WO2010101894A2 - Système et procédé d'imagerie microscopique à balayage laser à haute définition utilisant un éclairage cumulatif à motif spatial de champs de détection - Google Patents
Système et procédé d'imagerie microscopique à balayage laser à haute définition utilisant un éclairage cumulatif à motif spatial de champs de détection Download PDFInfo
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- WO2010101894A2 WO2010101894A2 PCT/US2010/025888 US2010025888W WO2010101894A2 WO 2010101894 A2 WO2010101894 A2 WO 2010101894A2 US 2010025888 W US2010025888 W US 2010025888W WO 2010101894 A2 WO2010101894 A2 WO 2010101894A2
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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
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- 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
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- 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
- G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
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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
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
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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
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
- G01N2021/655—Stimulated Raman
Definitions
- the present invention relates generally to microscopy, and relates in particular to high resolution laser scanning microscopy at resolutions that exceed the diffraction limit of the microscopy system.
- Super-resolution optical microscopy has attracted great interest among researchers in many fields, especially in biology where the scale of physical structures and molecular processes fall below the diffraction limit of resolution of light.
- the resolution of many conventional microscopy systems, such as fluorescence microscopy is limited by the diffraction limit of visible light.
- One example of a microscopy system to overcome this limitation, and provides microscopy beyond the diffraction limit of visible light is called stimulated emission depletion (STED) microscopy.
- STED stimulated emission depletion
- STED microscopy involves first providing a fluorescence-tuned excitation pulse, followed by a shaped (e.g., annular-shaped) depletion pulse that narrows the effective spot of a fluorescence spot to one having a point spread function that is far below the diffraction limit.
- a fluorescence-tuned excitation pulse followed by a shaped (e.g., annular-shaped) depletion pulse that narrows the effective spot of a fluorescence spot to one having a point spread function that is far below the diffraction limit.
- PAM photoactivated localization microscopy
- the images from the localized acquisitions are then tabulated and plotted to construct an image having a resolution that exceeds the diffraction limit. See Imaging intracellular fluorescent proteins at nanometer resolution, by E. Betzig, G.H.Patterson, R. Sougrat, O.W.Lindwasser, S. Olenych, J.S.Bonifacino, M.W.Davidson, J.Lippincott-Schwartz, and H.F.Hess, Science, Vol. 313(5793), pp. 1642-1645.
- STORM stochastic optical reconstruction microscopy
- Photo-switchable fluorescent probes to temporally separate the otherwise spatially overlapping image of individual molecules. See Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM), by MJ. Rust, M.Bates and X.Zhaung, Nature Methods, published on-line (March 9, 2006).
- STORM stochastic optical reconstruction microscopy
- a fraction of the fluorescent probes are turned on.
- the fluorophore positions obtained from a series of imaging cycles are then used to construct a high resolution image.
- the STORM technique has been disclosed to provide multicolor optical imaging at resolutions of 20 nm to 30 nm.
- FPALM fluorescence photoactivation localization microscopy
- a further non-diffraction limited microscopy system is structured illumination microscopy (SIM), which involves obtaining a set of wide-field images of a sample while the sample is illuminated with structured illumination from each of a variety of orientations with respect to the sample.
- the structured illumination may provide a beat between the structured illumination pattern and the frequency component, producing a resulting low frequency Moire fringe pattern.
- Mathematical analyses are then performed on the set of resulting low frequency Moire patterns to obtain high resolution images, In the SIM technique, however, the illumination light is not focused to a spot, but rather is distributed as a wide-field illumination.
- SSIM saturated structured illumination
- each of the above microscopy techniques may be used with one-photon fluorescence imaging, but would likely be almost impossible to employ in non- fluorescence microscopy.
- the STED technique utilizes stimulated emission, which is applicable only for fluorescent contrast.
- Both the FPALM and STORM techniques rely on single molecule detection, which is currently only possible with fluorescence detection.
- the wide field SIM (or SSIM) technique generally only applies to one-photon excited fluorescent samples since the wide field image of the sample needs to be sufficiently bright in response to the wide field structured excitation pattern.
