WO2011072175A2 - Procédé et système pour réalisation d'image de microscopie à éclairage structuré en trois dimensions rapide - Google Patents

Procédé et système pour réalisation d'image de microscopie à éclairage structuré en trois dimensions rapide Download PDF

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WO2011072175A2
WO2011072175A2 PCT/US2010/059779 US2010059779W WO2011072175A2 WO 2011072175 A2 WO2011072175 A2 WO 2011072175A2 US 2010059779 W US2010059779 W US 2010059779W WO 2011072175 A2 WO2011072175 A2 WO 2011072175A2
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image
plane
beams
illumination
pattern
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PCT/US2010/059779
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WO2011072175A3 (fr
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William M. Dougherty
Steven Charles Quarre
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Applied Precision, Inc.
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Priority to EP10836720A priority Critical patent/EP2510394A2/fr
Priority to JP2012543296A priority patent/JP2013513823A/ja
Publication of WO2011072175A2 publication Critical patent/WO2011072175A2/fr
Publication of WO2011072175A3 publication Critical patent/WO2011072175A3/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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • 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
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
    • G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
    • G02B26/10—Scanning systems
    • G02B26/105—Scanning systems with one or more pivoting mirrors or galvano-mirrors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/145—Beam splitting or combining systems operating by reflection only having sequential partially reflecting surfaces

Definitions

  • the present invention is related to fluorescence microscopy and, in particular, to an optical -electrical-mechanical method and subsystem for fast image collection for 3D-SIM imaging,
  • 3D-SIM Three-dimensional, structured illumination microscopy
  • 3D-S1M requires no specialized fluorescent, dyes or proteins, unlike certain competing super- resolution techniques.
  • Biologists achieve high resolution with 3D-S1M, but retain convenient and familiar fluorescence labeling techniques: .Multiple images of the subject are made with a shifting and rotating illumination pattern, Higher resoiution is achieved b solving a system of equations to restore the fine spatial detail normally blurred by diffraction.
  • a currently-available commercial 3D-S1M instrument uses a linearly polarized laser beam that is split into three orders by a binary phase grating.
  • the three diffracted orders (0-th and ⁇ V respectively) are focused onto the back focal plane of the microscope objective, and combine to form a three-dimensional interference fringe pattern in the sample volume.
  • 3D-SI.M data are acquired by taking a fluorescence image excited by the fringe pattern, moving the grating a fifth of a period, approximately five micrometers, then taking another image, and repeating these steps for a total of five images.
  • the grating is then rotated by 60 degrees, and the five image process is repeated, followed by another rotation and another five- images, for a total of 15 images per z-step, where a z-step is a fixed point in the. - Docket No. 096PCT axis, coincident with the optica! axis passing through the objective, Typically, at least eight z-steps are desired, for a tola! of 120 images per stack. These images are used to solve a system of linear equations to recover a 3D optically sectioned image with approximately double the resolution obtained by conventional wide-field microscopy. The image acquisition times involved are appreciable.
  • Laser exposure may span 5-100 ins
  • camera readout may span about 50 ins per full-frame
  • grating motion and settlings may span tens of milliseconds
  • rotation of the grating/polarizer assembly may span roughly one full second, leading to a 3D-S1M stack acquisition time of 10-20 seconds.
  • Embodiments of the present invention are directed to providing and controlling illumination for three-dimensional stnictured illumination microscopy.
  • Three phase-coherent beams referred to as a "beam triplet," are produced with planar beamsplitters.
  • the relative phases of the beams are controlled by piezo-coupled mirrors or other means.
  • the beams pass through the microscope objective and interfere to produce, the 3D structured illumination pattern.
  • the .spatial orientation and location of the pattern is manipulated fay adjusting the relative phases of the beams.
  • Figure 1 pro vides an abstract representation of an optical system, such as a standard optica! microscope or fluorescence microscope.
  • Figures 2-4 illustrate a mathematical model for optical imaging.
  • Figures 5-8 illustrate characteristics of the impulse-response function h(x > yjc y') discussed with reference to Figure 4, above.
  • Figures 9-10 illustrate a convolution-based mathematical model, discussed above with reference to Figure 4, for computing an output image of an optical system from the image input to the optical system and the impulse-response function for the optical system.
  • Docket No. G 6PCT
  • Figure 1 1 illustrates the diffraction limit associated with of optical microscopy.
  • FIGS. 12A-8 illustrate a basis for super-resolution microscopy.
  • Figure 13 illustrations relationships between the spatial domain and the spatial-frequency domain.
  • Figure 14 which uses the same illustration conventions as Figure 13, illustrates one approach to increasing the resolution of a fluorescence microscope.
  • Figure 15 illustrates a second problem associated with three- dimensional imaging using conventional microscopy
  • Figure 16 illustrates the volume of spatial-frequency domain, or reciprocal space, accessible to a conventional optical system attempting to image a real-space sample volume.
  • Figures 17A-B illustrate generation of a three-dimensional structured- illumination pattern, or grid, using three coherent . illumination beams.
  • Figure 18 illustrates a conventional 313-SIM apparatus.
  • Figure 19 shows a simulated cross-section of a typical 3D-S.IM intensity pattern.
  • Figures 20A-1 illustrate data collection for 3D-S1M imaging of a sample plane, as discussed above.
  • Figure 21 provides a schematic that illustrates many features of one embodiment Of the present invention.
  • Figures 22-2-9 further illustrate ' one embodiment of the presen invention.
