WO2016207881A1

WO2016207881A1 - Controlled optical focusing through flexible graded-index multimode fibers without distal end access

Info

Publication number: WO2016207881A1
Application number: PCT/IL2016/050653
Authority: WO
Inventors: Yaron Silberberg; Ori Katz; Doron GILBOA; Shamir ROSEN
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2015-06-23
Filing date: 2016-06-20
Publication date: 2016-12-29

Abstract

Methods, apparatus, and systems for focusing light and remote imaging through a multi-mode fiber without pre-calibration and without direct localized feedback from the distal end of the fiber. Focus control is performed by optimally shaping the wavefront input to the fiber's proximal end according to feedback information embedded in back- propagating fluorescence from the distal end, via processing that exploits the partial cylindrical symmetry of the fiber, and which optimizes according to a weighting mask. Also included is a method for efficient photo-bleaching of a fluorescently-tagged sample, which optimizes the speed and efficiency of the photo-bleaching while minimizing or avoiding damage to non-tagged areas of the sample.

Description

CONTROLLED OPTICAL FOCUSING THROUGH FLEXIBLE GRADED-INDEX MULTIMODE FIBERS WITHOUT DISTAL END ACCESS

BACKGROUND OF THE INVENTION

[001] Ultra- thin endoscopes are highly desirable for many applications involving remote imaging, and multimode fibers (MMF) potentially offer attractive ultra-thin lensless replacements for conventional endoscopes. One difficulty with imaging or focusing light through a multimode fiber, however, is phase-velocity dispersion and coupling between the fiber modes. This leads to phase randomization during propagation through the MMF and poses a major obstacle to imaging and focusing of light. An image, or even a single focused light beam launched into the MMF input facet, results in a complex speckle pattern at the fiber output.

[002] A current method for overcoming this problem is to shape the fiber's input beam wavefront in such a way that it compensates for the phase changes to effectively unscramble the complex speckle pattern, thereby allowing the fiber to be used for imaging. There are, however, shortcomings to this approach: For example, finding a correcting wavefront currently requires simultaneous access to both ends of the fiber for pre-calibration to obtain a compensation for the fiber's complex input-output mode relationships. The compensation, moreover, is highly sensitive to even slight fiber deformations and temperature variations. Such restrictions make this approach unsuitable in many applications.

[003] Thus, it would be advantageous to have a method and apparatus for determining the optimal wavefront for a fiber in situ and in-vivo, without having to access the distal end. This goal is attained by embodiments of the present invention.

SUMMARY OF THE INVENTION

[004] Embodiments of the present invention provide methods, apparatus, and systems for controlled in-situ and in-vivo focusing and scanning via ultrashort light pulses through a flexible non-precalibrated MMF, such as for an endoscope, where all the necessary instrumentation and associated optical elements are situated exclusively at the fiber's proximal end. In these embodiments, the fiber's distal end is used without the embodiments providing any additional discrete distal end optical elements, non-limiting examples of which include: lenses, mirrors, prisms, reflectors, refractors, waveguides, light sources, polarizers, waveplates, screens, optical filters, optical media, and the like; it being understood that optically-interactive features of the sample under examination or treatment by an embodiment of the invention are not parts of the embodiment itself. In related embodiments as detailed below, a 2PF coating is applied to the distal facet as a treatment of the distal facet, and is not an additional discrete optical element.

[005] In some embodiments, a beam of light input to the proximal end of the fiber is focused external to the distal end of the fiber, for uses including, but not limited to, scanning and targeting.

[006] In other embodiments, the beam of light illuminates a sample external to the distal end of the fiber in such a way as to form an image of the sample at the distal end, which propagates back to the proximal end for capture, such as by a camera.

[007] Related embodiments for application with graded-index (GRIN) fibers, provide light patterns at the proximal end for retrieving information about the distal light distribution. These properties, along with two-photon fluorescence, allow for robust focusing through GRIN fibers which undergo deformations and temperature changes during use. Certain embodiments of the invention provide methods and apparatus for lensless two-photon micro-endoscopy.

[008] Various embodiments provide nonlinear optical feedback in an epi-detection geometry, where a diffraction-limited focus is formed at the fiber distal end, whose position is deterministically controlled by exploiting wavefront correlations induced by the fiber's partial cylindrical symmetry.

