EP3186885A1 - Procédé et système de tomographie de fluorescence hétérodynée - Google Patents

Procédé et système de tomographie de fluorescence hétérodynée

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Publication number
EP3186885A1
EP3186885A1 EP14900413.7A EP14900413A EP3186885A1 EP 3186885 A1 EP3186885 A1 EP 3186885A1 EP 14900413 A EP14900413 A EP 14900413A EP 3186885 A1 EP3186885 A1 EP 3186885A1
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Prior art keywords
light
hft
sample
optical
fluorescence
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German (de)
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EP3186885A4 (fr
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Andreas G. NOWATZKY
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    • H—ELECTRICITY
    • H03—ELECTRONIC CIRCUITRY
    • H03F—AMPLIFIERS
    • H03F3/00—Amplifiers with only discharge tubes or only semiconductor devices as amplifying elements
    • H03F3/04—Amplifiers with only discharge tubes or only semiconductor devices as amplifying elements with semiconductor devices only
    • H03F3/08—Amplifiers with only discharge tubes or only semiconductor devices as amplifying elements with semiconductor devices only controlled by light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • 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

Definitions

  • the present invention relates to fluorescence tomography, and particularly to a novel fluorescence tomography modality referred to herein as "heterodyned fluorescence tomography" or "HFT"
  • Tomography of various kinds have been developed as valuable tools for a non-invasive imaging within a body of material, particularly as tools for imaging within a living organism such as the human body for diagnosis and bio-medical research.
  • images are produced by measurements of energy waves of one type or another that have passed through at least a portion of the body of material to be imaged and the measurements are employed to compute an image representative of the interior of the body.
  • the energy waves may be electromagnetic waves that pass through the body and are measured for intensity when they exit the body, as in the case of X-rays in computerized axial tomography, or a "CAT scan”; they may be electromagnetic waves induced within the body and measured for intensity when they exit the body, as in the case of radio waves in magnetic resonance imaging, or "MRI”; or they may be acoustical waves that pass into the body and are scattered back to a device outside the body which measures the intensity of waves scattered back from a particular depth within the body, as in ultrasound imaging.
  • CAT scan computerized axial tomography
  • MRI magnetic resonance imaging
  • ultrasound employs a much lower energy acoustical wave that does not present a known danger of DNA damage and has found valuable application in imaging larger features within the human body that do not require the high resolution that is obtained by higher energy waves.
  • Other considerations may be involved as well, depending on the nature of the material and the purpose of the methodology.
  • Electromagnetic waves of intermediate energy commonly, but imprecisely, known as "light waves", whether visible or not, offer the potential for tomography with good resolution and a low probability of DNA or other cellular damage in organisms.
  • OCT optical coherence tomography
  • coherent light waves typically having near infrared energy are launched into living tissue and the backscattered energy is allowed to interfere with the source so that, because of the short coherence length of the waves, the measured intensity for a given phase delay is representative of the backscattering at a given depth in the tissue.
  • An image of a volume within the object may be produced by scanning laterally over that volume while collecting interference intensity data. This technique provides good resolution for tissue density based on associated variations in the index of refraction of the tissue and is able to image tissue morphology, but it does so without any molecular specificity.
  • Confocal microscopy employs the idea of using the objective of a scanning microscope not only to produce an image of the interior of an object, but to project the image of a light source into, and illuminate only the focal point within, the object. That improves resolution by reducing the detection of scattering from other points within the object.
  • An image of a volume within the object may be produced by scanning over that volume while collecting back scattered intensity data.
  • confocal microscopy can also be made molecule specific.
  • the effective depth of confocal microscopy is only about 100 ⁇ , while OCT can resolve structures as deep as about 400 ⁇ , into the material due to the greater random backscatter rejection capability of this interferometry method.
  • Figure 1 A is a schematic diagram of a prior art optical coherence tomography system using a Michelson interferometer.
  • Figure IB is a schematic diagram of a prior art optical coherence tomography system using a Mach-Zehnder interferometer.
  • Figure 1C is a schematic diagram of a hypothetical optical coherence tomography system using a Mach-Zehnder interferometer, a fluorescent screen as an interference medium and a fluorescent light detector.
  • Figure ID is a schematic diagram of an HFT system according to the present invention.
  • Figure 2 is a plot of elliptical curves associated with OCT and hyperbolic curves associated with HFT.
  • FIG. 3 is a detailed schematic diagram of a single-objective HFT system.
  • Figure 4 is a diagram of the results of a simulation of a single-objective HFT system in which the back aperture of the objective lens is divided into central and surrounding annular zones illuminated by slightly different frequencies so as to cause a continuously changing relative phase between the respective wave fronts.
  • Figure 5A shows the magnitude and phase point spread functions of a simulated single- objective HFT system in which the back aperture of the objective lens is divided into a central and surrounding annular zones illuminated by slightly different frequencies so as to cause a continuously changing relative phase between the respective wave fronts.
  • Figure 5B shows the magnitude and phase point spread functions of a simulated single- objective HFT system in which the back aperture of the objective lens is divided into two semicircular zones illuminated by slightly different frequencies so as to cause a continuously changing relative phase between the respective wave fronts.
  • Figure 5C shows the magnitude and phase point spread functions of a simulated single- objective HFT system in which the back aperture of the objective lens is divided into two adjacent circular zones illuminated by slightly different frequencies so as to cause a continuously changing relative phase between the respective wave fronts.
  • Figure 6 shows the measured photo multiplier tube current, magnitude point spread function and phase point spread function for an experimental HFT system according to the present invention.
  • HFT is a new imaging modality that extends the capabilities of microscopy to penetration depths comparable to OCT. Increasing the ability to image molecules via fluorescence microscopy about 10 times deeper into biological tissues is a significant advance of the state of the art of molecular imaging. In particular, HFT enables research that is currently not practical or very difficult, for example in- vivo tracking of stems cells, and potentially enables medical diagnosis of conditions earlier than currently possible.
  • This disclosure is directed to a practical and versatile HFT scanner that can be deployed in a bio-medical laboratory to solve real problems. For example, with a scan depth of over 1 mm it will be possible to track cells in small organisms and embryos. It is a molecular imaging technology that is compatible with minimally invasive procedures, particularly to enable in-vivo, real-time, optical pathology. This may be used to determine the health of tissue during surgery without the need of extracting biopsy samples and analyzing them in a pathology laboratory, so as to improve cancer removal surgery. Theory of Operation
  • HFT heterodyned fluorescence tomography
  • a typical OCT system 10 employs a Michelson interferometer having a coherent light source 12 generating light at source wavelength K s , a beam splitter 14, a reference mirror 16, and a detector 18 sensitive to the source wavelength ⁇ 5 .
  • Light from the source 12 having a relatively short coherence length, travels along path 20 to the beam splitter 14 where it is split into two paths, path 22 and path 24.
  • Light along path 22 is directed to a sample 26 to be imaged and light along path 24 is directed to the reference mirror 16.
  • the light directed to sample 26 is scattered back along path 22 to the beam splitter 14, and light directed to the reference mirror 16 is reflected back along path 24 to the beam splitter 14, where the light scattered by the sample and the light reflected by the mirror are combined and directed along path 28 to the detector 18.
  • the optical path lengths of the path 22 and path 24 are within the light coherence length of one another interference will occur at detector 18, which produces a signal representative of the degree of such interference.
  • the reference mirror By moving the reference mirror back and forth as shown by arrow 17 so as to vary the path length 24, the intensity of light scattered at corresponding depths within the sample 26 can be measured by the intensity of the interference detector 18.
  • an OCT system 30 could employ a Mach-Zehnder interferometer.
  • light from the coherent light source travelling along path 20 encounters a first beam splitter 32 where it is split into two paths, path 34 and path 36.
  • Light along path 34 is directed to sample 26 and light along path 36 to a reference mirror 38, which reflects the light along path 40 to a second beam splitter 42, which passes that light there through and along path 44 to detector 18.
  • the reference mirror preferably is actually a mirror system that can vary the optical path length, as will be understood by a person having skill in the art.