  • Figure 30 shows an updating MatLab display used in tuning a D-SIM ' according to one embodiment of the present invention.
  • Embodiments of the present Invention are directed to devices and apparatuses that provide for fast 3D-S1M imaging, including a structured-illumination module ("SIM") included within, or added to, a 3D-3IM instrument to produce three- dimensional illumination patterns, the position and orientation of which are controlled b eieciro-optkal-mechanical control features of the SIM discussed below. Docket o. 096PCT
  • Figure I provides an abstract representation of an optical system, such as a standard optical microscope or fluorescence microscope.
  • the optical system 102 focuses and magnifies an input image 104 within the object plane 104 to form a magnified image. 108 on an image plane H O.
  • the object plane is, in microscopy, generally a plane normal to the optical axis within a sample and the image plane, also normal to the optical axis, is generally a CCD detector, the retina of a human eye, or an image plane within another type of detector.
  • Figures 2-4 illustrate a mathematical model for optical imaging.
  • Figures 2-4 use the same illustration conventions, or similar illustration conventions, as used in Figure i .
  • the input image at the object plane 202 can be described as a function i(x,y,z ⁇ 0) where x and ' are orthogonal dimensions in the image plane and z corresponds to the optical axis normal to the image plane and optical -system components.
  • the value of the function l ⁇ x,y) represents the intensity of light at each point in the object plane, and can be thought of as a surface 204.
  • the input image, /(.x v.r—O) can be mathematically modeled as the squared magnitude of a wave function:
  • the wave function can be expressed, in vector form, for monochromatic light of frequency v, as: where i/(r) « «(r)* w * - the complex amplitude;
  • the function U ⁇ v is a function that satisfies a differential equation known as the "wave equation:" ' Docket No. 096PCT
  • U(r) is the iime-in variant complex amplitude for the light wave and u ⁇ r,t) is a time- dependent model of the light wa ve.
  • the image J(x,y) is time- invariant, depending only on the complex amplitude u(r) .
  • the wave function is a scalar function of position and time, and is therefore not a complete mathematical for light, but is adequate for explaining many optical phenomena.
  • a lens 302 can be modeled as an optical system that transforms an input function J(x,y) ⁇ U(x,y.O) to an output function g(x,y ⁇ ⁇ V(x.yJ).
  • the input function /(rj-) arid output function g(x,y) are equivalent to the time-independent wave functions for a light wave at the object plane and image plane, respectively.
  • the input function flx,y) can he modeled as a superposition of harmon ic functions of the spatial dimensions x and y:
  • the output image is a frequency-domain image generated by focusing harmonic components of the input image /(A ' . V) to different points in the output image g(x,y).
  • the output image g(x,y) is a Fourier transform of the in put image Jlx,y).
  • Figure 4 illustrates a maihematicai model for an imaging optical system.
  • An input image 402 is transformed, by a first lens 404 to a frequency- domain image 406, as discussed above, and a second lens 408 transforms the Docket No. 096PCT
  • h ⁇ x,y;x',y) is the impulse-response function fo the optical system.
  • Figures 5-8 illustrate characteristics of the impulse-response function discussed with reference to Figure 4, above.
  • the impulse-response function. h(x,y; Q corresponding to the point (0,0) 502 on the •object plane 504 is a two-!obed intensity distribution 506 with circular cross-section 508 in the image, plane, a first lobe 510 of the two-lohed distribution is cylindrical! ⁇ ' symmetric about the ⁇ >axis. and. projects from the image plane back toward the object plane, and the second lobe 5 12, also cylindrical!' symmetric about the optical axis, projects outward, away from the object plane from the- circular cross-section 508 in the image plane.
  • the illustrated surface of the impulse-response- function is a surface of constant intensity.
  • the idealized impulse- response function extends without bound through real space. However, at a particular distance in any direction from the point (0,0) in output-image space, the intensity of the input-response function fails to an arbitrarily low value, so that, for example, a constant-intensity surface can be constructed to describe the impulse-response function for an intensity level below which intensity is undetectable.
  • the impulse- response function can be considered to be a. function that maps a point source in the object plane to an intensity distribution in output-image space, or, alternatively, as the image of the point source.
  • the output image becomes increasingly unfocused with distance, hi the z direction, from the image plane. Blurring of the image of the point source with distance is reflected in the initial increase in the radii of circular cross- sections of the impulse-response function with increasing distance from the output- Docket No. [!%P €T image plane. Subsequent decrease in the radii of circular cross-sections with greater distance from the image plane is related to the decreasing intensity with distance from the origin .(0, 0) in the image plane.
  • Figure 6 illustrates a constant-intensity surface of the impu!se- response function in three-dimensional output-image space.
  • fluorescence microscopists commoniy image a series of object planes 602-607 within a sample by changing the distance between the sample and the objective lens after acquiring each image of the series of images- at a fixed position with respect t the objective. This produces a corresponding set of output images 61 0-61 6.
  • the three-dimensional impulse-response function corresponding to the point (0,0,0) is a cyiindrieail symmetrical ellipsoid 620 in three-dimensional output-image space.
  • the impulse-response function h ⁇ x,y; 0,0 ⁇ is spherically symmetric about the point (0,0,0) in output-Image space.
  • the impulse-response function extends outward from the point (0,0,0) in output-image space through all of real space.
  • the intensity decreases with increasing- distance from the point (0,0,0), so that an ellipsoid or sphere of constant intensity can be constructed to represent the impulse-response function,
  • Figure 7 illustrates the impulse-response function in one dimension within the output-image plane.