[009] Therefore, according to an embodiment of the present invention, there is provided apparatus for remote imaging and focusing of light, the apparatus including: (a) a multimode fiber (MMF) having a proximal end and a distal end, for propagating light and images between the proximal end and the distal end; (b) a laser light source, for providing excitation light to the proximal end; (c) a spatial light modulator (SLM), to perform wavefront shaping of the excitation light from the laser light source to the proximal end; (d) a beam splitter, for separating light back-propagated from the distal end to the proximal end; (e) a primary light detector, for measuring an intensity of light excited at the distal end and back-propagated to the proximal end, as separated by the beam-splitter; and (f) a controller, for controlling the SLM to perform wavefront shaping according to a feedback signal from the primary light detector based on light back-propagated from the distal end to the proximal end.

[0010] In addition, according to another embodiment of the present invention, there is provided a method for optimizing remote imaging and focusing of light in an apparatus that includes: a multi-mode fiber (MMF) having a proximal end and a distal end, a spatial light modulator (SLM) at the proximal end, a primary light detector at the proximal end which measures an intensity of a back-propagated two-photon fluorescence (2PF) from the distal end; and a controller, the method including: (a) logically partitioning, by the controller, the SLM into a plurality of square partitions; (b) for at least one partition: (c) determining, by the controller, a phase for the partition that maximizes the intensity of the back-propagated 2PF; and (d) using a phase determined for the at least one partition in the SLM.

[0011] Moreover, according to a further embodiment of the present invention, there is provided a computer product for optimizing remote imaging and focusing of light in an apparatus that includes: a multi-mode fiber (MMF) having a proximal end and a distal end, a spatial light modulator (SLM) at the proximal end, and a primary light detector at the proximal end which measures an intensity of a back-propagated two-photon fluorescence (2PF) from the distal end; the computer product including a non-transitory storage containing executable instructions, which instructions, when executed by a data processing device cause the data processing device to perform: (a) logically partitioning the SLM into a plurality of square partitions; (b) for at least one partition: (c) detenriining a phase for the partition that optimizes the intensity of the back- propagated 2PF according to an optimization function; and (d) using a phase determined for the at least one partition in the SLM.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The subject matter disclosed may best be understood by reference to the following detailed description when read with the accompanying non-limiting drawings in which:

[0013] Fig. 1A illustrates an embodiment of the present invention that provides a two- photon lensless MMF-based system, such as for endoscopy.

[0014] Fig. IB is an image of the intensity pattern at the distal end of a fiber before optimization, in an embodiment of the present invention. [0015] Fig. 1C is an image of the intensity pattern at the distal end of a fiber after optimization, in an embodiment of the present invention.

[0016] Fig. ID is a graph showing the total number of proximally-detected two-photon fluorescences (2PF) during the optimization process, in an embodiment of the present invention.

[0017] Fig. 2A is an image of the intensity pattern and focus position at the distal end of a fiber after a first optimization, in an embodiment of the present invention.

[0018] Fig. 2B is an image of the intensity pattern and focus position at the distal end of a fiber after a second optimization, in an embodiment of the present invention.

[0019] Fig. 2C is an image of the intensity pattern and focus position at the distal end of a fiber after a third optimization, in an embodiment of the present invention.

[0020] Fig. 2D is an image of the intensity pattern and focus position at the distal end of a fiber after a fourth optimization, in an embodiment of the present invention.

[0021] Fig. 2E is an image of the intensity pattern at the proximal end of a fiber after the first optimization, corresponding to the distal end intensity pattern and focus position of Fig. 2A, in an embodiment of the present invention.

[0022] Fig. 2F is an image of the intensity pattern at the proximal end of a fiber after the second optimization, corresponding to the distal end intensity pattern and focus position of Fig. 2B, in an embodiment of the present invention.

[0023] Fig. 2G is an image of the intensity pattern at the proximal end of a fiber after the third optimization, corresponding to the distal end intensity pattern and focus position of Fig. 2C, in an embodiment of the present invention.

[0024] Fig. 2H is an image of the intensity pattern at the proximal end of a fiber after the fourth optimization, corresponding to the distal end intensity pattern and focus position of Fig. 2D, in an embodiment of the present invention.

[0025] Fig. 2J is a plot of the correspondence between the proximal end's fluorescence pattern angular orientation versus the distal end's focus position angle, in an embodiment of the present invention.

[0026] Fig. 2K is a plot of the correspondence between the proximal end's fluorescence pattern radial lobe separation versus the distal end's focus position radius, in an embodiment of the present invention. [0027] Fig. 3A shows an optimal SLM pattern found by a genetic algorithm to optimize a single speckle at a random location on the distal end, in an embodiment of the present invention.

[0028] Fig. 3B shows the optimal SLM pattern of Fig. 3 A rotated counter-clockwise by 12°, in another embodiment of the present invention.