  • light scattered by the sample 26 travels along path 46 to beam splitter 42, where it is reflected along path 44 and mixed with light from reference mirror 28 at the detector 18. While this illustrates the use of a Mach-Zehnder interferometer in OCT, that the light incident on and scattered from the sample 26 travel along different paths in this system means that it is not compatible with a confocal scanner as is ordinarily used in an OCT system of the type illustrated by Fig. 1 A.
  • a hypothetical OCT system 50 uses the Mach-Zehnder configuration, but the second beam splitter 42 in Fig. IB is replaced in system 50 with a fluorescent screen 52. If the two paths 34, 46 and 36, 40 are within the coherence length of one another, then the two respective wavefronts will produce an interference pattern on the screen 52, manifested by light produced at the fluorescence wavelength ⁇ f of the screen. In this case, a detector 54 is provided that is sensitive to the fluorescence wavelength f , but not necessarily to the source wavelength ⁇ 5 .
  • the gratuitous introduction of fluorescence in system 50 does not change the operating principle: the interference could be observed directly with, for example, a CCD array camera or a photographic plate in place of the fluorescent screen, or indirectly by observing the fluorescent light from the screen 52 either with the naked eye or the detector 54, depending on f .
  • the observed signal is proportional to the square of the sum of the two incident
  • an illustrative HFT system 60 also uses a Mach-Zehnder interferometer, but light travelling along path 34 encounters a second reference mirror 62, which reflects the source light along path 64, where it mixes with source light from path 40 within the sample 26, which is located where the fluorescent screen 52 is located in system 50.
  • the sample 26 is also the interference detector. More precisely, the sample includes fluorophores and those fluorophores serve as detectors of the interference between the source light wave-front arriving from path 64 and the source light wave-front arriving from path 40, respectively.
  • the fluorescent molecules within the sample 26 are excited by the coherent sum of the two wave-fronts and act as square-law detectors that produce an interference signal S ⁇ at wavelength X f .
  • the interference signal S is received by a fluorescent light detector 66, which is preferably blind to the source light at wavelength ⁇ 5 .
  • Interferometers compare optical path lengths. However, there is a difference between HFT and OCT in that HFT senses the optical path difference while OCT senses the round-trip optical path length. This provides advantages with HFT in some applications over OCT as described hereafter.
  • the first beam splitter 32 where the beam is split
  • the second beam splitter 42 where the light is recombined
  • focal points 70 and 72, and ellipsoids 74 for example, in Fig. 2.
  • the ellipsoid degenerates to a sphere with the center on the beam splitter. In the latter configuration, OCT can sense depth (Z), but only depth, while lateral resolution (X/Y) depends on a scanner.
  • the first reflective mirror 38 and the second reflective mirror 62 are both essentially emitters and the sample itself is the detector. All fluorophores that are located on hyperbolic shells that are defined by focal points located at mirrors 38 and 62 will produce the same signal. In other words, any point within the sample volume that has the same distance difference from mirror 38 and mirror 62 will generate the same signal.
  • This relation is also illustrated in Fig. 2, where the points 70 and 72 correspond to the locations of the focal points, that is, the locations of the mirrors 38 and 62, and lines 76, for example, correspond to the hyperboloids.
  • HFT has good lateral (X Y) resolution but cannot resolve depth (Z) along the line of symmetry.
  • X Y lateral
  • Z depth
  • Each source light wavefront will also produce a fluorescence signal, Sw f .
  • the difference between the optical path lengths along paths 34, 64 and along paths 36, 40 is deliberately changed periodically, which causes the interference pattern within the sample 26 to move. Consequently, the amplitude of light from a stationary fluorophore within a sample is modulated. This amplitude modulated signal is used to form the HFT image. Provided that the period of the modulation is long compared to the fluorescence lifetime of the fluorophore, the fact that fluorescence is a stochastic process does not limit the resolution of HFT.
  • the image reconstruction algorithm decomposes the problem into two steps. First, a wave-length sweep is performed for each sample point, which yields the discrete Fourier transform (DFT) of the fluorophore density distribution over the set of hyperbolic shells that are defined by the illumination of geometry. Therefore, the first step is simply to apply the inverse DFT to obtain the density distribution in the spatial domain. The second step is then to apply the inverse Radon transform to the collection of all sample points to obtain a 3D image of the sample volume. This is the outline for reconstructing images where the two illumination points are relatively far apart, which is the case for the HFT configurations disclosed herein.
  • DFT discrete Fourier transform
  • fluorescence data from a sample may be obtained and three-dimensional image of the sample may be reconstructed by obtaining data at multiple angles ( ⁇ , ⁇ ) of beams 64 and 40, respectively, according to the following steps:
  • detector position which represents a projection on the plane of the detector for mirror angles (a -m,PM-m) a d wavelength ( ⁇ - ⁇ ) of a one- wavelength-dimensional Fourier transform of the fluorophore density distribution in the three-dimensional sample over the set of hyperbolic shells that are defined by the illumination geometry.
  • the resultant images IMN represent projections on the plane of the detector of a N- wavelength-dimensional discrete Fourier transform of the fluorophore density
  • FIG. 3 A schematic diagram of a preferred embodiment of a single objective HFT system 80 incorporating the principles of the invention is shown in Fig. 3.
  • light from a tunable, long coherence length laser 82 is directed through an acousto-optical crystal modulator ("AOM") 84, which is used to achieve frequency shifting.
  • AOM acousto-optical crystal modulator
  • the deflected, first order beam 86 is skewed with respect to the direction of propagation of the acoustic wave within the AOM crystal, shown by arrow 88, thus its frequency is altered with respect to the direction of propagation of the zero order beam 90 due to the Doppler effect.
  • the frequency of the first order beam is shifted with respect to the zero-order beam by 70 Mhz.
  • a 70 Mhz shift is normally insignificant.
  • this frequency shift also means that the interference pattern within the sample volume changes 7 million times per second, and that the amplitude of the fluorescence will be modulated with this 7 Mhz frequency.
  • Detecting modulation frequencies in the radio frequency ("RP") domain has a number of advantages. The most important of which is that heterodyning preserves phase information in the RF signal, so that the HFT signal carries more information than ordinary square wave detectors directly sensing the mixed light beams can provide.
  • the RF power into the AOM, provided by RF generator 92 is preferably adjusted so that the zero and first order beams are of equal intensity.
  • the zero order beam is directed to a first beam expander 94, in this case for example by three mirrors 96, 98 and 100.
  • the first order beam is directed to a second beam expander 102, in this case for example by two mirrors 104 and 106.
  • Beam expander 94 illuminates aperture 108 and beam expander 102 illuminates aperture 110.
  • the beams 112 and 114, that is, portions of expanded beams 90 and 86, that pass through the respective apertures 108 and 110 are then recombined by beam splitter cube 116.
  • This beam splitter plays no role in the Mach-Zehnder interferometer because the beams 112 and 114 that propagate through apertures 108 and 110, respectively, enter the beam splitter cube 116 from different facets.
  • the beam splitter only serves to combine the two beams in two different combined-beam branches 118 and 120.
  • the beam outputs from the beam expanders 94 and 102 were shaped with two respective apertures, that is ? a circular aperture and an annular aperture, and combined with a beam splitter cube 116.
  • a portion of beam 120 from the beam splitter 116 was directed by another beam splitter to the reference detector 134 which comprises a photomultiplier with a 1 ⁇ pinhole and a neutral density filter.
  • the reference detector 134 comprises a photomultiplier with a 1 ⁇ pinhole and a neutral density filter.
  • most of beam 120 was routed to a 800 mm telescope lens and focused on a CCD camera whose output was used to align the two combined wave-fronts in beam 120 so that they were parallel within a pointing error or less than 6 ⁇ rad.
  • the beams are combined so that they both can fill the back aperture of the same objective 122.
  • the combined beams pass through a dichroic mirror 124 that reflect fluorescent light from the sample 126 to a signal detector 128 such as a photomultiplier tube ("PPT").
  • PPT photomultiplier tube
  • the combined beams are focused by lens 130 onto a pinhole 132 in front of a reference detector 134. Ideally, the reference detector is unnecessary.
  • the output of the RF- generator could be used as a reference.