  • the horizontal axis 702 is a line in. the output-image plane passing through the origin (0,0),
  • the theoretical impulse-response function has a tail, relatively narrow central peak 704 with: secondary peaks of decreasing height 705-714 extending in both directions away from the central peak.
  • the height of the impulse-response curve corresponds to intensit and the horizontal axis 702 corresponds to linear distance from the origin in the output-image plane.
  • the theoretical impulse-response function is proportional to the square of the J j Bessel function.
  • Figure 8 provides a representation of the impulse-response function in three-dimensional space, where the two horizontal axes 802 and 804 lie in the plane of the output-image plane and cross at the origin (0,0) and the height, at an point on the surface of the impulse-response function corresponds to the intensity observed at a corresponding position on the image plane.
  • An image of the impulse-response Docket No. 096PCT An image of the impulse-response Docket No. 096PCT
  • Figures 9-10 Illustrate a convolution-based mathematical model, discussed above with reference to Figure 4, for computing an output image of an opiicai system from the image input to the optical system and the impulse-response function for the optical system.
  • the impulse-response function illustrated in Figures 5-8 is a ' theoretical impulse-response function of an aberration-free optical system.
  • the impulse- response function is experimentally determined by imaging tiny Sight sources in the object plane.
  • the impulse-response function may vary with respect to location of point sources in the input-image plane.
  • the impulse-response function is generally assumed to be position independent.
  • Figure illustrates the basic operation thai is repeated over the entire area of the input image plane in order to compute the output ' image by the convolution method discussed above with reference to Figure 4.
  • the output image i(x,y) is computed as convolution of the impulse-response function, or point-spread function ("PSF"), s(x-x', y-y% with the input image o(x',y
  • PSF point-spread function
  • This convolution operation becomes a ultiplication operation in the frequency domain: where I(u,v) is the frequency-domain transform of the output image i(x,y); x( , v) is the optical transfer function thai is the Fourier transform of the PSF; and 0( ,v) is the • frequency-domain transform of the input image o(x,y).
  • I(u,v) is the frequency-domain transform of the output image i(x,y)
  • x( , v) is the optical transfer function
  • thai is the Fourier transform of the PSF
  • 0( ,v) is the • frequency-domain transform of the input image o(x,y).
  • the estimated input image can then be altered, by any of various techniques, such as a Newton-descent optimization technique, to produce a subsequent, better estimate of the input image, from which a corresponding output image can be computed and a new R factor generated.
  • the process is iterated until the R factor fells below a threshold vaiue, indicating that the estimaied input image sufTtcieniSy closely represents the actual image input to the optical system.
  • Figure 1 1 illustrates the diffraction limit associated with of optical microscopy.
  • the images of these two points output from an optical system are two point-spread functions 1 110 and 1 1 1 2 centered at. the output- image points (Xi. i) and corresponding to points and (.vj,. ⁇ ) -
  • the spreading of Sight from point sources of the input image into poim-spread ut eiion images at the output image is a diffraction-related phenomenon.
  • di * is sufficiently large that the corresponding distance between the centers of the point- spread functions dj in the output image separates the point-spread-function distributions so that the sum of the two point-spread functions, represented in Figure !
  • NA is. the numerical aperture for the optical system
  • the minimum spacing, in the input image corresponds to spacing between output point-spread functions at which the first left-hand zero point of the right-hand point- spread function coincides with the first right-hand zero point of the left-hand point- spread function.
  • the minimum separation of features that can be imaged corresponds to approximately 200 nm for optical microscopy systems.
  • the minimum spacing, or maximum resolution, is referred to as "'the di fraction limit,' 1 since the point-spread- function images of point sources in the output image arise as a result of diffraction.
  • Figures 12A-B illustrate a basis for super- resolution microscopy.
  • Figure 12A illustrates the effect of an optical system 1202 on Docket No, 0 6PCT
  • the optica! system spreads the intensity of the light over a disk- shaped point-spread function 1206- in the image plane.
  • the point-spread function is thus viewed as a real-time smearing, or diffusion, of a point source by the optical system in output-image space. This smearing of point-like source light occurs for a number of different reasons.
  • optical components such as lenses, have finite apertures, and thus receive only a lower-angle portion of the non-coherent light emitted from a point source.
  • optica! components act. as a spatial-frequency filter. Focusing of light by a second Sens, modeled as an inverse Fourier transform, produces a spatial-domain ' image that is somewhat blurred, due to removal -of high- frequency frequency-domain signals by optical components. Many additional factors contribute to dispersion of light in an output image represented by the point-spread function, including- various types of aberrations inherent, in optical components and other factors.
  • a second wa to consider the point-spread function is thai the point-spread function represents a probability distribution, with the intensities associated with points by the point-spread function corresponding to probabilities that individual photons will be deflected to that, point by an optica! system.
  • the point of highest accumulated intensity, in the output image can be located to a precision equal to that of the resolution of the detector after accounting for the magnification factor of the optical system, when output light is collected for a sufficient period of time to generate a well-formed distribution of accumulated intensity in the image plane.
  • This point corresponds to the object-plane location of a point, source corresponding to the PSF in the image plane. It is theoretically possible to determine the location of point sources, using the ceniroids of corresponding PSF distributions in art output image, to Docket No. 096PCT
  • the light-emitting point sources must be separated by sufficient distance that, their point-spread-function images, in the output image, do not appreciably overlap.