[0029] Fig. 3C shows the single speckle at the distal end, as optimized by the SLM pattern of Fig. 3A, in the embodiment of the present invention.

[0030] Fig. 3D shows the single speckle at the distal end, as optimized by the SLM pattern of Fig. 3B, and rotated counter-clockwise by 9.2°, in the other embodiment of the present invention.

[0031] Fig. 3E is a plot of the angular rotation (φ) of the focus position at the distal end versus the angular rotation (Θ) of the SLM pattern, in certain embodiments of the present invention.

[0032] Fig. 3F is a plot of the peak intensity of the focus at the distal end versus the angular rotation (Θ) of the SLM pattern, in certain embodiments of the present invention.

[0033] Fig. 4A shows the weighting mask for the intensity pattern (Fig. 2F) at the proximal end used in the second optimization, in an embodiment of the present invention.

[0034] Fig. 4B shows the weighting mask for the intensity pattern (Fig. 2G) at the proximal end used in the third optimization, in an embodiment of the present invention.

[0035] Fig. 4C shows the weighting mask for the intensity pattern (Fig. 2H) at the proximal end used in the fourth optimization, in an embodiment of the present invention.

[0036] Fig. 4D shows the fluorescence pattern at the proximal end, on which the weighting mask of Fig. 4A is applied, in an embodiment of the present invention.

[0037] Fig. 4E shows the fluorescence pattern at the proximal end, on which the weighting mask of Fig. 4B is applied, in an embodiment of the present invention.

[0038] Fig. 4F shows the fluorescence pattern at the proximal end, on which the weighting mask of Fig. 4C is applied, in an embodiment of the present invention.

[0039] Fig. 4G shows the intensity distribution at the distal end, corresponding to the proximal end fluorescence pattern shown in Fig. 4A, in an embodiment of the present invention. [0040] Fig. 4H shows the intensity distribution at the distal end, corresponding to the proximal end fluorescence pattern shown in Fig. 4B, in an embodiment of the present invention.

[0041] Fig. 4J shows the intensity distribution at the distal end, corresponding to the proximal end fluorescence pattern shown in Fig. 4C, in an embodiment of the present invention.

[0042] Fig. 5 is a flowchart for a method of optimization according to an embodiment of the present invention.

[0043] Fig. 6 is a flowchart for a method of selectively photo-bleaching a fluorescently- tagged target according to an embodiment of the present invention.

[0044] For the following figures: Fig. IB, 1C, Fig. 2A - Fig. 2H; Fig. 3C, Fig. 3D; the excitation field is at laser 121 wavelength.

[0045] For the following figures: Fig. 4D - Fig. 4J; two shortpass filters (670 nm, 600 nm) are used filter out the laser wavelength and image the 2PF.

[0046] For the following figures: Fig. IB, Fig. 1C; Fig. 2A - Fig. 2H; Fig. 3C, Fig. 3D; Fig. 4A - Fig. 4C, and Fig. 4D - Fig. 4J; the figures are rendered in pseudo-grayscale to indicate spatial forms and distributions for focused light, fluorescence, and weighting masks relative to a fiber cross-section. Where applicable, cross-section boundaries and other points of note are indicated as described in the text.

[0047] For simplicity and clarity of illustration, elements shown in the figures are not necessarily drawn to scale, and the dimensions of some elements may be exaggerated relative to other elements. In addition, reference numerals may be repeated to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0048] Embodiments of the present invention utilize wavefront shaping for focusing and imaging through random media. The main approaches for determining an optimal correcting wavefront include measuring the transmission matrix, phase conjugation, and iterative adaptive algorithms. As previously noted, these approaches currently require simultaneous access to both ends of the fiber during a calibration procedure, which must be repeated after any movement or bending of the fiber, albeit slight, as well as after a change in temperature. [0049] According to various embodiments of the invention, focusing light through a flexible fiber in an endoscopic fashion (i.e., from the fiber's proximal end to the fiber's distal end without having to access the distal end) is accomplished via a non-linear excitation response at the distal end that results in a back-propagated light detected at the proximal end. When the excitation response is suitably non-linear, a focus condition at the distal end is detected at the proximal end by an increase in the intensity of the back-propagated light for a constant excitation intensity. In certain embodiments, this back-propagated light is indicative of the distal peak intensity and focus position. Non-linear excitation response is provided by a number of different mechanisms (e.g., Raman scattering). According to a related embodiment, an «-Photon Fluorescence (nPF) provides the non-linear response. In another related embodiment, the nPF is a spatially-distributed two-photon fluorescence (2PF). In certain embodiments, this is provided by fluorescent tagging of specimen samples; in other embodiments, this is provided by applying a fluorescent treatment to the distal facet. In further embodiments, partial cylindrical symmetry of the MMF is exploited to obtain additional information about the focus condition at the distal end.