  • commercially available AOMs often exhibit a large and very temperature dependent phase shift. Therefore, it is preferred to sense the actual beat signal between the two beams explicitly to avoid random phase drift from AOM 84.
  • the objective lens 122 focuses the light to a spot within the sample 126.
  • the same objective also collects the fluorescent emission from the sample, which is mounted on a precision actuated X/Y/Z stage 136.
  • This light is directed by the dichroic mirror 124 through an emission filter 138 that blocks the laser light from the signal detector 128.
  • the detector does not form an image in this case; rather, a beam from the infinity corrected objective reaches the detector substantially collimated.
  • a signal 140 from signal detector 128 is processed with an RF lock-in amplifier 142 that yields a phase component 144 of the HFT signal and a magnitude component 146 of the HFT signal.
  • a three-dimensional image of the sample may be reconstructed according to the following steps:
  • n being the maximum number of wavelengths used, which represents a projection on the plane of the detector of a n- wavelength-dimensional discrete Fourier transform of the fluorophore density distribution in the over the set of hyperbolic shells that are defined by the illumination geometry. 6. Perform an inverse Fourier transform to get the density distribution in the spatial domain.
  • two objectives could be used to illuminate the sample volume and, perhaps, a third objective could be used to collect the fluorescent emissions. While this could provide more flexibility, achieving stability would present a greater challenge.
  • two light beams illuminating the sample may be produced by dividing the back aperture of the objective into two regions, each of which is illuminated by a different wavefront, thereby producing two distinct output beams that exit the front aperture of the objective and are focused to the same spot in the sample. Because the two output beams are distinct and have sufficient coherence length, they interfere in the sample. Because, as explained above, they have slightly different frequencies, which is equivalent to continuously changing their relative phase, they produce an HFT signal for the illuminated spot.
  • Fig. 4 One example of this approach is illustrated by Fig. 4, where the back aperture is divided into a concentric pattern 150 having a central circle 152 that receives one wavefront and a surrounding annulus 154 that receives the other wavefront. Both wavefronts are focused to the same spot 156 in the sample 26, but each has a different angular spectrum a and ⁇ , respectively. Consequently, they interfere with one another. In addition, because each beam has a different frequency, the detected amplitude of the two beams varies at a given spot at the lower beat frequency of the two beams which, preferably, is a radio frequency as explained above.
  • the average intensity 170 of the fluorescent light corresponds to the signal that a conventional imaging system would produce.
  • the amplitude of the modulation and its phase are the signals that the HFT system uses to form an image.
  • the phase is also a function of position along the optical, Z-axis of the objective.
  • Fig. 5 In addition to the back aperture division configuration shown in Fig. 4, two other configurations are shown in Fig. 5, along with their resulting interference patterns the X-Z plane (A-Scan) and a (X-Y) plane perpendicular thereto (C-Scan) and their corresponding phase distributions. More specifically, Fig.
  • FIG. 5 shows the concentric pattern 150 and corresponding A- Scan 172 and its phase distribution 174, and C-Scan 176 and its phase distribution 178; a double- D pattern 180 and corresponding A-Scan 182 and its phase distribution 184, and C-Scan 182 and its phase distribution 188; and a double-circle pattern 190 (wherein a portion 192 of the aperture is dark) and corresponding A-Scan 194 and its phase distribution 196, and C-Scan 198 and its phase distribution 200.
  • the concentric configuration is asymmetric in the sense that the two beams arrive at the focal point with different angles relative to the optical axis, as opposed to the double-D and double-circle configurations, where both beams have the same shape and arrive at the focal point with the same angular distributions.
  • the concentric configuration also differs from the double-D and double circle configurations in that the concentric configuration has an intrinsic ability to sense position along the Z-axis, while the double-D and double-circle configurations yield no Z-resolution in the Y-Z plane (B-Scan), that is, the plane including the optical axis of the objective that separates the two illuminated regions.
  • B-Scan Y-Z plane
  • the symmetric configurations have higher resolution in the X-Y plane.
  • the illumination pattern should be rotating so that each point is sampled with two or more configurations to achieve isotropic resolution.
  • the concentric configuration yields a simpler system with better Z-resolution without relying on a Radon transform as explained below.
  • the Test System [046] The system was implemented with conventional free-space optics on a vibration isolated optical table.
  • the tunable laser (corresponding to element 82 in Fig. 3) comprised a dye-laser that was pumped with an argon-ion laser.
  • the dye-laser was modified for electronic tuning. It included a custom Yag-etalon with a free spectral range of 79.225267 Ghz. Stable operation was possible on 570 distinct lines, from 579.9 nm to 635.5 nm, each about 6 Ghz wide. They dye laser produced about 250 mW when pumped with 4 W.
  • the output of the dye laser was fed through an electro-optical amplitude stabilization system.
  • the beam was then directed through an AOM (84).
  • the zero-order beam was reflected off two mirrors on a movable stage to the beam expander (94).
  • the first-order beam was directed to the beam expander (102), which included a 6 ⁇ pinhole that acted as a spatial filter for the laser beam.
  • the second order beam was directed at a beam position sensor for alignment purposes.
  • the beam position sensor is also used to determine the dispersion of the AOM. Because the system swept the laser over a range of about 55 nm, the wavelength dependent AOM deflection angle could cause misalignment. Compensation for this was accomplished by changing the AOM operating frequency.
  • Compensation for dispersion was also accomplished by electronically changing the AOM frequency (about 67 to 73 Mhz).
  • a delay adjustment stage was used to equalize the optical path- lengths. By sweeping the laser and recording the phase between the reference signal and the AOM drive, the optical path was equalized programrnatically to below 1/10 wavelength.
  • the fluorescence signal detector (128) was a PMT protected from the excitation light with an interference filter (138).
  • the signal from the PMT was amplified with a combined low- noise and an RF-lock-in amplifier (142).
  • the point spread function was measured by imaging 0.2 ⁇ microspheres with a voxel size of 0.33 ⁇ m/side and averaging over 22 isolated spheres that were manually selected.
  • Example results are shown in Figure 7.
  • Image 152 of Figure 7 is the PSF obtained by recording the overall PMT current at a wavelength of 587.3 nm. It is essentially equivalent to the PSF of an ordinary light microscope. Given a numerical aperture of 0.65, the expected Rayleigh resolution was 551 nm in the XY plane at the focal point. The resolution measured from the average PMT current is only about 2 ⁇ . This degradation by a factor of 4 is due to the fact that it is averaged over all possible phase shifts. Also, there was somewhat more power in the central zone than in the outer zone.
  • Image 154 in Figure 6 is the phase of the HFT signal, which shows the gradient along the X-axis.
  • the total phase change of the usable portion of the PSF was about 2.5 rad. This was in line with the simulation, but it may be desirable to increase this range. For example, deliberately focusing the two beam expanders in opposite directions will have the effect of moving the two virtual emission points apart along the Z-axis while maintaining rotational symmetry.
  • Applicant hereby incorporates Appendix I entitled “Additional HFT Disclosure” as part of the specification of this application.
  • Applicant hereby incorporates Appendix II, which is a copy of a United States Patent Application No. 13/298,066, entitled LOW NOISE PHOTO-PARAMETRIC SOLID STATE AMPLIFIER as part of the specification of this application.
  • Applicant hereby incorporates Appendix III, entitled “Quad-Galvos-Synthesized Beam Rotation/Scanning” as part of the specification of this application.
  • Appendix III entitled “Quad-Galvos-Synthesized Beam Rotation/Scanning” as part of the specification of this application.
  • HFT Like confocal microscopy and OCT, the idea behind HFT is quite general and can be implemented in a number of different ways.
  • the research presented in this proposal is specifically aimed at biological and medical applications where imaging the locations of fluorescently labeled molecules or cells is needed and where the operating depth of confocal microscopy is insufficient.
  • the practical operating depth of confocal microscopy is about 100 ⁇ «?, while OCT can resolve structures up to about 20x deeper.
  • OCT images are formed from back-scattered light that can only show morphology
  • confocal microscopy can employ fluorescent labels to show the locations of specific molecules
  • HFT promises the best of both technologies: high resolution fluorescent imaging at penetration depths comparable to that of OCT.