- the invention provides a method for performing high-resolution microscopy.
- the method includes the steps of providing an illumination beam, selectively directing and focusing the illumination beam substantially toward a first of a plurality of spots in a sample volume, receiving a first radiation signal from the sample volume at a wide field array detector, scanning the illumination beam such that the illumination beam moves from being substantially directed toward the first of the plurality of sports in the sample volume to be substantially directed toward a second of the plurality of spots in the sample volume, directing the illumination beam substantially toward the second of the plurality of spots in the sample volume, and receiving a second radiation signal from the sample volume at the wide field array detector.
- the invention provides a method of performing high-resolution microscopy that includes the steps of providing an illumination beam, selectively directing and focusing the illumination beam toward the sample volume during a first scan period of time such that a first orientation of a spatially patterned cumulative illumination field is provided to the sample volume over the first scan period of time, receiving first radiation signals from the sample volume cumulatively at a wide field array detector during the first scan period of time, selectively scanning the illumination beam toward the sample volume during a second scan period of time such that a second orientation of the spatially patterned cumulative illumination field is provided to the sample volume over the second scan period of time, receiving second radiation signals from the sample volume cumulatively at the wide field array detector during the second scan period of time, and determining an image corresponding to Moire fringe patterns associated with the first and second radiation signals.
- the invention provides a microscopy system that includes an illumination source for providing an illumination beam, optics for directing and focusing the illumination beam toward a sample volume, a controller and a wide fieid array detector.
- the optics include a scanning system that selectively directs the illumination beam toward any of a plurality of spots in the sample volume.
- the controller is coupled to the scanning system and provides the illumination beam to the sample volume such that a cumulative illumination field at the sample volume over a scan period of time is spatially patterned.
- the wide field array detector is for imaging a radiation signal radiated from the sample volume, and the processing of the radiation signal results in an image being formed that is beyond the diffract limit.
- the invention provides another method of performing high-resolution microscopy comprising the steps of providing an illumination beam, selectively directing and focusing the illumination beam toward a sample volume during a first scan period of time, receiving first radiation signals from the sample volume cumulatively at a single element detector during the first scan period of time, wherein said first radiation signals are directed toward the single element detector through a mask that is modulated to be spatially variably absorptive over the first scan period of time, selectively directing and focusing the illumination beam toward the sample volume during a second scan period of time, receiving second radiation signals from the sample volume cumulatively at the single element detector during the second scan period of time, wherein said second radiation signals are directed toward the single element detector through the mask that is modulated to be spatially variably absorptive over the second scan period of time, and determining an image corresponding to Moire fringe patterns associated with the first and second radiation signals.
- the invention provides a microscopy system that includes an illumination source system for providing an illumination beam, optics for directing and focusing the illumination beam toward a sample volume, a controller and a single element detector.
- the optics include a scanning system that selectively directs the illumination beam toward any of a plurality of spots in the sample volume.
- the controller is coupled to the scanning system and is for providing the illumination beam to the sample volume.
- the controller is also coupled to a mask that is spatially variably absorptive over a scan period of time such that cumulative illumination field is provided to the sample volume.
- the single element detector is for receiving a radiation signal radiated from the sample volume through the mask such that processing of the radiation signal results in an image may be formed that is beyond the diffraction limit.