[0050] Fig. 1A illustrates an embodiment of the present invention that provides a two- photon lensless MMF-based system 100, non-limiting applications of which include endoscopy, wherein an ultrashort pulse is delivered to a target 101 through a fiber 103.

[0051] In related embodiments, target 101 is a fiuorescently-tagged sample, specimen, or screen, non-limiting examples of which include tissue or other material under examination and/or treatment.

[0052] The term "lensless" herein refers to the ability of fiber 103 to image objects at a distal end (or facet) 105 and faithfully transmit the images along the length of fiber 103 utilizing only wavefront shaping without lenses or other focusing elements at distal end 105 or within the image guide, i.e., without any additional discrete optical elements at distal end 105, as previously discussed. In various embodiments, however, external to fiber 103 at a proximal end (or facet) 107 there are additional optical elements, such as lenses, mirrors, modulators, etc., to condition the light input to and/or manage the received images output from fiber 103. In further related embodiments, as disclosed below, a 2PF coating is provided as a treatment of fiber distal end 105. [0053] Light (such as 2PF) excited at distal output end 105 by the spatially- and temporally- distorted pulse is collected and back-propagated by fiber 103, and detected at proximal input end 107 by a primary light detector 109. In an embodiment of the invention, primary light detector 109 has a scalar value output, and the proximally-detected back-propagated fluorescence (light) value is then used as feedback signal 113 to be maximized by a wavefront shaping optimization algorithm that controls a phase-only spatial light modulator (SLM) 111 for input to proximal fiber end 107.

[0054] In another embodiment, primary light detector 109 is a primary camera 109, a non- limiting example of which includes an Electron Multiplying Charge Coupled Device (EMCCD), which, in addition to measuring intensity of the back-propagated light (e.g., back-propagated fluorescence) is also capable of forming an image thereof, to obtain more information about the back-propagated light. Primary camera 109 images the Fourier plane of proximal fiber end 107.

[0055] In various embodiments SLM 111 is a reflective device. In other embodiments, SLM 111 is a transmissive device. References herein to SLM 111 pertain to the active optical area thereof. In a related embodiment, a controller 115 provides a transformation from feedback signal 113 output by primary camera 109 into appropriate signals and data for controlling SLM 111. In another related embodiment, controller 115 executes optimization algorithms according to various embodiments of the present invention. Controller 115 receives data from primary camera 109 and provides control for SLM 111. In a further related embodiment, controller 115 accesses mask data 127 for optimization. In certain embodiments, controller 115 is a programmable device, such as a data processor or computer.

[0056] The most straightforward approach to optimizing the input wavefront is to maximize the total back-propagated nonlinear fluorescent response (light), which, for an Nphoton process, scales with the Nth power of the exciting field intensity. For thin fluorescent target 101, the total detected nonlinear fluorescence power 7_det varies according to:

(1) where I^_ut (x, y, t) is the instantaneous optical intensity at distal end 105, at each spatial position (x,y) and time (t), and a(x,y) is the fiuorophore absorption distribution. For a linear optical process (Ν = 1) and homogeneous fluorescent target 101, the integrated intensity is simply the total launched energy, and does not contain information about the output field spatial distribution. However, by optimizing the total intensity from a high order nonlinear process (N > 1), such as 2PF, a focused beam enhancing a single speckle grain intensity would be formed and the pulse would be focused to a diffraction limited spot (Fig. IB and Fig. 1C), where the fiber boundary is shown as a dashed line 131 in Fig. IB. Most importantly, the position of the formed focus can be deterministically established, controlled, and scanned utilizing information embedded in the spatial distribution of the back-propagated 2PF at proximal end 107 (Fig. 2A-2K - Fig. 3A-3F).

[0057] Pulse focusing via proximally detected 2PF according to the embodiment illustrated in Fig. 1A is as follows (the specific parameter values presented are for purposes of illustration and are non-limiting):

[0058] Pulses of 810 nm light lasting approximately 100 fs from a laser 121 attenuated to 15 mW are expanded to cover a circle bounded by the dimensions of SLM 111. The output waveform shaped by SLM 111 is coupled to graded-index MMF 103 having a core diameter of 50 μπι, a numerical aperture of (NA) of 0.2, and approximately 200 spatial modes at each polarization, via optics equivalent to a lOx microscope objective with NA 0.25. In a related embodiment target 101 is a fluorescent screen made of a 50 μπι thick capillary filled with disodium fluorescein in ethanol, which is positioned immediately against fiber distal end facet 105. The pulses excite 2PF from target 101, and the 2PF is then collected at distal end 105 and back-propagated by fiber 103 to proximal end 107. The back-propagated fluorescence is separated from the excitation light at proximal end 107 by a beam-splitter 117, a non- limiting example of which is a dichroic mirror (DM). Then the Fourier- transformed image at proximal facet 107 is captured by primary camera 109.