  • OCT has been described as radar that uses infra-red light instead of microwaves [3].
  • the distance from the instrument to the location of the structure within the sample that causes light to be reflected is measured by the optical path length induced delay of the reflected light.
  • the reflected light must maintain coherence back to the interferometer, thus the image formation mechanism is back-scatter from discontinuities in the refractive index of the sample (Fresnel reflection), which yields images of the sample morphology without any molecular specificity.
  • HFT can be compared to radar that uses transponders, which are devices that receive the radar signal and then transmit a response on a different frequency [14].
  • the fluorescent organic dye molecule plays the role of the transponder,
  • the fiuorophore is excited by the interrogating light at one wavelength and responds with a fluorescent emission at a different, lower frequency.
  • this analogy is flawed by the fact that a radar transponder replies with a precisely defined delay, while the process of fluorescence involves an unpredictable delay ( ⁇ -l-10ns) that is very long compared to the required temporal resolution to form a good image ( ⁇ 3-10fs).
  • HFT overcomes this problem by using an intermediate frequency (IF) that has a period that is much longer than the temporal jitter of fl
  • FIG 1 illustrates the basic principles behind OCT and HFT.
  • the classic OCT setup (A) employs a Michelson interferometer where the light from a source (LS) with short coherence length is split (BS) into two paths. The first path is directed at the sample (S) while the second path is reflected off a reference mirror (RM), The reflected light from both paths is recombined in (BS) and directed at a detector (D). The detector will sense an interference signal only if the optical path lengths of the reference and sample arm are within the coherence length of the light source. Thus it is possible to obtain a range profile by moving the reference mirror.
  • Panel B) of Figure 1 depicts a hypothetical OCT system that uses a Mach-Zehnder interferometer instead of the Michelson configuration.
  • Panel C) of Figure 1 uses the same Mach-Zehnder configuration, but it replaces the beam splitter (BS2) with a fluorescent screen (FLS). If the two paths of the interferometer are within the coherence length, then the two wave-fronts will create an interference pattern on the screen that can be observed with the detector (D).
  • BS2 beam splitter
  • FLS fluorescent screen
  • the fluorophore within the sample volume will serve as a detector in this interferometer: the light from the source (LS) is split into two paths that are both directed at the sample via the mirrors RM1 and RM2.
  • the fluorescent molecules within the sample volume are excited by the coherent sum of both wave-fronts and act as square-law detectors.
  • the emitted light from the fluorescence is subsequently received by the external detector (D) which is blind to the excitation light.
  • the optical path-lengths difference is deliberately changed in a periodic fashion, which in rum causes the interference pattern within the sample volume to move. Therefore the amplitude of the light from a stationary fluorophore within the sample is modulated.
  • This amplitude variation is the signal that is used to form the HFT image.
  • the period of the modulation is long compared to the fluorescence lifetime of the fluorophore, the fact that fluorescence is a stochastic process does not limit the resolution of HFT.
  • coherence only needs to be maintained along the path into the sample. The light emitted from the sample does not need to maintain coherence. Therefore any fluorescent light, scattered or otherwise contributes to the HFT image, which increases penetration depth compared to OCT where both the incident and reflected light must maintain coherence.
  • Section 3 has a detailed description of the proposed research and development. It is followed by a discussion of the proposed new system, its application and capabilities. This technical description will conclude by describing the intellectual merits and the broader impact of this research.
  • FIG. 2 OCT vs. HFT Sensing damental difference between OCT and HFT, namely that OCT can sense the round-trip optical path length, while HFT senses the optical path length difference.
  • any scatterer S that is located on an elliptical shell whose two focal points are the points in BS 1/BS2 where the light is split/recombined will produce the same signal.
  • the ellipsoid degenerates to a sphere with the center on the beam splitter.
  • OCT senses depth (Z) while lateral resolution (X Y) depends on a confocal scanner.
  • HFT requires multiple acquisition steps with different emitter pairs and an inverse Radon transform to resolve Z (and X/Y). It should be noted in this discussion that the amplitude or sensitivity of either system strongly depends on how the light is focused into the sample volume. Illuminating the sample with unfocused, spherical waves would be rather inefficient in either case.
  • Figure 1 suggests the use of two objectives to illuminate the sample volume and perhaps a third objective to collect the fluorescent emissions. While such a set-up would provide the most flexibility to explore the HFT design space, it is also quite complex and fraught with stability problems. Therefore a single objective configuration was considered, where both illumination beams are projected into the sample through a single objective lens.
  • FIG. 3 shows one single objective HFT
  • AOM acousto-optical modulator
  • AOMs are used to deflect laser beams, filter out particular wavelengths or modulate the
  • the deflected, first order beam is not perpendicular to the direction
  • Detecting modulation frequencies in the RF domain has a number of advantages. The most important of which is that heterodyning preserves phase information, thus the HFT signal carries more information than ordinary light detectors can provide.
  • the RF power into the AOM is adjusted so that the zero and first order beams are of equal intensity, These beams are then directed to two beam expander BEl and BE2 that illuminate the apertures Al and A2.
  • the expanded beams are then recombined with the beam-splitter (BS).
  • This beam splitter plays no role in the Mach-Zehnder interferometer because the apertures Al and A2 do not overlap, thus each part of the beamsplitter cube is illuminated by at most one branch. It serves only to combine the beams so that both can fill the back aperture of the same objective (Obj).
  • the combined beams pass through a dichroic mirror (DCM) that will reflect the fluorescent light from the sample to the signal detector (SD).
  • DCM dichroic mirror
  • SD signal detector
  • the beam-splitter produces two combined beams.
  • the second beam is focused with a lens onto a pin-hole in front of the reference
  • the location of the detector is irrelevant for HFT. In fact, there can be multiple detectors or area detectors in order to capture more light. detector (RD). Ideally, the reference detector is unnecessary. The output of the RF-generator could be used as a reference. However, the commercially available AOMs exhibit a large and very temperature dependent phase shift. Therefore it is better to sense the actual beat signal between the two beams explicitly to avoid the random AOM phase drift.
  • the objective lens (Obj) focuses the light onto a spot within the sample (S).
  • the same objective also collects the fluorescent emission from the sample.
  • This light is directed by the dichroic mirror (DCM) through an emission filter (EF) that blocks the laser light from the signal detector (SD).
  • the detector does not form an image in this case, rather all light from the infinity corrected objective reaches the detector.
  • the signal from SD is processed with an RF lock-in amplifier which yields both phase and magnitude of the HFT signal.
  • the sample is mounted on an actuated precision X/Y/Z stage.
  • Figure 4 illustrates this idea: in this case, the back aperture of the objective lens is divided into two concentric zones, a central circle surrounded by a ring. The two planar wave-fronts filling each zone have slightly different frequencies, which is equivalent to continuously changing their relative phase.
  • Figure 4 shows the instantaneous magnitude of the EM-field near the focal point of an objective lens.
  • the first image simply shows the point spread function of the objective because a phase difference of 0 between the two planar waves entering the back aperture of the objective is equivalent to just one uniform planar wave, As the phase difference increases, the focal point of the objective appears to move up.
  • the white point illustrates the location of one fiuorophore and the graph below shows the intensity at this point as a function of time.
  • the average intensity corresponds to the signal that a conventional imaging system would produce.
  • the amplitude of the modulation and its phase are the signals that the HFT system uses to form an image. It should be noted that the phase is a function of the position along the Z-axis. A configuration that produces a large change in the phase when a point is moved through the sample volume can be expected to yield better resolution.
  • Figure 5 shows the simulated HFT point spread function for three configurations, concentric rings, two half circles and two circular sub-apertures.
  • the top row of pictures show the color coded magnitude of the HFT signal in the XZ and XY planes (A and C-scan).
  • the second row shows the corresponding phase, where the angular range of 0-360 degrees is mapped to a color ring, where black represents 0, blue 90, white 180 and red 270 degrees.
  • the first configuration is asymmetric in the sense that the two beams arrive at the focal point with different angles relative to the optical axis, as opposed to the later two configurations where both beams have the same shape and arrive at the focal point with the same angular distribution.