- Figure 1 shows an illustrative diagrammatic view of a microscopy system in accordance with an embodiment of the invention
- Figures 2A and 2B show illustrative diagrammatic views of spatially patterned cumulative illumination patterns for use in system in accordance with certain embodiments of the invention
- Figures 3A - 3C show illustrative diagrammatic views of different orientations of an illumination pattern in accordance with certain embodiments of the invention
- Figures 4A - 4C show illustrative diagrammatic views of cumulative images from the illumination patterns oriented as shown in Figures 3A - 3C respectively;
- Figure 5 shows an illustrative diagrammatic view of a microscopy system in accordance with another embodiment of the invention
- Figure 6 shows an illustrative diagrammatic view of a microscopy system in accordance with a further embodiment of the invention that employs a forward detection direction
- Figures 8A and 8B show illustrative diagrammatic views of the point spread function and the optical transfer function respectively of image data using numerical apertures of 1.4 and 0.5 in a system in accordance with an embodiment of the invention
- Figure 9 shows illustrative diagrammatic graphical representations of illumination and imaging signals during scanning
- Figure 10 shows an illustrative diagrammatic view of cumulative illumination as a sinusoidal wave and as a combination of three delta functions
- Figure 11 shows illustrative diagrammatic graphical representations of emissivity in Fourier space for convolved transfer functions in a system in accordance with an embodiment of the invention
- Figure 12 shows an illustrative diagrammatic view of a microscopy system in accordance with a further embodiment of the invention employing scanning patterned detection in an epi-direction
- Figure 13 shows an illustrative diagrammatic view of a microscopy system in accordance with a further embodiment of the invention employing scanning patterned detection in a forward direction.
- the invention provides a high-resolution microscopy system and method that may be used with fluorescence microscopy (one or two photon fluorescence) as well as many non-fluorescence microscopy techniques such as spontaneous Raman microscopy, coherent anti-Stokes Raman scattering microscopy, second harmonic generation microscopy, and third harmonic generation microscopy, among others.
- the invention is built on the discovery that if a spot of illumination is modulated as it is scanned over a sample wherein the modulation provides that illumination occurs sequentially at discrete points of an excitation pattern (such as a structured illumination pattern used in SIM or SSIM) during a scan period of time, then summing the resulting wide field radiation signals from each spot of illumination cumulatively will yield a resulting low frequency Moire fringe pattern.
- the invention utilizes detection modulation together with spatially cumulative imaging using a non-descanned single element detector, again yielding low frequency Moire fringe patterns.
- FIG. 1 shows a spatially patterned cumulative illumination microscopy system 10 in accordance with an embodiment of the invention.
- the system 10 includes an illumination source system 12 such as a laser, a controller 14, an illumination modulator 16 and an x-y scanner 18 that scans the modulated illumination beam in x and y directions with respect to a sample.
- An optical system including optical elements 20, 22 and 24, direct focused illumination onto the sample 26, and a detected radiation signal is directed by optical element 28 (via a beam splitter 30) onto a wide field array detector such as a charge couple device (CCD) array 32. While the detector 32 is employed in the reflected direction with respect to the sample, in other embodiments, the detector may be positioned a forward transmitted light position with respect to the sample.
- CCD charge couple device
- the modulator 16 changes an optical property (such as intensity) of the illumination beam as the beam is moved from a first spot of a plurality of spots to a second spot etc.
- the optical property of the illumination beam is modulated during the scanning such that for each scanning position, the sample volume receives a portion of a spatially patterned illumination field such that the net effect of the modulation and scanning is to provide a patterned cumulative illumination field over a scan period of time.
- the spatially patterned cumulative illumination field is chosen to be parallel lines, as shown for example in Figure 2A at 40, then for each individual spot 42, the laser would be either on or off, depending on whether the spot was aligned with an illumination line 44 or a dark line 46 of the cumulative illumination pattern.
- Figure 2 shows using one spot per illuminated line 44 and one spot per non-illuminated line 46, any combination of patterns may be employed.
- the system may provide lines 44 having widths of, for example, 5 - 10 spots, and the same or a different number of spots per width for each non-illuminated line 46, as long as the cumulative illumination field is spatially patterned.
- the scanning system may provide raster scanning or wrap-around scanning as shown.
- Each point of what would be a structured illumination field is therefore sequentially illuminated to provide a cumulative illumination field.
- the detector array is left on during a scan period, and receives the radiation associated with each illumination spot of the cumulative illumination field.
- the modulator 16 may be omitted, and instead, the controller may direct the x-y scanner to skip over areas where no illumination is to occur.
- the spatially patterned cumulative illumination field 40' is provided by having illumination appear at spots 42', and to jump from one spot to another without modulating the illumination beam. Illumination is therefore provided in areas 44', and not provided in areas 46', yielding the same spatially patterned cumulative illumination field over the scan period of time.