[0059] For a flat input wavefront a speckled intensity pattern is present at fiber distal end 105 (Fig.lB). However, after optimizing SLM 111 output wavefront phase pattern to maximize the total 2PF using a genetic algorithm (GA), the pulses are tightly focused at fiber distal end 105 (Fig.lC).

[0060] For purposes of verifying the internal operation of the above embodiments, in a related embodiment a secondary camera 119 is used for inspecting the resulting intensity pattern at distal end 105. A non-limiting example of secondary camera 119 includes a charge-coupled device (CCD). In this embodiment, secondary camera 119 is used only for test and verification operation by capturing images of the intensity pattern at distal end 105, to confirm proper operation of the apparatus and the optimization thereof. For example, secondary camera 119 captures images of the intensity pattern at distal end 105 before an optimization (Fig. IB) and after an optimization (Fig. 1C), thereby verifying that embodiments of the invention are properly focusing to a single speckle grain. In Fig. IB and Fig. 1C the dashed circle indicates the borders of fiber distal output facet 105. Figs. 2A, 2B, 3C, and 2D; Figs. 3C and 3D; and Figs. 4G, 4H, and 4J also include verification images captured by secondary camera 119, as detailed below. It is emphasized that in embodiments of the invention intended for in situ and in-vivo use (i.e., for actual practical uses, such as endoscopy), secondary camera 119 is not used.

[0061] According to a non-limiting embodiment of the invention: laser 121 is equivalent to a Spectra-Physics "Tsunami" laser, with a repetition rate of 80 MHz; SLM 111 is equivalent to a Hamamatsu LCOS-SLM X10468-02; MMF 103 is equivalent to a Thorlabs GIF50C; target 101 is imaged via an optical device equivalent to a microscope objective (X10/0.25 NA); primary camera 109 is equivalent to an Andor iXon3; secondary camera 119 is equivalent to an iDS uEye LE; and beam-splitter 117 is equivalent to a Semrock FF720-SDi01 with two bandpass filters equivalent to a Chroma D525-250.

[0062] The above-described focusing process is robust and reproducible, yielding intensity enhancements of η « 50 relative to the initial background (Fig. 1C), but the position of the formed focus with this simple approach is not predetermined; focus position depends on the initial speckle pattern and optimization algorithm. However, additional embodiments of the invention, as disclosed below, provide for scanning and controlling the focus position, based on the nature of light propagation and the circular cross-section of MMF 103.

[0063] In step-index fibers the back-propagating fluorescence is practically evenly distributed over the entire core. In GRIN fibers the transmitted fluorescence carries focusing and imaging information in the patterns. For a point source input, the self-imaging properties of the GRIN fiber create, in a periodic fashion, a series of images and mirrored- images as it propagates along the fiber. While the quality of these images deteriorates with length, they are localized enough to retrieve the input point location even after propagation. The periodicity of the imaging depends on the point source location. There are two symmetric distal focus locations that correspond to nearly identical proximal fluorescence patterns. An embodiment of the invention provides unambiguous retrieval of the focus position by using only half the fiber facet, such as by creating a permanent obstruction on half of the fiber output facet.

[0064] In certain embodiments of the invention, rotations of an optimized SLM pattern around the fiber axis directly controls, to an extent, the azimuthal position of the focus. Fig. 3A-3F shows that rotation of the optimized SLM pattern of over approximately 10 degrees is possible before an increasing loss of wavefront correlation reduces the focus intensity. According to these embodiments, only a few positions of the focus require optimization, after which it is possible to scan between them.

[0065] For in situ and in-vivo applications, it is important to keep certain limitations in mind, particularly wavefront shaping speed. A liquid-crystal based SLM has a limited refresh rate of approximately 10Hz, which extends the amount of time needed to perform the optimization process to the order of 30 minutes. A micro-electro-mechanical system (MEMS) -based SLM can reduce this time by more than three orders of magnitude, but requires a brighter non-linear signal.