  • the first configuration has an intrinsic ability to sense position along the Z-axis, while the later configurations yield no Z-resolution in the YZ-plane (B-scan).
  • the symmetric configurations have higher resolution in the X/Y plane.
  • the illumination pattern should be rotating so that each point is sampled with two or more configurations to achieve isotropic resolution.
  • the first configuration is interesting because it yields a simpler system with better Z-resolution without relying on the inverse Radon transform.
  • the proof-of concept system ( Figure 6) was implemented with conventional free-space optics on a vibration isolated optical table.
  • the optical layout mostly follows the schematic from Figure 3.
  • the light source is a tunable dye-laser that is pumped with a Coherent IONOVA-100 argon-ion laser.
  • the dye-laser is based on the Spectra-Physics SP375B folded cavity laser, which was modified for electronic tuning. It also has a custom Yag-etalon with a free spectral range of 79.225267 Ghz.
  • the line-width is monitored with a Candela LS-1 laser spectrometer and the wavelength is measured with an Advantest Q8326 wavelength meter, Using Rhodamine 6G, stable operation is possible on 570 distinct lines, from 579,9nm to 635.5nm, each about 6Ghz wide.
  • a fraction of the output beam from the dye laser is monitored with a Coherent beam-profiler
  • the output of the dye laser is fed through an electro-optical amplitude stabilization system (Canop- tics LASS-II Noise-Eater, NE).
  • This system had to be modified for broadband operation because it uses a wavelength-depended bias voltage.
  • the circuit determining this voltage was very slow and caused large fluctuations when the wavelength was changed.
  • the modification consisted of supplying an external bias voltage that is controlled from the computer that also controls the rest of the HFT system and which collects the image data (a high end white-box PC running Linux).
  • the beam is then directed though a NEOS Te02 AOM (AOM), which is mounted in a machined aluminum box which also includes the RF-power amplifier and a frequency doubler. Hermetic sealing is necessary because the AOM requires about 1 W of RF-power.
  • the signal detector is essentially a very sensitive radio-receiver that operates on the same frequency. Therefore even minute leakage from the AOM driver could cause interference.
  • AOM is a variable attenuator (AT).
  • the first order beam is directed to the beam expander BE1, which also includes a 6 ⁇ ⁇ pinhole that acts as a spatial filter for the laser beam.
  • the second order beam is directed at a beam position sensor (BPS) for alignment purposes.
  • BPS beam position sensor
  • the wavelength dependent AOM deflection angle would cause miss-alignment. This is compensated by changing the AOM operating frequency accordingly.
  • a dispersion compensator was computed which would allow constant intermediate frequency (IF) operation over the whole wavelength range at the expense of two custom prisms, but electronically changing the AOM frequency (about 67 to 73 Mhz) is the simpler and cheaper solution to this problem.
  • the zero-order beam is reflected off two mirrors on a movable stage (DA) to the beam expander BE1.
  • the delay adjustment stage is used to equalize the optical path-lengths.
  • the beam outputs from the beam expanders are shaped with two apertures and combined with a beam splitter cube on an electronically adjustable stage (BSS).
  • BSS electronically adjustable stage
  • the output towards the bottom of the picture is directed to the reference detector (RD) , which is a Hamamatsu photomultiplier with a ⁇ pinhole and a neutral density filter.
  • RD reference detector
  • TL 800mm telescope lens
  • C CCD camera
  • the objective is mounted horizontally in a Physics-Instruments piezo-stage that allows rapid Z-scans over a ⁇ range with 0.7nm resolution.
  • the fluorescence is detected with a Hamamatsu H6780-01 pho- tomultiplier rube (PMT), which is protected from the excitation light with an interference filter (Semrock LPD01-633RU-25).
  • the signal from the PMT is amplified with a custom, low-noise amplifier (LNA) and send to a Stanford Research Systems SR844 RF-lock-in amplifier.
  • LNA low-noise amplifier
  • the sample is mounted on a Newport XYZ stage that uses 3 Physics Instruments M222-20 motorized micrometers.
  • This stage has a resolution of about 0.1 ⁇ , which is sufficient for the proof of concept.
  • this prototype system has surprisingly high resolution.
  • the stage resolution is currently insufficient for this experiment.
  • the left picture in Figure 6 shows the sample holder, PMT and BSS.
  • the central obstruction to form the ring zone aperture stems from an Avery counting dot an a microscope slide, which limits the choice of inner beam diameters and is one aspect of this set-up in need of refinement.
  • the point spread function was measured by imaging 0.2 ⁇ micro-spheres (Invitrogen TetraSpeck T14792) with a voxel size of and averaging over 22 isolated spheres that were manually selected.
  • the right image of Figure 7 is the PSF obtained by recording the overall PMT current at a wavelength of 587.3nm. It is essentially equivalent to the PSF of an ordinary light microscope. Given a numerical aperture of 0.65, the expected Rayleigh resolution is 551nm in the XY plane at the focal point. The resolution measured from the average PMT current is only about 2 ⁇ . This degradation by a factor of 4 is due to the fact that it is averaged over all possible phase shifts.
  • Integrating the PSF over cylindrical shells around the focal point should yield a 1/r decrease of the signal per voxel due fact that the volume of a cylindrical shell with constant thickness increases proportional to the radius,
  • the magnitude of the HFT signal in the current configuration decreases more rapidly and is proportional to r ⁇ LS .
  • the right image in Figure 7 is the phase of the HFT signal, which shows the expected (See Figure 5) gradient along the Z-axis.
  • the total phase change of the usable portion of the PSF is about 2.5rad. This is in line with the simulation, but it is desirable to increase this range. For example, deliberately defocusing the two beam expanders in opposite directions will have the effect of moving the two virtual emission points apart along the Z-axis while maintaining rotational symmetry.
  • the most direct image reconstruction algorithm decomposes the problem into two steps.
  • a wavelength sweep is performed for each sample point, which yields the discrete Fourier transform (DFT) of the fluorophore density distribution over the set of hyperbolic shells that are defined by the illumination geometry. Therefore the first step is simply to apply the inverse DFT, to get the density distribution in the spacial domain.
  • the second step is then to apply the inverse Radon transform to the collection of all sample points to get a 3D image of the sample volume. This is the outline for reconstructing images where the two illumination points are relatively far apart, which is the case for the HFT configurations proposed below.
  • a quad-processor PC with 32Gbyte of memory running Linux (FC-7) is capable of reconstructing a 300 by 300 by 300 voxel image in about 30minutes.
  • the computational requirements are no real disadvantage for HFT because of the rapid advance of PC hardware.
  • NVIDIA [10] that their next generation graphics processing unit (GPU) will support 64bit floating point arithmetic will make it likely that HFT image reconstruction can be erformed in real time using the GPU.
  • FIG 8 is an example of a raw HFT image.
  • Each panel is a 300x300 pixel slice of an 100x100 micrometer area.
  • the top row is a C-scan (X Y-plane) of structures about ⁇ below the surface of a plant leaf taken from our office decoration. Leaves make nice test samples because they have plenty of natural auto- fluorescence and because their cuticle and upper epidermis form a diffuse scattering layer which normally impedes microscopy.
  • the test samples were unmodified leaves embedded in water and covered with a normal 0.17mm cover slip, Because the image acquisition time is rather long due to the slow stage mechanism, bleaching is severe, which can be seen in the image formed by the average PMT current (left panel).
  • the scan proceeds from the top to the bottom and the first row is much brighter than the rest.
  • the lower image set is an A-scan (X/Z plane).
  • About 2/3 from the top is a bright line, which was caused by a glitch in the laser amplitude stabilization system that briefly increased the laser power.
  • This is interesting because the impact was much less notable in the HFT magnitude image (center column) and had practically no effect on the phase image (right column).
  • Another interesting aspect of this image is that it was obtained with slightly missaligned beams. This means that the focal points of the wave entering the objective through the center circle and the focal point for the ring wave did not line up on the optical axis. This case had been simulated and produced the expected asymmetry of the HFT PSF. But it also shows the increased spatial resolution in the corresponding direction in the X/Y plane. This effect was discussed in section 2.1.