- the array detector 32 remains on for the entire scan period, and captures a cumulative radiation signal from the sample volume, which is stored in a computer system such the controller 14 or in another computer system.
- a computer system such the controller 14 or in another computer system.
- separate images may be obtained (and separately stored) for each scan spot in a scan period of time. This may be done, for example, if further information is desired that is not recorded by the single cumulative recording, such as certain phase information that varies for the different illumination spots (42 or 42') during a scan. The separate recorded images would then be combined mathematically to provide an effective cumulative recording.
- the position and orientation of the illumination pattern with respect to the sample volume is then changed (e.g., either rotationally and/or linearly) from a first position and orientation as shown at 50 in Figure 3 A, to a second position and orientation as shown at 52 in Figure 3B 1 and then to a third position and orientation as shown at 54 in Figure 3C.
- a cumulative image 60 as shown in Figure 4 A, 62 as shown in Figure 4B and 64 as shown in Figure 4C respectively
- the change in position and orientation is provided by rotating one of the sample volume or the scanning system
- the change of position and orientation may be provided by linearly shifting (translating) one of the sample volume or the scanning system
- the position and orientation may be changed by both rotation and translation.
- the fringe patterns in the images 60, 62 and 64 are then analyzed in the time domain in accordance with Fourier analyses (as applied in SSIM) to obtain a super resolved final image of the sample as discussed in more detail below.
- the illumination source system may include two lasers that together provide a single collinear beam having signals with two center frequencies (e.g., pump and Stokes), either of which may be modulated as desired for certain applications.
- a spatially patterned cumulative illumination microscopy system 60 includes an illumination source system 62 includes a first laser 64 and a second laser 66.
- one laser and an optical parametric oscillator may be used to provide the two laser output frequencies.
- a controller 68 is coupled to an optional modulator 70 and to an x - y scanner 72. The modulator and scanner, or the scanner alone causes the spatially patterned cumulative illumination field to be directed to a sample 74 via optics 76, 78 and 80.
- a radiation signal from the sample is received by the objective 80 and is directed via beam-splitter 82 and optics 84 toward a CCD detector array 86.
- detection may be achieved in a forward direction, as shown for example in Figure 6.
- the microscopy system 80 includes an illumination source 82, and a controller 84 that is coupled to an optional modulator 86 and to an x -y scanner 88.
- the modulator and scanner, or the scanner alone causes the spatially patterned cumulative illumination field to be directed to a sample 90 via optics 92, 94 and 96.
- a radiation signal from the sample is received by the objective 98 and is directed via beam-splitter 100 and optics 102 toward a CCD detector array 104.
- the microscopy system may move the stage rather than scan the illumination beam, as shown for example in Figure 7.
- the microscopy system 110 includes an illumination source 112, and a controller 114 that is coupled to an optional modulator 116.
- the sample 118 is provided on an x - y movable stage 120.
- the modulator 116 and movable stage 120, or the movable stage 120 alone causes the spatially patterned cumulative illumination field to be directed to the sample 118 via optics 122, 124, 126 and 128.
- a radiation signal from the sample is received by the objective 128 and is directed via beam-splitter 130 and optics 132 toward a CCD detector array 134.
- the derivation of the image is achieved in accordance with the following analysis.
- t, r, and x be a 2D vector indicating the coordinate of any point, and k be the corresponding 2D spatial frequency.
- s ⁇ r be the density distribution of fluorescent molecules in the object
- psf ⁇ r be the intensity point spread function of the microscope
- mod ⁇ r be modulation pattern of the scanning beam intensity
- em ⁇ r be the emissivity of the object upon illumination
- im(x) be the image recorded.
- Figure 9 which shows an illumination signal 160 and an image signal 162
- Let t indicate the center of a particular scanning spot, and r be any point in the specimen plane. Fluorophores at r are illuminated with light intensity mod ⁇ i) ⁇ ps ⁇ r - t). The image of these fluorophores is a/ ⁇ s/with peak intensity mod ⁇ t) • ps ⁇ r - t) • s(r).