[0066] In another embodiment of the present invention, light is selectively focused onto fluorescent-tagged targets, providing a minimally-invasive targeting and burning ("photo- bleaching") of 2PF tagged targets without harming the immediate environment, such as in medical applications for destroying malignant cells with minimal or no damage to surrounding tissue. An advantage of this embodiment is that it does not require active positioning or steering of the focus - a beam optimized according to embodiments of the present invention will automatically focus on the fluorescent-tagged targets and will move on to new tagged targets as soon as the previous tagged targets are destroyed. In related embodiments, this process is enhanced as disclosed below and illustrated in Fig. 6.

[0067] In other embodiments, rather than using 2PF in target 101, a fluorescent or second- harmonic coating 123 is applied to fiber distal facet 105 as a treatment of distal facet 105 to produce a thin nonlinear medium for the focusing procedure, after which various types of scanning microscopy could be performed. To avoid ambiguity in the retrieval of the focus position, in a related embodiment, as an alternate treatment of distal facet 105, the two halves of distal facet 105 are coated with coating 123 on one half, and a coating 125 on the other half which has a different color of fluorescence from that of coating 123. [0068] Due to a self-imaging property of graded index fibers, some information regarding the position of the focus is retained in the excited fluorescence during propagation from distal end 105 back through fiber 103 to proximal end 107.

[0069] Fig. 2A - Fig. 2D show images of the focused intensity on the 2PF screen at distal end 105 after four different runs of the optimization with different feedback metrics for each run. The fiber boundary is shown by a dashed line 201, and a typical focal point 203 is shown in Fig. 2A. Fig. 2E - Fig. 2H show typical fluorescent patterns as detected at proximal end 107 corresponding to the focal images of Fig. 2A - Fig. 2D. Typical lobes are indicated by dashed lines 211 in Fig. 2F. The fluorescent patterns feature a pair of bright lobes at a distance and azimuth, which correlate to the position of the distal focus.

[0070] Fig. 2J is a plot of the correspondence in radius, and Fig. 2K is a plot of the correspondence in angular orientation of Fig. 2A - Fig. 2D versus Fig. 2E - Fig. 2H, showing that, up to constant rotation angle, all the information necessary to focus at any arbitrary location at distal end 205 is available in the fluorescent patterns seen at proximal end 107. According to various embodiments of the invention, these correlations are used as a precise feedback signal for deterrninistically controlling the position of the formed focus.

[0071] Certain embodiments use a spatially-modified cost function for the optimization feedback to obtain the different focus positions in Fig. 2B, 2C, and 2D, rather than by simply maximizing the total 2PF. The cost function was created by multiplying the detected fluorescence image at proximal facet 107 by a weighting mask set to match the fluorescence lobes corresponding to desired focus positions (dotted circles in Fig. 2E - Fig. 2H, with weighting masks as shown in Fig. 4A - Fig. 4C). These correlations hold during bending the fiber with a radius of curvature as small as 5cm.

WEIGHTING MASKS FOR FOCUSING TO A DETERMINISTIC POSITION

[0072] According to an embodiment of the present invention, a weighting mask is a double- lobed mask symmetrically-positioned around a center-point (r = 0). In a related embodiment, each lobe has the form e ⁸ - 1.3 · e ⁵⁰ . The orientation angle and distance between the lobes uniquely define a mask, and corresponds to two possible focus positions at fiber distal end 105 (Fig. 1A). Optimization feedback is calculated by summing the products of each image pixel (from primary camera 109) multiplied by the corresponding pixel of the weighting mask. Fig. 4A - Fig. 4C illustrate the masks, Fig. 4D - Fig. 4F illustrate the corresponding proximal 2PF images, and Fig. 4G - Fig. 4J illustrate the distal intensity distribution (such as obtained via test and verification using secondary camera 119).

[0073] Fig. 2J and Fig. 2K illustrate the relationships between the intensity distributions at fiber proximal end 107 and fiber distal end 105, utilizing secondary camera 119 to record the focus at distal end 105 and primary camera 109 to record the corresponding 2PF pattern at proximal end 107.