  • Figure 9 shows the same sample imaged with a Leica scanning confocal microscope. However it was not
  • HFT has much higher spatial resolution at the depth of 70 ⁇ and beyond. However the true
  • the image reconstruction software is fully operational.
  • Figure 10 is one slice of a more recent 300 3 oxel scan
  • the image acquisition via stage scanning is too slow and must be replaced with a faster scanner , Also, this scanner should have higher resolution so that possible improvements in the optical resolution beyond the Rayleigh criterion could be demonstrated unambiguously.
  • the free-space optics should be replaced with a fiber-optic implementation.
  • One of the stability issues stems from the fact that the longitudinal modes of the dye-laser also cause some spatial divergence, so that the mode mixture in the two beam-expanders can differ unpredictably. Coupling the laser into a single mode fiber before the AOM would clean up the beam.
  • the laser should be replaced with a ring-laser that has no longitudinal modes. There are several other desirable refinements of the laser, including faster wavelength switching times and temperature stabilization of the etalon.
  • the zone geometry should be controllable to optimize the HFT signal. It also should allow a dark zone between the zones and other patterns besides concentric rings.
  • the detector sensitivity, linearity and dynamic range should be improved.
  • the current PMT is not optimal for operation in the red and near IR region.
  • the goal of this proposal is to build a practical and versatile HFT scanner that will be used and evaluated in ongoing bio-medical research at the Cedars-Sinai medical center.
  • This device will be based on the experience gained from the proof-of concept HFT system described above.
  • the current system is large, slow and requires a great deal of care to be used.
  • the proposed system will be much smaller and can be deployed in a bio-medical laboratory to solve real problems. For example, with a scan depth of over 1 mm it will be possible to track cells in small organisms and embryos.
  • the primary motivation for this work is the development of molecular imaging technology that is compatible with minimally invasive procedures. In particular to enable in-vivo, real-time, optical pathology.
  • the proposed system will consist of two parts: 1. the laser and AOM subsystem and 2. the scan head.
  • the AOM subsystem will be connected to a laser with a single mode, polarization maintaining, wide-band fiber (photonic crystal). Besides allowing easy switching of lasers, this use of fiber optics also mitigates the beam-pointing and mode-structure problems.
  • the AOM subsystem will take care of amplitude stabilization, programmable attenuation, blanking, optical path-length equalization and frequency shifting,
  • the optical output of the AOM subsystem is connected to a pair of matched fibers, which deliver the laser light to the scan-head,
  • the scan-head is a compact unit that can be attached to various stages and imaging optics. This ranges from dedicated objective lenses to the use of existing optical platforms, in particular surgical microscopes, stereo microscopes and the telecentric optics of a locally developed black-box for live animal investigations.
  • the scan head includes the two beam expanders, the apertures, the beam combiner (aperture wheel), the detector and dichroic mirror, and the reference detector.
  • the beam-splitter cube that was used in the proof of concept system is sub-optimal, because it adds two planar optical surfaces into the beam path. Even with a good anti-reflective coating, it caused extra interference and ghosting.
  • the new HFT system will replace this optical element with an aperture wheel which intersects the beam path diagonally. This wheel also has 26 patterned mirror zones that form the aperture and that will replace the Avery dot.
  • the galvo scanner replaces the stage scanning mechanism.
  • the beam- rotating prism serves two functions. The first is to allow the use of apertures like the second and third of Figure 5. The second purpose is to allow rotation of the plane of polarization. It is also planned to use a detector pair that can sense the polarization of the fluorescence. By allowing the rotation of the excitation polarization, it is possible to sense the organization of certain biological structures, for example muscle fibers,
  • HFT depends on a CW laser that can rapidly switch wavelengths and that has a long coherence lengths .
  • Some swept frequency lasers have been developed for OCT, for example from Thorlabs. But these lasers operate too far in the IR region to excite fluorescence and have too low coherence length for HFT.
  • Most commercially available lasers that are tunable over a wide range and that have long coherence length rely on mechanically actuated wavelength selectors, that are intrinsically too slow for HFT.
  • KD*P Pockels cell was used to rapidly Dye Jet , ⁇ oc tune a SP375B dye laser.
  • KD*P is a bad material for this purpose because it will be
  • Figure 11 EO Tunable Laser that overcome this problem.
  • Figure 11 shows this configuration.
  • the custom crystals have been polished so that the entrance and exit faces admit the laser beam at Brewster's angle to minimize cavity loss and to enhance the selectivity of the Lyot filter.
  • the cavity was initially simulated with the
  • PMTs photomultiplier tubes
  • Solid state detectors have vastly more dynamic range, which is important for HFT because the detector sees a lot of fluorescence that stems from scattered light that does not contribute to the HFT image.
  • Photodiodes also have higher quantum efficiency and nicely cover the near IR range. However they have no built-in gain and require amplification, which generates far more noise than the electron multiplication process in an PMT.
  • FIG 13 shows the schematic and implementation of a 40MHz OPA.
  • the key point is that the junction capacitance of a photo-diode depends on the reverse bias voltage. This means that the photo-diode can be used to periodically alter the resonant frequency of an LC network, which can act as a negative resistance that offsets the losses of the output resonator.
  • the main property of an OPA is that it uses only reactances, which do not generate noise. This circuit was evaluated and compared to the performance of a PMT. It achieved a gain of over 60db. The gain of an OPA is bandwidth dependent, higher gain comes at the expense of reduced bandwidth.
  • an OPA provides about 25db of gain, which is more than sufficient because the noise figure of an RF amplifier is mostly determined by the first amplification stage.
  • the circuit in Figure 13 outperforms a PMT by nearly a factor of 10 in signal to noise ratio. It is also vastly superior with respect to sensitivity in the IR, linearity, and lack of short-term gain fluctuations.
  • This device uses a high resolution optical
  • this galvo senses absolute position with practically no
  • HFT is a Objective more general 3D sensing methodology that can be adapted to
  • Figure 14 Galvo Configurations non-traditional applications.
  • Figure 15 shows the proximal end
  • the two light sources of the HFT system illuminate a pair of fibers of an image-preserving fiber bundle.
  • Such imaging fiber bundles typically have 15000 to 30000 individual fibers and an outer diameter of 1 mm or less.
  • the fiber bundle must consist of single mode fibers. While these are available, bundles of polarization maintaining, single mode fibers are not. Thus one of the branches of this system needs a polarization controller or scrambler.
  • this HFT micro-endoscope is shown in figure 16. It consists simply of a grin-lens that is fused to the fiber. Unlike ordinary endoscopes where the objective lens is supposed to form an image on the face of the fiber bundle, this system does not form an image, rather each fiber is intended to form a certain wavefront just ahead of the tip. Sim ulations have shown that it is quite easy to configure the tip so that a cylindrical volume Figure 15: Endoscopic HFT Scanner of about 1 mm in diameter and 1 to 2 mm in length is illuminated. Image acquisition is accomplished by cycling through a large set of fiber pairs and performing one wavelength sweep for each pair. The fluorescent light from the sample volume is collected through all fibers simultaneously to maximize sensitivity. The 3D image is then computed using the inverse DFT followed by inverse Radon transform approach.
  • the HFT endoscope can sense a complete 3D volume. It is also expected that this system
  • HFT Endoscope Tip end of an HFT endoscope is mucn simpler that that of other endoscopes, so t3 ⁇ 4s it could be smaller, like the tip of a needle. Because it consists purely of glass, it is also easier to steril i ze.
  • the proof of concept system has shown the feasibility of HFT.
  • HFT we are refining HFT to a practical instrument and to show its utility in real bio-medical applications.
  • the first device to be developed is a versatile and compact HFT scan-head that can be attached to existing microscopes. It will also be used in conjunction with an objective lens as a stand-alone system.
  • the overarching aim of this group is to develop new technology for bio-medical applications.
  • One demonstration application for HFT is noninvasive, real-time pathology during surgical procedures, which require molecular specificity, deep optical penetration, and high resolution. This application also motivates the proposed endoscopic implementation of HFT.
  • Mauna Kea's CellVizio system has shown the utility, including clinical, of micro-endoscopy.