  • the fiuorophores must be positioned within the sample so that the distance between any two fiuorophores is greater than the diffraction-limit distance of between 180 and 200 nm, according to currently- practiced super-resolution imaging techniques, Em bod i roent of the Present, invent ion
  • Figure 13 illustrations relationships between the spatial domain and the spatial-frequency domain.
  • an object or sample being imaged by a .fluorescence microscope is considered to be in a real, spatial domain 1302.
  • the emitted fluorescent ' light passes through an optical lens 1304 to produce a Fourier transform of the image in & spatial-frequency domain 1306. Because of the limitations of the optical, system and the diffraction limit associated with .
  • the signal produced by the lens in the spatial-frequency domain 1306 is confined to a disk-like region 1308 with a radiu r 13 10, where r is inversely related to ci the ' smallest distance between two features that can be resolved by the optical system, in other words, the -distances of positions of spatial-frequeney- domain signals from the intersection of the optica! axis with the spatial-frequency- domain plane 1312 is inversely related to, or reciprocal, to spacing* between features in the spatial-domain fluorescence-emission pattern.
  • Figure 14 which uses the same illustration conventions as Figure 13, illustrates one approach to increasing the resolution of a fluorescence microscope.
  • patterns of light and dark lines of different orientations may include a set of lines 1404- 1408, shown as dashed lines in Figure 14, that are too closely spaced to the neighboring solid lines, such as solid line 1410. to be imaged by the optical system shown in Figure 13.
  • the information contained in that larger region could be transformed, by an inverse Fourier transform, to produce an image of the sample in which the illumination-pattern features 1404-1408 are resolved.
  • Figure 15 illustrates a second problem associated with three- dimensional imaging using conventional microscopy
  • three-dimensional Imaging multiple, closely spaced, planar images are recorded for each of multiple closely spaced planes 1502- 1509 within a sample volume normal to the z axis, or optical axis, in certain cases, the three-dimensional information may be processed in order to produce s single, higher-resolution two-dimensional image 1510 representative of the central plane of the stack of closely spaced images.
  • unfocused fluorescent emission from nearby image planes is recorded along with the Focused illumination from a particula image plane. For example, as.
  • the detector will generally receive, unfocused fluorescent emission from -nearby sample planes, represented in Figure 15 by unshaded disks 1514-1517 above the image sample plane and unshaded disks 1515- 1520 below the sample plane.
  • This unfocused illumination falls onto the detector along with the focused illumination from the considered feature 1 512, leading to an inability to record image information within a region of the sample image surrounding the intersection of the optical axis with the sample plane, and leads to blurring of a surrounding region .
  • Figure 16 illustrates the volume of spatial-frequency domain, or reciprocal space, accessible to a conventional opticai system attempting to image a real-space sample volume-.
  • a real-space sample volume 1602 generally includes emission patterns from fluorophores distributed at microscale and nanoscale dimensions within the real-space sample.
  • emission patterns from fluorophores distributed at microscale and nanoscale dimensions within the real-space sample.
  • region 1604 of the spatial-frequency domain is accessible to the optical system.
  • the lack of accessible spatiakfrequeney-doniain information and at greater distances from the origin than the external surface of the torus limits the resolution of features in the sample that can be imaged, and the absence of spatiai-frequency-doniain signal in the invagination 1606 within ihe toroidal shape coaxial with the optical axis, referred to as the "missing cone," prevents imaging of portions of the sample domain near to the optical axis.
  • FIGS I 7A-B illustrate generation of a three-dimensional structured-illumination pattern, or grid, using three coherent illumination beams.
  • three coherent beams .1702- 1704 are introduced onto the back focal plane of a high magnification, high numerical aperture objective lens 1705.
  • the three coherent beams 1702-1704 are plane, waves in which the phases of all component waves of each beam are identical across any plane, such as plane 1706, normal to the beam direction.
  • each illumination beam may have a different phase displacement than the other two illumination beams. Focusing of the incident beams by the objective lens 1 02 to a focal point 1710 changes the direction of the two non- axial illumination beams 1 02 and 1704, as shown in Figure I 7A, as a result of which the wave vectors k of the three plane waves are no longer parallel. As a result the three sets of plane waves, intersect to form a grid-like pattern of bright spots, due to constructive interference, and dark surrounding regions, due to destructive interference.
  • a complex stationary three- dimensional wave 1720 is obtained, which features a three-dimensional lattice of bright cylindrically elliptical regions at lattice points separated by dark regions.
  • the positions of the grid points and orientation of the lattice axes can he selected by varying the phase relationships of the incident, illumination beams .1702-1704, As explained below, data is recorded for a large number of different positions and orientations of the structured-illumination pattern in order to reconstruct a three-dimensional image for a sample volume. Docket No. 0 6PCT
  • Figure 18 illustrates a -conventional 3D-SI apparatus.
  • Laser light from a multi-mode fiber 1802 is eoSlsroated onto a linear phase grading 1804.
  • the linear phase grading generates three different illumination beams 1 06- 1808 as the - I , 0, and +1 order diffracted beams from the linear phase grading.
  • These three illumination beams are refocused onto the back focai plane, of the objective lens 181 0. They interfere with one another to generate a simetured- ⁇ lamination pattern within a volume of the sample at the focal plane of the objective lens 1812.