OPTIMIZATION

[0074] Fig. 5 is a flowchart of a method 500 according to an embodiment of the present invention for optimizing remote imaging and focusing of light in an apparatus as described herein, via optimized wavefront shaping. According to additional embodiments, method 500 is used to perform an optimized scan of samples and specimens in situ and in-vivo. In a related embodiment, the method is carried out by a data processing device, such as controller 115. In a step 501, a parameter K 503 is set to an integer value. Then, in a step 505, SLM 111 (see Fig. 1A) is logically partitioned into K square partitions. The phase of each partition is stored in a data structure 507, where the phase of partition 1 is stored in a data object 507A, the phase of partition 2 is stored in a data object 507B, ellipsis 507C represents the phases of partitions 3 through K- 1 , and the phase of partition K is stored in a data object 507D. In a loop starting at a point 521 and ending at a point 529, an index is iterated from 1 to K, inside which optimizing processing is done for each partition as follows:

[0075] For each iteration, a phase for partition is obtained which maximizes the value of an optimization function 533 of 2PF data 531 recorded by primary camera 109 (see Fig. 1A). In a related embodiment optimization function 533 also takes weighting mask data 127 into account, wherein an inter-lobe distance 537 and an inter-lobe angular orientation 539 are also arguments of optimization function 533. Weighting mask data 127 is for a weighting mask with properties as previously disclosed and discussed.

[0076] In an embodiment of the present invention, optimization function 533 is simply the total intensity of the back-propagated fluorescence, such as in the case where primary light detector 109 has a scalar output that is to be maximized. In another embodiment, the sum of the 2PF image pixels is used as optimization function 533 in the optimization for Fig. 1C, Fig. 3C, and Fig. 3D. As another non-limiting example, pixel-wise multiplication of the 2PF image by a weighting mask prior to summing the values is used as optimization function 533 in the optimization for Fig. 2B, Fig. 2C, and Fig. 2D.

[0077] According to another related embodiment, a genetic optimization algorithm 535 (such as from the Matlab Genetic Algorithm toolbox) is used to obtain the desired phase for each partition .

[0078] The quality of optimization is evaluated periodically, to determine when the optimization process should be terminated. In an embodiment of the invention, this is done at a decision point 525. If the optimization is satisfactory, decision point 525 branches to a terminating step 527, in which the determined phases for the K partitions are used in the SLM for wavefront shaping to optimize the optical performance of the apparatus as herein described. Otherwise, loop end 529 proceeds. If < K, control resumes at loop starting point 521 for further iteration. In a related embodiment, if loop end 529 finishes processing all partitions (i = K) without satisfactory optimization, a step 541 increases K and continues with partition step 505. In an optimization according to a particular embodiment of the invention, K= 300, and after 300 iterations, K is quadrupled (i.e., each square partition is broken down into 4 smaller squares). Optimizations involving between 1 ,000 to 2,000 iterations may be encountered.

[0079] Fig. 6 is a flowchart for a method 600 of selectively photo-bleaching a fiuorescently- tagged target 101 (non-limiting examples of which include an in situ or in-vivo sample or specimen - see Fig. 1A) according to an embodiment of the present invention. In a related embodiment, method 600 is carried out by controller 115 (see Fig. 1A), which stores values for a scan intensity 601 and a bleach intensity 603. Scan intensity 601 is a low intensity whereas bleach intensity 603 is a high intensity. These intensities are relative to one another and are adjusted as appropriate for their intended purposes, as detailed below.

[0080] In operation, scan intensity 601 and bleach intensity 603 are input as values into a selector function 605, whose output goes to an excitation intensity control 607, which controls the photon excitation intensity of laser 121 (see Fig. 1A). In practice, the relatively low scan intensity 601 is used for scanning target 101 (a fluorescently-tagged sample or specimen) to identify the locations and distributions of the fluorescently-tagged areas for photo-bleaching.

[0081] Thus, in a step 611, scan intensity 601 is input into selector function 605, which selects low intensity in excitation intensity control 607 for laser 121. Then method 500 is used to perform an optimized scan of target 101. Next, in a step 615, detector (or camera) 109 (see Fig. 1A) is activated to detect back-propagated fluorescence, and at a decision point 617 the output of detector (or camera) 109 is compared against a target acquisition threshold 619, and if the back-propagated fluorescence meets threshold 619 (e.g., equals or e.g., exceeds threshold 619), then in a step 623 the higher bleach intensity is selected at selector function 605 for further optimized scanning via method 500.

[0082] However, if at decision point 617 the back-propagated fluorescence does not meet threshold 619 (e.g., is less than threshold 619), then in a step 621, scan intensity 601 is used for setting the intensity of laser 121.

[0083] In a related embodiment, selector function 605 outputs an arbitrary predetermined function to excitation intensity control 607, based on the inputs from steps 611, 621, and 623, and the values of scan intensity 601 and bleach intensity 603.

[0084] In the above-described manner, method 600 minimizes the scan intensity in areas where there are no fiuorescently-tagged areas of target 101, thereby minimizing or avoiding photo- bleaching damage to untagged areas; while maximizing the scan intensity in areas where there are fiuorescently-tagged areas of target 101, and thereby photo-bleaching the tagged areas more rapidly and efficiently.