  • HFT is a new imaging modality that extends the capabilities of confocal microscopy to penetration depths comparable to OCT. Increasing the ability to image molecules via fluorescence about 10 times deeper into biological tissues is clearly a significant advance of the state-of-the-art in molecular imaging. The viability of the principle behind HFT has been demonstrated. We seek to develop HFT to the point where its capabilities can be demonstrated in real, bio-medical research projects, and eventually clinical settings. HFT stands at the beginning of its development cycle and it is very likely that during the course of this research a number of significant refinements will be discovered. For example it was surprising to find evidence that HFT can potentially improve optical resolution beyond the ayleigh criterion. This observation begs to be verified and - if confirmed - exploited.
  • HFT human immunodeficiency tom-vtvo tracking of stem cells.
  • a successfully developed HFT system also has direct medical applications. Cancer margin determination during surgery is one such example.
  • the request of a neurosurgeon who treats brain-tumors in infants was one motivation for allocating precious research resources for the construction of the proof of concept HFT system, which has received no external funding to date.
  • the concrete problem in this case is to determine the structure of the brain tissue in front of a micro-endoscope, which is opaque to conventional optics.
  • NVIDIA Nvidia glOO: Teraflops visual computing. In Hot Chips Conference, Stanford, 2008.
  • the present disclosure relates to optical amplification, and particularly relates to a photo-parametric amplifier that uses the properties of solid state detectors for very low noise amplification of weak, pulsed, high frequency optical signals.
  • the internal gain is the significant advantage for PMTs over solid state photo detectors which have no built-in gain mechanism and which have to rely on external electronics to amplify the photo current to usable levels.
  • PMT optical amplifiers outperform those based on photo diodes (solid state photo detectors) in terms of overall sensitivity and signal to noise ratio, even though photodiodes are actually much better at sensing light.
  • the quantum efficiency i.e., the probability for one photon to generate one electron
  • a photodiode often exceeds 80% while it is rare that a PMT has a quantum efficiency that approaches 30%.
  • Photo diodes are extremely linear devices, e.g., the current output is strictly proportional to the light input for over 12 decades while a PMT barely maintains linearity over 3 decades.
  • Photodiodes are very rugged devices that are not harmed by exposure to high light levels, while PMTs are fragile and easily destroyed by exposure to room light levels while powered on.
  • Photodiodes are available that operate well with IR light, while PMTs can barely detect light in the near IR spectrum. Photons with wavelength in excess of 1 micrometer do not have enough energy to free an electron, thus there are no practical PMTs that can cover the 1 - 2 micrometer wavelength range, while solid state detectors exist that can operate up to 10 micrometer wavelengths.
  • PMTs are large, fragile, expensive and require high voltages to operate while solid state detectors are small, rugged and relatively inexpensive.
  • the photo diodes outperform PMTs in most aspects other than built-in amplification by a wide margin.
  • FIG. 1A illustrates an optical transfer function (OTF) provided by a plane mirror standing wave microscope.
  • FIG. IB illustrates a point spread function (PSF) provided by the microscope of FIG. 1A.
  • PSF point spread function
  • FIG. 2 illustrates a conceptual omnidirectional standing wave microscope in accordance with an aspect of the disclosure.
  • FIG. 3 illustrates a conceptual processor system configured with the omnidirectional standing wave microscope of FIG. 2, in accordance with an aspect of the disclosure.
  • FIG. 4 illustrates a conceptual apparatus for super-resolution optical microscopy in accordance with an aspect of the disclosure.
  • FIGS. 5A-5F illustrate the 3-D resolution enhancement that may be obtained in operation of a a OSW microscope equipped with an RDOE for super-resolution optical microscopy in accordance with an aspect of the disclosure.
  • FIG. 6 is a flow diagram describing a method for obtaining an image using super- resolution optical microscopy in accordance with an aspect of the disclosure.
  • a structure and method is disclosed using photo-parametric amplification (PPA) to detect light in biomedical applications that use pulsed lasers for excitation.
  • PPA photo-parametric amplification
  • Figure 1 is a conceptual representation of a system 100 for PPA sensing and/or imaging.
  • Light 107 originates from a pulsed laser 104 and is directed mostly to an optical system 106.
  • a small f action of the laser output 107 is diverted to a reference detector 108 that is used to synchronize a photo -parametric amplifier 110 to the laser 104.
  • the reference signal is amplified P T/US2014/052589
  • the phase locked loop (PLL) circuit 112 includes a variable delay mechanism 116 to adjust the phase of a pump signal 117 with respect to the laser output 107.
  • the PLL circuit 112 includes a phase frequency detector (PFD) 118, a low pass filter (LPF) 120, the VCO 114 and a frequency divider (f/2) 122. Typically, this functionality may be integrated into one commercially available integrated circuit.
  • the laser light 107 may be passed through various optical filters (excitation filter 130 and/or emission filter 132) and is directed at a specimen 133 under investigation.
  • scanned point sensing may provide an image.
  • the collected light from the specimen 133 under investigation is directed to the photo-parametric amplifier 110, where it may first pass through the emission filter 132, where the emission filter may include one or more spectrometers (not shown).
  • the sensed light is not the light that was used to illuminate the specimen 133. Rather, processes such as fluorescence, secondharmonic generation, and Raman scattering may used to measure molecular properties of the specimen 133.
  • the most common of these methods is the use of fluorescence from certain dye molecules that are attached to antibodies which in turn selectively bind to specific proteins, e.g., at specific sites.
  • the fluorescence light reports the location of the protein of interest.
  • the periodic modulation of the excitation signal is preserved by the imaging process and is present in the emission signal. This is the case for the non-linear processes such as second-harmonic generation and Raman scattering.
  • the fluorescent lifetime of most organic dyes is in the sub- 10 nano-second range, which means that the fluorescent signal from these dyes also retains most of the time varying structure of the laser illumination source 104.
  • a light detector 205 is a C30971BFC fast silicon photodiode from Perkin Elmer, which is integrated into a fiberoptic FC-type connector. It should be noted that this is a typical silicon photodiode and not a varactor diode specifically designed for microwave applications. Hence the diode is optimized for light sensitivity and not for its controllable junction capacitance.
  • the photodiode 205 may be optimized for speed, which means that the internal series resistance is low, which in turn reduces its Johnson noise contribution. However, when the photodiode 205 is operated in the photo- conduction mode, that is with a reverse bias applied, its shot-noise dominates.
  • An inductor LI and a capacitor CI form a high-Q resonator 210 circuit that resonates at 80 Mhz, and which is excited from the pump input 117 via a capacitor C3.
  • One or more capacitors that make up C5 provide DC isolation of the pump-resonator 210 while blocking an RF signal path to ground.
  • a connection B may be used to apply a reverse bias voltage to the photodiode 205 and also to measure the sum of a light induced DC photo-current and a dark current.
  • the cathode of the photodiode is connected an inductor L2 and a capacitor C2, which form a LC-resonator 220 tuned to 40 Mhz.
  • a case 230 of the photodiode 205 may preferably be grounded.
  • the case 230 may be attached to RF-shielding (not shown) of the circuit.
  • a signal from the resonator 220 may be coupled to the output of the PPA 110 via a capacitor C4.
  • the entire PPA 110 may preferably be enclosed in an soldered RF-shield.
  • the two tank circuits 210, 220 may preferably be located in separate shielded chambers and have very low inductive coupling. [0024] Controlling gain is important for biomedical applications, and because of the high sensitivity of the gain to the pump power level, it is preferable that the PPA 110 include facilities to control and stabilize the power of the input pump 117.
  • the output of the VCO 114 is fed to a variable gain amplifier (VGA) 124 which in turn drives the PPA 110.
  • VGA variable gain amplifier
  • a fraction of the pump input 1 17 power is diverted to a stable sensor to measure the power delivered to the PPA 110. This value is compared to gain setting and the difference is used to adjust the gain of the VGA by a gain control 140.
  • This is a form of open loop stabilization that relies on measuring the relation between PPA gain and pump power and that assumes that this calibration remains stable.