  • Fluorescent emission from illuminated fiuorophores passes back through the objective lens .and is reflected into a CCD camera 1 814 via a dichroic mirror 1 16 for data collection,
  • tildes (- ⁇ indicate the Fourier transform of the corresponding real-space quantities
  • 0(k) - H(k) is the optical transfer function
  • k is a position vector in the spatial-frequency domain.
  • the desired information is the density distribu tion of fiuorophores in the sample, S(r).
  • the distribtrtion of fluorescent emission is givesi by:
  • the convolution operation is nonlocal, in particular, the convolution can make the observed, data within the observable region of A ' (k) depend on normally unobservable components of S ⁇ k) from other parts of reciprocal space. That. information is then observable, in principle, but must be computationally extracted from image data.
  • the struciured l!umination pattern is the sum of a finite number of components, , each of which is separated into an. axial and Iateral functiono :
  • each .lateral-component function J m is a simple harmonic wave, and when the axial functions / m are harmonic or the structured-i llumination pattern is fixed, in the axial direction, with relation to the focal plane of the microscope during multiple two-dimensional-image data collection., extraction of the normally unobservable components of S( ) is facilitated.
  • the observed data ca then be expressed as:
  • each lateral frequency component in of the illumination structure corresponds to a separate optical transfer function (3 ⁇ 4,. which is given by a convolution of the conventional detection OTP with the axial illumination structure of the m'" pattern component, and applies to a component of object information that has been translated in reciprocal space by the lateral wave vector p m of that pattern component.
  • a single raw data image is a sum of several different information components, one for each index m.
  • these information components are separated. This can be done by acquiring additional data, sets with different known values of the phases 3 ⁇ 4 , Changing the phase values by phase shifts ⁇ ⁇ alters the coefficients ? "" ''"" in Eq. 8 from e ⁇ ** to er- leading to a linearly independent combination of the unknown information components.
  • Each phase- shifted image thus supplies one independent linear equation in N unknowns (where N is the number of frequency components in Eq. 8). If data are acquired with at least N different phases, the number of equations is at least equal to the number of unknowns, allow ing the N information components to. be separated by solving the system of equations via matrix inversion.
  • the separated information components can be computationally moved back, by a distance m , to their true positions in reciprocal space, reeombined into a single extended-resolution data set, and retransformed into real space.
  • the total effective, observable region with this method is given by the support of the convolution of the conventional QTF O with the total illumination structure ⁇ .
  • the maximal resolution increase in a given dimension is therefore equal to the maximum illumination spatial frequency in thai dimension.
  • the maximum possible spatial frequency in the illumination equals the conventional resolution limit of the detection, sealed by the ratio of emission and excitation wavelengths.
  • Embodiments of the present invention efficiently use laser light to greatly increase the time efficiency for 3D-SIM data collection.
  • Current 3D-SIM microscopes require relatively powerful lasers, greater than 1.00 mW, which are expensive, but which still entail long exposures to obtain adequate image signal to 5 noise ratios, indeed, the fraction of laser light reaching the back of the microscope objective in existing designs is only a few per cent.
  • Embodiments of the present invention increase efficiency of laser-light use by more than an order of magnitude higher and deliver high quality beam waveftonts which produce high contrast interference fringes. This is accomplished by replacing the inefficient shifting and
  • the mirrors attached to precision piezo actuators, move rapidly with, fractional- wavelength displacements, settling in from a. few milliseconds to less than a millisecond.
  • the beam polarization geometry employed in embodiments of the present invention, allows the use of dielectric, mirrors, which generally exhibit more
  • Figure 19 shows a simulated cross-section of a typical 3D-SIM intensity pattern.
  • the pattern features elliptical cylinders of high intensity light within a matrix of lower-intensity light.
  • the elliptical cylinders are shown in cross-section, with each light-shaded ellipse, such as ellipse J 02, D representing a cross section of an elliptical cylinder.
  • the intensity pattern generated by interference of the three beams, is projected onto a sample plane, represented in Figure i 9 by horizontal line 1 04, the interference pattern is seen, on Docket No, Q96PCT the sample plane, as a set of parallel high-intensity lines spaced apart ai a fixed distance.
  • the shape and size of the structured-illumination pattern is governed by the laser wavelength arid the angle and amplitudes of the respective waveironis,
  • the location of the structured-il lumination pattern with respect to the sample is, in embodiment ' s of the present invention, determined by the phase relationships between the three illumination beams.
  • the pattern can be displaced, axiaSly up and down by advancing and retarding the relative phase of central beam.
  • a phase change of ⁇ radians moves the pattern vertical iy by half a period.
  • a 2 ⁇ change reproduces the original pattern.
  • advancing and retarding the phase of the peripheral beams by equal and opposite amounts displaces the pattern laterally.
  • a single 3D-SI pattern lateral displacement step is performed by displacing two mirrors by ⁇ .1 / 10 , approximately ⁇ 50 am, respectively.
  • each of the three beams- can be represented as plane wave with a complex amplitude E m ,m : ⁇ 1,2,3.
  • a m is the amplitude of beam m
  • k m and x are the vector wave number and position in the sample plane, ' respectively:, and ⁇ 3 ⁇ 4
  • a beam propagating in the direction of k m accumulates 2n radians- of phase for each laser wavelength of displacement along that direction. is the (interfering) sum of the three field amplitudes at point in the sample.
  • the optical intensity. / is proportional to the squared magnitude of the field:
  • the laser beams are linearly polarized. Maximum fringe contrast, or fringe visibility, is obtained when the three beams are in a relative s-po!arization state.