- Image intensity at point x contributed by the fluorophores at r is given by mod ⁇ t) ⁇ ps ⁇ r - t) ⁇ s(r) ⁇ psflx - r). So all fluorophores illuminated by the illumination spot give rise to psf and contribute to image intensity at point x in a similar fashion, the overall intensity at x in the image is obtained by integrating over all specimen coordinates:
- ® stands for convolution.
- the image is given therefore, by the convolution of emissivity and the psf.
- the illumination pattern is:
- OTF optical transfer function
- emissivity the Fourier transform of the psf.
- a scanning patterned detection system utilizes detection modulation together with spatially cumulative to image using a non- descanned single-element detector.
- the single- element detector may comprise a photomultiplier tube (PMT) with a large detection area.
- a controllable mask having spatially variable absorptions is positioned before the single-element detector, and the mask sums up all transmitted signals through the mask, and assigns the sum to the pixel corresponding to the current scanning position.
- FIG 12 shows a scanning patterned detection system 200 in accordance with an embodiment of the invention.
- the system 200 includes an illumination source system 202 (comprising one or more lasers) and an x-y scanner 204 that scans illumination from the illumination source 202 in x and y directions with respect to a sample.
- An optical system including optical elements 206, 208 and 210, direct focused illumination onto the sample 212. Reflected illumination from the sample is directed by a beam splitter 214 and an optical element 216 toward a single element detector 220 (e.g., a PMT) having a large detection area.
- a single element detector 220 e.g., a PMT
- a controllable mask 218 In front of the detector 220 is a controllable mask 218 having spatially variable absorption.
- a controller 222 is coupled to both the mask 218 as well as the x-y scanner 204 such that spatially cumulative imaging is achieved using the non-descanned single-element detector 220, providing low frequency Moire fringe patterns.
- a scanning patterned detection system may also be employed together with a movable stage instead of a fixed mirror as discussed above with reference to the scanning patterned illumination system of Figure 7.
- FIG. 13 shows a scanning patterned detection system 240 in accordance with another embodiment of the invention employing forward detection
- the system 240 includes an illumination source system 242 (comprising one or more lasers) and an x- y scanner 244 that scans illumination from the illumination source 242 in x and y directions with respect to a sample.
- An optical system including optical elements 246, 248 and 250, direct focused illumination onto the sample 252.
- Forward directed illumination from the sample 252 is directed by a beam splitter 254 and an optical element 256 toward a single element detector 262 (e.g., a PMT) having a large detection area, in front of the detector 262 is a controllable mask 260 having spatially variable absorption.
- a controller 264 is coupled to both the mask 260 as well as the x-y scanner 244 such that spatially cumulative imaging is achieved using the non- descanned single-element detector 262, providing low frequency Moire fringe patterns.
- such a scanning patterned detection system may also be employed together with a movable stage instead of a fixed mirror as discussed above with reference to the scanning patterned illumination system of Figure 7.
- This scanning patterned detection system produces a picture p ⁇ f) as discussed above, and in order to recover out-of-band frequencies in s(k) , M(x) needs to be shifted to generate additional images. Again, the same algorithmic manipulations are then applied to obtain super-resolved s(k) as for the scanning patterned illumination systems above.
- Both scanning patterned illumination and scanning patterned detection depend on the fact that light intensity from different incoherent point sources is linearly summed. This property of incoherent imaging enables spatial structures to be first decomposed and encoded into a temporal sequence and subsequently synthesized from the sequence through summation.
- the mathematical formalisms of scanning patterned illumination and scanning patterned detection stem from the symmetry between x and t in the above equation for I ⁇ m (x,t) .
- the above equations for the pictures p(x) and p ⁇ t) are weighted sums over t and x with the temporal and spatial modulation terms being the weights, respectively.
- Each approach therefore, may be viewed as linear filtering approaches to the difficult problem of solving for s(r) in the above equation for I ⁇ m (x,t) .
- the spatial or temporal integration utilized in scanning patterned illumination and scanning patterned detection digitally rather than physically.
- individual pictures corresponding to I,,,,(x,t) must be taken for each scanning position t.