  • closed loop stabilization may be preferable because of the high sensitivity of the PPA gain to pump power and bias voltage.
  • the reference light source can be a stabilized light emitting diode.
  • the reference light source adds a veiy small signal at a frequency that is just outside of the operating bandwidth. This signal is subsequently detected at the output of the amplifier chain via synchronous demodulation and provides a direct measure of the total gain of the entire amplifier chain. While the extra light will add some noise to the system, it also provides a direct, stable end-to-end measure and permits long-term, reliable calibration of the instrument; (c) Add an electronic pilot signal. This method is identical to the method described above, except that it uses a loosely coupled probe to inject the pilot signal electronically into the resonant circuit of the PPA 110.
  • the gain of the PPA 110 also depends on the reverse bias of the photo diode 205. This bias voltage is essentially a mechanism to fine tune the PPA 110. The sensitivity of the PPA 110 on the bias voltage is modest; hence a digital to analog converter that produces 0-10V with 8 to 12 bits of resolution may be adequate. The PPA 110 performance can be optimized by changing the bias voltage depending on the pump-power level.
  • the overall sensitivity of the PPA 110 is limited by the dark current of the photodiode 205 and its associated shot noise. In order to achieve the full potential of this light detection system, it may be preferable to cool the photo diode 205. Because the photodiode 205 generates no significant amount of heat, a conventional thermoelectric cooler may suffice.
  • the gain of a degenerate parametric amplifier, such as the PPA 110 described in this disclosure is phase sensitive, which means that it may be necessary to adjust the phase of the pump oscillator with respect to the excitation pulses.
  • Controlling the phase of a phase locked loop, such as the one described above is easily done using any of a plurality of standard solutions for this type of problem, including electronic phase shifters, variable delay element or direct digital synthesis (DDS) in the PLL loop.
  • DDS direct digital synthesis
  • an AD9540 integrated circuit from Analog Devices may be considered a suitable element for this purpose.
  • phase sensitive is beneficial for this application because out-of phase noise is attenuated, which effectively halves the noise contribution of this detection system (110).
  • varying the phase of the detection system deliberately can be used as a sensing mechanism to measure the fluorescent lifetime of a molecule that is being sensed. The lifetime information can be used to discriminate between several signals with otherwise similar optical properties.
  • a solid state detection system comprising:
  • PPA photo-parametric amplifier
  • an automatic gain control system configured to adjust a level of power of the pump waveform based on measuring a photo-current and an intensity of shot noise of the photo diode within a narrow bandwidth of the PPA.
  • an automatic gain control system configured to adjust a level of power of the pump waveform based on measuring the signal from a small optical pilot signal directed at the photo diode, wherein the signal is sensed via a narrow-band, synchronous demodulation of the output from one of the PPA or a subsequent amplification stage.
  • the small optical pilot signal is replaced by an electronically injected pilot signal.
  • variable delay circuit coupled to the periodically pulsed light source
  • PFD phase frequency detector
  • a low pass filter coupled to the PFD
  • VCO voltage controlled frequency oscillator
  • a frequency divider coupled to the VCO and the PFD
  • LA7480192.1 a variable gain amplifier (VGA) coupled to the VCO and the frequency divider.
  • VGA variable gain amplifier
  • RF radio f equency
  • a method for generating arbitrary centers of beam rotation in free space that permit the coupling of an optical system of arbitrary characteristics to a laser-detector assembly, or that adapt a pair of arbitrary optical systems to one another through software selectable modifications alone.
  • This approach is of special interest for high-resolution retinal scanner for the early, low cost detection of Alzheimer's disease.
  • Other applications of interest include coupling laboratory optical systems in situation where flexibility is of great importance and a time delay of some minutes in image transfer is acceptable.
  • Mirror positions a 3 and a 4 of the last two mirrors in the QG sequence are completely determined functions of the first two mirror positions. These dependencies permit the control system to be relatively simple.
  • Mirror angles and 2 are a function of R0 beam angles ⁇ 0 and ⁇ 0 .
  • Mirror angles a 3 and ct 4 are calculated from ⁇ - ⁇ and a 2 . Once an initial position is fixed, the relative locations of the mirrors are perturbations from this local datum. The result is very direct.
  • the following pages are of code from a program in MathCAD14 that solves for the independent mirror positions a ? and a 2 , from inputs defining the source ray R c .
  • the two inputs ⁇ 0 and y 0 are shown in amber at the top, as are the four outputs show in amber at the bottom. Active portions of the code are shown in blue.
  • the remaining dependent mirror positions a 3 and a 4 are calculated from the first two mirror positions, t and 2 .
  • mirror M3 is constrained to lie on a straight line, on the face of the mirror, which is also
  • Pr3z L1.
  • the third coordinate varies with the angular positions of M1 and 2.
  • Equation for Pr3y completes the set of constraints needed to define all four mirror angles.
  • the preliminary design based on Nowatzyk's original concept comprises a pair of two galvos based beam steering assemblies, mounted back to back. This is the design configuration shown below. This configuration is sufficient to demonstrate the concept. And the configuration may be assembled with off-the-shelf two galvos beam steering assemblies. Because the packing density off-the-shelf arrangement has been optimized for two rather than four galvos the possibilities from such configurations are limited.
  • the following image constructed in SolidWorks11 shows a Quad Galvos arranged for retinal scanning.
  • the human eye shown in cross-section in the center lower right is to normal adult human scale with respect to the actuators.
  • Control systems for scanning mirror assemblies tend to be based on the premise that axes of mirror rotation are truly orthogonal and that the mirror surfaces are identically the axes of rotation. In practice each axis will be slightly skew from an ideal coordinate reference frame. To obtain a system of the highest accuracy incorporating the tolerances of assembly into the control system is necessary. A non-contact method of identifying the as-built axes of the assembly in the final configuration of use is intended, such that these values may be entered into a 'look-up' table for the controller. The controller can then adapt to calculate each beam rotation based on the 'true' rather than the 'nominal' axis of each galvos mirror.
  • axis correction is a 'solved problem' in the world of industrial robot controllers. This is especially true in applications for which robot arms are used in semi-static mode for pick and place tasks requiring extremes of accuracy.
  • the kinematic chain of a common robot arm and a Quad Galvos are similar in basic ways.
  • the pivot bearings of both are nominally orthogonal. Errors in the bearings nearest the ground plane have a cumulative effect on the linkage further out.
  • An approach 1 developed for KUKA robots at the Fraunhofer Deutschen IPK, Berlin is applicable for the QG case.
  • the calibration method is an iterative one.
  • the method is one of taking the partial derivatives of the equations of end-effector (in this case projected exit beam) motion with respect to axis alignment; selecting cases in the range of permissible motion where the influence of one bearing axis is greater than others; approximating the axis error from that axis; inserting a corrective transformation matrix in the equation of motion, and repeating to convergence.
  • end-effector in this case projected exit beam
  • a remote center compliance (RCC) gripper system including: an erectable RCC device including a compliance unit having a plurality of compliance members; and apparatus for applying a force to the centrally disposed joint to fix the joint and erect the RCC device.”
  • the above is a block diagram summarizing the incorporation of our scanner into the complete instrument.
  • the quad galvos is fiber fed from the broadband source. This reduces the weight and complexity of the hand held elements meaningfully.
  • a Fianum "WhiteLase Micro" compact super- continuum fiber-fed laser produces useful power over 450 nm - 1800 nm range, averaging > 2 mW / nm. Pulsed around 40 MHz. A recent price reduction in this class of components for lower power applications makes them more commercially attractive.

Abstract

L'invention concerne un système de détection à l'état solide comprenant un amplificateur photo-paramétrique (PPA) dégénéré, le PPA comprenant une photodiode, et une source lumineuse à pulsation périodique, l'amplificateur photo-paramétrique (PPA) étant synchronisé avec la source lumineuse pulsée par une boucle à verrouillage de phase qui génère une onde de forme de pompage pour le PPA à deux fois la fréquence du taux d'impulsions d'excitation.
EP14900413.7A 2014-08-26 2014-08-26 Procédé et système de tomographie de fluorescence hétérodynée Withdrawn EP3186885A4 (fr)

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