  • S-polarixation occurs when the electric field in each beam is perpendicular to the plane in which the three beams enter the back of the objective. This condition is created in the conventional-grating scheme by mixing the polarization state of the original laser beam, and then by passing it through a linear polarizer that co-rotates Docket No. 0 6PCT with the grating thereby losing half the light.
  • Other SIM instruments .employ active phase rotators, such as liquid crystal devices, but beam efficiency and wavefroni quality and cost issues arise.
  • Embodiments of the present invention avoid the use of polarizers, because the linear polarization of the input illumination beam is maintained as the beam is split and directed through the SIM optical components, and has proper S-polarization at each optical interface at which non-S-polarized light would otherwise be absorbed.
  • the optical paths of the three beams are controlled by the designer.
  • the optical path lengths of the three, beams are made equal to within the coherence length of the laser for each of the three angular orientations.
  • Many solid state. lasers suitable for fluorescence microscop have coherence lengths of a t3 ⁇ 4w millimeters.
  • Three- beam layouts on optical tables comprising beamsplitters and multiple mirrors with total path lengths exceeding one meter achieve excellent and stable fringe contrast with such lasers.
  • a 3 D.-SIM ' microscope embodying the present invention is designed or adjusted to support beam path differences of a . millimeter or less. Lasers with long coherence lengths are avoided to prevent parasitic interference between unavoidable reflections from beam path components.
  • phase shift mirrors used in the described SIM move a few tens of nanometers, clearly thermal expansion, vibration, and other factors must be faced and dealt with,
  • the optics must be enclosed in a compact, thermally-stable and air-current free environment supported by a vibration-isolating floating table or the equivalent. Components that span the optical paths are machined from Invar. Measurements with an enclosed microscope prototype via interference fringe tracking have shown that thermally induced phase drift and residua! air currents are negligible on a time scale often seconds ( ⁇ 2 degrees rms). Ordinary levels of sound and vibration in the room containing out test enclosure cause no disturbances, however, jumping on the floor causes a noticeable effect. Instrument vibration, which will tend to smear the contrast Docket No. 096PCT
  • a method is incorporated into the disclosed SIM to measure the phase relationship of the three beams by picking off beams samples between the beam align/focus ("BAF') lens and objective, and coHimatmg and crossing the beams via a lens onto the face of a CCD or other pixilated optical detector, ideally, three htferferograms comprising different overlapping beam combinations are captured simultaneously, e.g., Center-Left, Center-Right, and all three.
  • BAF' beam align/focus
  • Beam pairs are selected by blocking the unneeded beam wiih a mask in a focal plane. These are acquired for each beam triplet in all three arms of the instrument.
  • the phase computed from the data would be used to optically close the feedback loop via corrections issued to the phase mirrors.
  • Well-executed, with temperature-stable CCDs, such, a -scheme obviates the need for in situ position feedback mechanisms or the phase mirrors, such as expensive capacitance sensors.
  • Explicit control over the relative phases of the three beams provides another significant advantage over grating-based and some other 3D SIM architectures.
  • Lateral displacement of the grating advances and retards the phases of the outer beams respectively, but does not affect the zero-order (center) beam passing through the grating.
  • the depth (in z) of the 3D interference pattern at the sample is fixed for a grating which has a iixed-z location, in the optical train. The depth of the 3D pattern must be tuned.
  • the relation of the pattern to the sample plane is studied by acquiring 3D SIM image stacks of a single sub-di fraction (e.g., ⁇ 00 nm) fluorescent bead and measuring the modulation depth at the main fringe spatial frequenc but also at the higher spatial frequency, which derives from the angles of the two outer beams.
  • a single sub-di fraction e.g., ⁇ 00 nm
  • the modulation depth at the main fringe spatial frequenc but also at the higher spatial frequency, which derives from the angles of the two outer beams.
  • Figures 2.0 A -I illustrate data collection, for 3D-S.IM imaging of a sample plane, as discussed above.
  • Figure.20 A shows the geometry of the imaging technique.
  • an interference pattern 2002 is focused at the focal plane behind the objective lens 2004 causing- the Interference pattern to be projected through the objective and focused on the plane of the sample 2006 to be imaged.
  • the mathematical technique for 3D.-S1M data processing reconstructs the higher-resolution image for the sample plane from data collected from a .sample volume, shown by the dashed lines 2008 in Figure 20A, that includes the sample plane and volumes both above and below the sample plane.
  • the interference pattern is focused onto the top of the sample volume (2008 in Figure 20A), A first image is recorded with the interference pattern positioned as shown in Figure 20B. Then, as shown in Figure 20C, the interference pattern is shifted, in a direction orthogonal to the lines of the interference pattern 2010, and a second image collected, In one embodiment of the present, invention, five lateral shifts, such as that shown in Figure 20C, are carried out to produce five different, laterally shifted interference patterns from which five images are collected, in other words, the spacing between interference-pattern lines is divided by six to generate a finer spacing distance, with five shifts needed to sample the finer-spacing distance. Then . , as shown in Figure 20D, the interference pattern is rotated 2-012 by 60 degrees 9
  • the interference pattern is ( hen laterally shifted 2014 in a direction perpendicular to the lines of the interference pattern and another image is recorded.
  • the interference pattern is shifted again by 60 degrees, and a next image is recorded.
  • the interference pattern is laterally shifted 2018 from the position shown in Figure 20F and another image: recorded.