- Modulations and integrations are then carried out as weighted sums over these pictures. The sum may be performed either over different pictures, following the integration over t in the equation for p(x) above, or over different pixels in each picture, following the integration over x in the equation for p( ⁇ above.
- This digital implementation is essentially a linear filter approach to inversion of the pictures.
- physical integration is much faster in the image acquisition process, digital processing may carry one advantage; when the illumination pattern is physical, it cannot take negative values.
- the mask's transmittance cannot be negative either, unless special detection configurations are utilized.
- Systems of the invention may be used for linear optical processes such as spontaneous Raman scattering, confocal reflectance, phase contrast (differential interference contrast), an absorption such as with hematoxylin and eosin (H & E) staining or Golgi staining.
- the system of the invention may also be used for non- linear optical processes such as two photon fluorescence, coherent anti-Stokes Raman scattering (CARS), second harmonic generation, third harmonic generation, and stimulated Raman scattering.
- CARS coherent anti-Stokes Raman scattering
- a system of the invention may be provided therefore, by adding two new instrument elements onto a standard laser scanning microscope which uses a focused laser spot to raster scan the sample.
- a nonuniform scanning approach was employed.
- the scanner can actively choose to scan the regions of interest (ROI) while jumping over the rest areas.
- the optical property of the excitation beam can be temporarily modulated while the laser spot is scanning continuously over the sample.
- a patterned and non-uniform laser intensity map can be written onto the sample.
- the modulation can be intensity on-and-off switching. Therefore, effectively, this results in a scanning pattern in which certain regions of interest (ROI) are illuminated with full laser power while the other regions are not excited. Due to the diffraction-limited finite size of the focused laser spot, the final illumination pattern cannot have arbitrary high spatial frequency.
- a de-scanned single-point intensity detector such as photo multiplier tube (PMT) in the standard laser scanning confocal microscope
- a non-de-scanned array detector such as a CCD camera was used to image the overall emission pattern generated from the sample. In the whole process of modulated scanning, this camera is kept exposed to register the signal until a complete frame is scanned over.
- the experimental procedure used to obtain an image with doubled spatial resolution was as follows. First, a temporal modulation sequence was created that corresponds to a spatial illumination pattern which consists of densely packed on-and- off stripes. The effective spatial frequency of these stripes is designed such as it is close to the highest possible passed frequency of the microscope depending on the objective's numerical aperture and the wavelength of the light, e.g. typically on the order of 200nm.
- the phase of the above modulation sequence i.e., the spatial illumination pattern, relative to the fixed sample position, was shifted into two other positions. So, a total three illumination patterns are created.
- the phase of the above modulation sequence i.e., the spatial illumination pattern, relative to the fixed sample position, was shifted into two other positions. So, a total three illumination patterns are created.
- the non-descanned array detector such as a charge coupled device
- the fast and slow scanning axis of the x-y scanning mirror were switched, i.e., by rotating the scanning field of view by 90 degree.
- the above procedure was repeated along the perpendicular direction in order to collect another 3 cumulative images.
- all 6 images (3 along x and 3 long y directions) were analyzed. A super-resolved image in the Fourier domain was then reconstructed.
- Systems of certain embodiments of the invention therefore, provide that the diffraction-limited spatial resolution of fluorescent and many non-fluorescent optical microscopies are exceeded through a non-uniform laser scanning microscopy.
- a set of images that are recorded under a set of corresponding different non-uniform scanning patterns are used to reconstruct the final super-resolved image.
- the non-uniform scanning pattern can be achieved by an active mode in which the laser beam scanner can actively choose to scan the regions of interest while jumping over the rest of the areas.
- the non-uniform scanning pattern can also be achieved by a passive mode in which the optical property (e.g., intensity) of the excitation beam can be temporarily modulated while the laser spot is scanning continuously over the sample.
- a non-de- scanned array detector such as a CCD camera to image the overall emission pattern generated from the sample.
- this camera is kept exposed to register the signal until a complete frame is scanned over.
- the imaging process is repeated under a set of different non-uniform scanning patterns that have different modulation direction and phases.
- the resulting set of images is used to computationally reconstruct the final image which has twice high spatial resolution as the original ones.
