821A1 (en) * 2013-01-31 2014-07-31 Hewlett-Packard Development Company, L.P. Viewing-angle imaging
CN203323713U (zh) * 2013-06-28 2013-12-04 湖南长重机器股份有限公司 一种无执行机构的曲面形状精密测量装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAMANAKA K ET AL: "AN ARTIFICIAL COMPOUND EYE USING A MICROLENS ARRAY AND ITS APPLICATION TO SCALE-INVARIANT PROCESSING", OPTICAL REVIEW, SPRINGER VERLAG, TOKYO, JP, vol. 3, no. 4, 1 July 1996 (1996-07-01), pages 264 - 268, XP008047158, ISSN: 1340-6000, DOI: 10.1007/S10043-996-0264-6 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017013054A1 (fr) * 2015-07-17 2017-01-26 Leica Microsystems Cms Gmbh Microscope à feuille de lumière pour la représentation simultanée de plusieurs plans d'objet
US10877254B2 (en) 2015-07-17 2020-12-29 Leica Microsystems Cms Gmbh Light sheet microscope for simultaneously imaging a plurality of object planes
US11067781B2 (en) 2016-05-03 2021-07-20 Leica Microsystems Cms Gmbh Microscope and method for localizing fluorescent molecules in three spatial dimensions
WO2017191121A1 (fr) * 2016-05-03 2017-11-09 Leica Microsystems Cms Gmbh Microscope et procédé de localisation de molécules fluorescentes dans trois dimensions spatiales
WO2018167786A1 (fr) * 2017-03-13 2018-09-20 Technion Research And Development Foundation Ltd. Système et procédé fondés sur la ptychographie
US11347045B2 (en) 2017-03-13 2022-05-31 Technion Research And Developmen T Foundation Ltd. Ptychography based system and method
CN107037576B (zh) * 2017-05-03 2023-08-15 中国科学院苏州生物医学工程技术研究所 光学显微成像设备及其成像方法
CN107037576A (zh) * 2017-05-03 2017-08-11 中国科学院苏州生物医学工程技术研究所 光学显微成像设备及其成像方法
US11009499B2 (en) 2017-06-09 2021-05-18 Roche Diagnostics Operations, Inc. Method and apparatus for determining properties of a laboratory sample contained in a laboratory sample container by tomographic reconstruction
CN109030425A (zh) * 2017-06-09 2018-12-18 豪夫迈·罗氏有限公司 用于确定容纳在实验室样品容器中的实验室样品的性质的方法和设备
JP2019002915A (ja) * 2017-06-09 2019-01-10 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 実験用試料容器に含まれる実験用試料の性質を決定するための方法及び装置
EP3413033A1 (fr) * 2017-06-09 2018-12-12 Roche Diagnostics GmbH Procédé et appareil de détermination des propriétés d'un échantillon de laboratoire contenu dans un récipient d'échantillons de laboratoire
CN110799829A (zh) * 2017-07-06 2020-02-14 特拉维夫大学拉莫特有限公司 用于在环境室中的生物细胞样品的三维无标记光学成像的系统和方法
CN108036989B (zh) * 2017-12-07 2020-11-06 福建赛福食品检测研究所有限公司 一种食品安全检测用制样装置
CN108036989A (zh) * 2017-12-07 2018-05-15 金恩升 一种食品安全检测用制样装置
WO2019153530A1 (fr) * 2018-02-06 2019-08-15 西安电子科技大学 Système bimodal d'imagerie par tomographie à projection optique et par effet raman
US10365219B1 (en) 2018-02-06 2019-07-30 Xidian University Two-mode Raman optical projection tomography system
GB2589327B (en) * 2019-11-26 2023-09-13 Andor Tech Limited Differential phase contrast microscope

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