  • five lateral shifts are carried out in the third orientation shown in Figures 20F and G,
  • the volume. enclosing the sample plane generaies 120 different recorded images. 15 images per plane in the s direction generated from three different orientations of the interference pattern in each plane.
  • FIG. 2 provides a schematic that illustrates many features of one embodiment of the present invention.
  • the laser employed in one embodiment of the present invention is a Sapphire-488 (20 mW) made by Coherent.
  • the light is
  • the "Kmeflex" system manufactured, by Qioptkj (formerly Point Source) is used in this embodiment of the present invention, which additionally expands the beam using a dual-mirror 4X reflective beam expander (Thor!abs).
  • a fast tilt mirror 2104 directs the beam to one of three optical trains
  • This mirror need not be as fast as the phase shifters.
  • Fused silica dielectric mirrors employed were ⁇ /10 flatness, 5-10 scratch-dig (Thorlabs).
  • the beam is directed, by directional mirrors such as directional Docket No, 096PCT mirror 21 10, into a set of beam splitters 21 1 6-2121.
  • the fast tilt mirror essentially selects one of three structured-illumination -pattern orientations, each orientation representing a rotation of 120* with respect to each of the other two orientations.
  • the three beams 120-2122 are directed via mirrors to a common beam aligner module 2130.
  • the relative intensities of the three beams may be optimized for maximum 3D-SIM reconstruction efficacy by careful design of the beam splitter ratios at the laser wavelengths of interest.
  • the beam aligner module comprises severs small mirrors., six 2140-2145 in a circle, and one 2146 at the center. The six outer mirrors are adjusted and then fixed. A given triplet lies in a plane, and reflects from the center mirror and two outer mirrors. The triplets can be generated in three different orientations, depicted schematically as Angle 1, Angle 2 » and Angle 3, 2150-2152, respectively.
  • the outer two mirrors are aimed so that the three beams comprising a triplet overlap at the back aperture of the BAF lens 2154.
  • the outer mirrors are separated to each subtend an angle, ⁇ , of approximately 20 mR,
  • the center mirror aims to direct the center beam straight down the optical axis. Because the center beam from each triplet must arrive at the center mirror from a different angle, the center mirror needs to be a fast tilt mirror, adjusted onee for each triplet.
  • a single tilt mirror may be used to accomplish the initial aiming and triplet-aiming tasks simultaneously,
  • the phase-shifting module 21.60 comprises three small mirrors affixed to three independent piexo displacement modules. This module introduces relative phase shifts among the beam triplet to change the position of the structured- iilummation pattern, The pattern can be shifted laterally, in the x and y directions, and vertica lly, in the z direction, where the directions x, y, and z are instrument-frame directions.
  • a commercially available closed-loop pieato actuator has a stroke of 2 pm, a resolution of 0.03 nm, and a natural resonance frequency of 23 kHz.
  • the mirrors are shaped such that one mirror controls the central beam phase, and the other mirrors control the phases of the outer beams for each triplet, as depicted schematically.
  • the three mirrors need not lie in a plane, as long as total beam path lengths are respected.
  • the angle of incidence on the mirrors is not normal (perpendicular), to avoid reflecting back to the beam aligner module, it is kept near-perpendicular, however, to avoid coupled beam translations, and to permit the use of efficient dielectric mirrors, Docket No. 096PCT which tend to induce undesirable eliiptical poiarization in beams that are a mixture of s and /? polarization. If preferred, piezo control of only the two outer mirrors can be sufficient to control the relative phases of the three beams.
  • the three beams come to a focus at distance with the outer focal spots displaced by a distance d -/ ⁇ .
  • the separation of the outer mirrors is engineered such that the displacement d is optimized for the selected microscope objective.
  • the choice of the BAF lens focal length is determined by the beam diameter., the required illumination spot size at the sample, and mechanical design considerations for the. mirror mounts.
  • the three beams enter the objective 2170 and create the 3. -S1 pattern. Unlike the grating- based schemes, the system is highly achromatic, with the outer beam positions naturally optimized for each wavelength.
  • Figures 22-29 further illustrate one embodiment of the present invention. These figures provide mechanical illustrations of the SIM that represents one embodiment of the present invention.
  • Figure 22 illustrates the SIM within the contest of the optical bench of a 3D-5SM fluorescence microscope.
  • the optical bench 2202 supports a central optics stage 2204 that includes the wide-angle objective 2206,
  • the SIM that represents one embodiment of the present invention 2208 is mounted below the optical bench to direct the three incident beams thai produce the structured-illumination pattern upward vertically-through ' aperture 2210 to the back of the focal plane of the objective lens.
  • FIG 23 illustrates the SIM, The polarized laser light is received via a phase-maintaining optical cable 2302 that is input to the beam expander 2304.
  • a tilt .mirror (2104 in Figure 21) is located underneath a vertical plate 2306 at the top of the SIM The tilt mirror serves as the central mirror (2146 in Figure 21 ) of the beam aligner module, also located below plate 2306.
  • Figure 24 illustrates the expanded, polarized light 2402 (2102 in Figure 21 ), impinging on the tilt mirror 2404 (2104 in Figure 21 ) and being reflected towards one of three different directional mirror 2406 (2106 in Figure 2-1 ).
  • the tilt mirror 2404 essentially selects one of three directional mirrors ⁇ 23. 0-23 12 in Figure 23) to which to direct the incident polarized, expanded input beam .