- Scanning patterned illumination microscopy therefore, synthesizes an effective structured illumination field through modulation of the peak intensity of the scanning spot and temporally cumulative imaging on a spatially resolved wide-field detector.
- Scanning patterned detection microscopy exploits the formal symmetry between x and t in the image field I im , and uses a mask at the image plane to accomplish the spatial frequency shift.
- both schemes may be combined with multiphoton excitation to image thick samples with optical sectioning, as well as non-fluorescent incoherent optical processes such as spontaneous Raman scattering. Neither process requires special fluorophores or special light sources to generate extreme light intensities.
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Abstract
L'invention porte sur un procédé pour effectuer une microscopie de haute définition. Le procédé comprend les étapes consistant à délivrer un faisceau d'éclairage, à diriger et à focaliser de façon sélective le faisceau d'éclairage sensiblement vers un premier d'une pluralité de points dans un volume d'échantillon, à recevoir un premier signal de rayonnement à partir du volume d'échantillon au niveau d'un détecteur de groupement à champs large, à faire effectuer un balayage au faisceau d'éclairage de telle sorte que le faisceau d'éclairage se déplace à partir d'une position sensiblement dirigée vers le premier de la pluralité de points dans le volume d'échantillon vers une position sensiblement dirigée vers un deuxième de la pluralité de points dans le volume d'échantillon, à diriger le faisceau d'éclairage sensiblement vers le deuxième de la pluralité de points dans le volume d'échantillon, et à recevoir un deuxième signal de rayonnement à partir du volume d'échantillon au niveau du détecteur de groupement à champ large.
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
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DE102011051086A1 (de) * | 2011-06-15 | 2012-12-20 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Verfahren und Vorrichtung zur Abbildung einer mit einem Fluoreszenzfarbstoff markierten Struktur in einer Probe |
JP2013080024A (ja) * | 2011-10-03 | 2013-05-02 | Nikon Corp | 走査型顕微鏡 |
JP2014021487A (ja) * | 2012-07-19 | 2014-02-03 | Sony Corp | 顕微鏡法における被写界深度(dof)をシミュレートするための方法及び装置 |
WO2014106657A1 (fr) | 2013-01-04 | 2014-07-10 | University Of Limerick | Système et procédé de nanoscopie infrarouge différentielle |
DE102015116598A1 (de) | 2015-09-30 | 2017-03-30 | Carl Zeiss Microscopy Gmbh | Verfahren und Mikroskop zur hochauflösenden Abbildung mittels SIM |
US9664614B2 (en) | 2011-07-11 | 2017-05-30 | University Of Limerick | Method for high resolution sum-frequency generation and infrared microscopy |
DE102016007839A1 (de) | 2016-06-28 | 2017-12-28 | Horst Wochnowski | Hochauflösendes Verfahren zur Mikroskopie und Nanostrukturierung von Substratoberflächen basierend auf dem SIM-Verfahren (Structured Illumination Microscopy) |
CN110007453A (zh) * | 2019-05-13 | 2019-07-12 | 中国科学院生物物理研究所 | 一种多照明模式的荧光信号测量装置及其测量方法和应用 |
EP3712670A1 (fr) * | 2019-03-21 | 2020-09-23 | Carl Zeiss Microscopy GmbH | Procédé de microscopie à balayage haute résolution |
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WO2023109625A1 (fr) * | 2021-12-13 | 2023-06-22 | 深圳先进技术研究院 | Système d'imagerie microscopique à deux photons à double couleur in vivo |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3210654B1 (ja) * | 2001-05-02 | 2001-09-17 | レーザーテック株式会社 | 光学式走査装置及び欠陥検出装置 |