  • Each of the three directional mirrors represents the starting point for a Docket No. 0 PCT
  • the central horizontal plate (2380 in Figure 23) includes three arms 2382-2384 with axes that are orientated with one another at angles of 120 degrees.
  • the directional mirrors (2310-231 in Figure 23) each directs light to a different one of these three arms, to each of which a series of beamsplitters (2 i 16- 21 18 in ' Figure 21) are attached.
  • Figure 25 shows splitting of a polarized, expanded beam by three beam splitters associated with one arm of the central horizontal: plate- of ' the stem.
  • the three beam splitters 2516-2518 split the incident beam 25 ( 2 to form a triplet of three coherent beams 2513-251 5 that are directed to three corresponding directional mirrors 2532-2534 that direct the three coherent beams upward to the beam aligner module bounded below the top horizontal plate (2306 in Figure 23) of the SIM.
  • Three different directional-mirror assemblies, such as directional-mirror assembly 2536 in Figure 25, can be seen at. the base of the SIM in Figure 23, 2336-2338.
  • Figure 26 shows the beam triplet reflected from the directional mirrors 2532-2534 in Figure 25 .
  • phase-shifting module can be seen mounted at the base of the SIM 2339 in Figure 23.
  • Figure 27 illustrates the phase- shifting module in greater detail.
  • a triplet impinges on a central mirror 2788 and onto side mirrors 2789 and 2790.
  • the phase-shifting mirrors introduce relative phase shifts between the incident beams of the triplet beam in order to change the position of the ' structured-illumination pattern with respect to an instrument reference frame.
  • the phase-shifted beam triplet is reflected from the phase-shift module 2339 through an aperture in the top horizontal plate 2306 of the SIM and, as shown in Figure 29, directed, at proper orientation and separation, to the back focal plane of the objective 2206.
  • Figure 30 shows an updating MatLab display used in tuning a 3D-SI according to one embodiment of the present invention.
  • the pitch and orientation of the 3 D interference pattern at the sample is tuned by altering the angle of the outer Docket No. 0%! J CT
  • the middle panel 3004 displays the phase offsets for the three rows and the bottom displays the inferred fringe angle with respect to the camera horizontal and the fringe pitch.
  • the left beam is blocked and the right beam is set to a satisfactory angle with respect to the cetrier beam; .the angle and pitch of the interference pattern are recorded, The beam blocker is swapped, and then the left beam is carefully tuned, adjustin multiple mirrors, to match the recorded values;
  • the .mirrors can be aimed in pitch and yaw by turning 100 TP! screws in custom mirror fixtures which are either flexure-mounted or kmematically-ntounted. with springs. The process is repeated for the other arms. Rather good pitch agreement between the three arms can be obtained.
  • the result is a tilt of the interference plane with respect to the sam le plane.
  • Mirror-tuning precision is such that this can be rendered negligible. I.f the angles are not commensurate, this can complicate the 3D interference pattern.
  • the beam is aimed such that angle-mismatch subtends a small fraction of a fringe pitch across ' the whole field of view.
  • the present invention has been described in terms of particular embodiments, it is not intended that the invention be limited to these embodiments. Modifications will be apparent to those skilled in the art.
  • a variety of alternative embodiments of the SIM can be designed and constructed to provide the functionality of the S M illustrated in paragraphs 21 -29.
  • the tilt mirror, (2104 hi Figure 21) is also used as the central mirror of the beam aligner module (2150 in Figure 21),
  • a separate tilt mirror may Docket No. 0 PCT
  • FIG. 31 be employed for tilt mirror 2104.
  • Many different electro-optical-mecliantcal components from many differe t vendors can be used for the various components shown in Figures 21 -29.
  • the dimensions, geometry, and configuration of the SIM may be modified to accommodate alternative types of optical components and alternative light-paths and light-path geometries.
  • a variety of additional calibration and control components and functionality may be employed , in a 3D-SI fluorescence microscope, in order to calibrate and control operation of the SIM, including software components that execute on a computer system coupled to the 3D-SIM fluorescence microscope.
  • the calibration and control systems and functionality- can be implemented by using many different physical and computer- software components .

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Abstract

La présente invention, selon des modes de réalisation, porte sur la réalisation et la commande d'un éclairage pour une microscopie à éclairage structuré en trois dimensions. Trois faisceaux à phase cohérente, désignés sous le nom de « triplet de faisceau », sont produits avec des diviseurs de faisceau plans. Les phases relatives des faisceaux sont commandées par des miroirs piézo-couplés ou par d'autres moyens. Les faisceaux traversent l'objectif de microscope et interfèrent de façon à produire le motif d'éclairage structuré en trois dimensions. L'emplacement et l'orientation spatiale du motif sont manipulés par réglage des phases relatives des faisceaux.
PCT/US2010/059779 2009-12-09 2010-12-09 Procédé et système pour réalisation d'image de microscopie à éclairage structuré en trois dimensions rapide WO2011072175A2 (fr)

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DE102012103459B4 (de) * 2011-10-19 2016-03-31 National Synchrotron Radiation Research Center Optisches abbildungs-oder bildgebungssystem mit strukturierter beleuchtung
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WO2014005193A1 (fr) * 2012-07-05 2014-01-09 Martin Russell Harris Appareil et procédé d'endoscopie ou de microscopie
WO2018202466A1 (fr) * 2017-05-05 2018-11-08 Carl Zeiss Microscopy Gmbh Microscope optique et procédé de fourniture de lumière d'éclairage structurée
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