JP2004163198A (ja) * | 2002-11-12 | 2004-06-10 | Lasertec Corp | 欠陥検査装置、欠陥検査方法、光学式走査装置、半導体デバイス製造方法 |
US20040156102A1 (en) * | 2001-11-16 | 2004-08-12 | Leica Microsystems Heidelberg Gmbh | Method for generating a multicolor image, and microscope |
JP2005345221A (ja) * | 2004-06-02 | 2005-12-15 | Hitachi High-Technologies Corp | 検出光学装置及び欠陥検査装置 |
US20060012785A1 (en) * | 2004-07-16 | 2006-01-19 | Joerg-Michael Funk | Light scanning electron microscope and use |
-
2010
- 2010-03-02 WO PCT/US2010/025888 patent/WO2010101894A2/fr active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3210654B1 (ja) * | 2001-05-02 | 2001-09-17 | レーザーテック株式会社 | 光学式走査装置及び欠陥検出装置 |
US20040156102A1 (en) * | 2001-11-16 | 2004-08-12 | Leica Microsystems Heidelberg Gmbh | Method for generating a multicolor image, and microscope |
JP2004163198A (ja) * | 2002-11-12 | 2004-06-10 | Lasertec Corp | 欠陥検査装置、欠陥検査方法、光学式走査装置、半導体デバイス製造方法 |
JP2005345221A (ja) * | 2004-06-02 | 2005-12-15 | Hitachi High-Technologies Corp | 検出光学装置及び欠陥検査装置 |
US20060012785A1 (en) * | 2004-07-16 | 2006-01-19 | Joerg-Michael Funk | Light scanning electron microscope and use |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102011051086A1 (de) * | 2011-06-15 | 2012-12-20 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Verfahren und Vorrichtung zur Abbildung einer mit einem Fluoreszenzfarbstoff markierten Struktur in einer Probe |
US9664614B2 (en) | 2011-07-11 | 2017-05-30 | University Of Limerick | Method for high resolution sum-frequency generation and infrared microscopy |
JP2013080024A (ja) * | 2011-10-03 | 2013-05-02 | Nikon Corp | 走査型顕微鏡 |
JP2014021487A (ja) * | 2012-07-19 | 2014-02-03 | Sony Corp | 顕微鏡法における被写界深度(dof)をシミュレートするための方法及び装置 |
WO2014106657A1 (fr) | 2013-01-04 | 2014-07-10 | University Of Limerick | Système et procédé de nanoscopie infrarouge différentielle |
US9897536B2 (en) | 2013-01-04 | 2018-02-20 | University Of Limerick | Differential infra red nanoscopy system and method |
DE102015116598A1 (de) | 2015-09-30 | 2017-03-30 | Carl Zeiss Microscopy Gmbh | Verfahren und Mikroskop zur hochauflösenden Abbildung mittels SIM |
DE102016007839A1 (de) | 2016-06-28 | 2017-12-28 | Horst Wochnowski | Hochauflösendes Verfahren zur Mikroskopie und Nanostrukturierung von Substratoberflächen basierend auf dem SIM-Verfahren (Structured Illumination Microscopy) |
US11300767B2 (en) | 2019-03-21 | 2022-04-12 | Carl Zeiss Microscopy Gmbh | Method for high-resolution scanning microscopy |
EP3712670A1 (fr) * | 2019-03-21 | 2020-09-23 | Carl Zeiss Microscopy GmbH | Procédé de microscopie à balayage haute résolution |
CN110007453A (zh) * | 2019-05-13 | 2019-07-12 | 中国科学院生物物理研究所 | 一种多照明模式的荧光信号测量装置及其测量方法和应用 |
CN110007453B (zh) * | 2019-05-13 | 2023-11-21 | 中国科学院生物物理研究所 | 一种多照明模式的荧光信号测量装置及其测量方法和应用 |
WO2021113421A1 (fr) * | 2019-12-06 | 2021-06-10 | Illumina, Inc. | Appareil et procédé d'estimation de valeurs provenant d'images |
US11768364B2 (en) | 2019-12-06 | 2023-09-26 | Illumina, Inc. | Apparatus and method of estimating values from images |
CN113495062A (zh) * | 2020-04-07 | 2021-10-12 | 旭臻科技有限公司 | 用于大面积显微光致荧光扫描与测绘测量的*** |
WO2023109625A1 (fr) * | 2021-12-13 | 2023-06-22 | 深圳先进技术研究院 | Système d'imagerie microscopique à deux photons à double couleur in vivo |
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