[0085] A further embodiment of the present invention provides a computer product for optimizing including a non-transitory data storage containing executable instructions, which instructions, when executed by a data processing device (such as controller 115) cause the data processing device to perform a method of the present invention, including the method illustrated in Fig. 5 and described herein; and the method illustrated in Fig. 6 and also described herein.

Claims

CLAIMS What is claimed is:

1. An apparatus for remote imaging and focusing of light, the apparatus comprising:

a multimode fiber (MMF) having a proximal end and a distal end, for

propagating light and images between the proximal end and the distal end;

a laser light source, for providing excitation light to the proximal end;

a spatial light modulator (SLM), to perform wavefront shaping of the

excitation light from the laser light source to the proximal end;

a beam splitter, for separating light back-propagated from the distal

end to the proximal end;

a primary light detector, for measuring an intensity of light excited at

the distal end and back-propagated to the proximal end, as separated by the beam-splitter; and

a controller, for controlling the SLM to perform wavefront shaping

according to a feedback signal from the primary light detector based on light back-propagated from the distal end to the

proximal end.

2. The apparatus of claim 1 , wherein the primary light detector is a primary camera for capturing images of light back-propagated to the proximal end.

3. The apparatus of claim 2, wherein the primary camera includes an electron multiplying charge coupled device (EMCCD).

4. The apparatus of claim 1, wherein the SLM is a liquid-crystal SLM.

5. The apparatus of claim 1, wherein the SLM is a micro-electro-mechanical system (MEMS) SLM.

6. The apparatus of claim 1, wherein the beam-splitter is a dichroic mirror (DM).

7. The apparatus of claiml, wherein the controller is a programmable device selected from a group consisting of a data processor and a computer.

8. The apparatus of claim 1, wherein the distal end is coated with a fluorescent coating.

9. The apparatus of claim 7, wherein the fluorescent coating is a two-photon fluorescence (2PF) coating.

10. The apparatus of claim 1 , wherein a first half of the distal end is coated with a first fluorescent coating having a first fluorescence color, and wherein a second half of the distal end is coated with a second fluorescent coating having a second fluorescence color.

11. The apparatus of claim 1, wherein the controller is adapted to modify the feedback signal according to a weighting mask.

12. The apparatus of claim 11, wherein the weighting mask is a double-lobed mask symmetrically-positioned around a center-point.

13. The apparatus of claim 12, wherein the weighting mask is characterized by a radius and an angular orientation.

14. The apparatus of claim 1 for in situ and in-vivo use, wherein imaging and focusing of light at the distal end is accomplished without the use of any additional optical elements at the distal end.

15. The apparatus of claim 8 for in situ and in-vivo use, wherein imaging and focusing of light at the distal end is accomplished without the use of any additional optical elements at the distal end.

16. The apparatus of claim 10 for in situ and in-vivo use, wherein imaging and focusing of light at the distal end is accomplished without the use of any additional optical elements at the distal end.

17. The apparatus of claim 1, further including a system for test and verification of the apparatus, wherein the apparatus further comprises:

a fiuorescently-tagged sample screen at the distal end; and

a secondary camera for capturing an image of fluorescence directly

from the fiuorescently-tagged sample screen.

18. The apparatus of claim 17, wherein the fiuorescently-tagged sample screen is a two- photon fluorescence (2PF) tagged sample screen.

19. The apparatus of claim 17, wherein the fiuorescently-tagged sample screen includes a capillary filled with disodium fluorescein in ethanol.

20. The apparatus of claim 17, wherein the secondary camera includes a charge-coupled device (CCD).

21. A method for optimizing remote imaging and focusing of light in an apparatus that includes: a multi-mode fiber (MMF) having a proximal end and a distal end, a spatial light modulator (SLM) at the proximal end, a primary light detector at the proximal end which measures an intensity of a back-propagated two-photon fluorescence (2PF) from the distal end; and a controller, the method comprising:

logically partitioning, by the controller, the SLM into a plurality of

square partitions;

for at least one partition:

determining, by the controller, a phase for the partition that

maximizes the intensity of the back-propagated 2PF; and using a phase determined for the at least one partition in the SLM.

22. The method of claim 21, wherein determining a phase for the partition is according to an optimization function.

23. The method of claim 21 , wherein the primary light detector is a primary camera for capturing images of the back-propagated 2PF.

24. The method of claim 21, wherein determining a phase for the partition is according to weighting mask data of a weighting mask.

25. The method of claim 21, wherein the weighting mask is a double-lobed mask symmetrically-positioned around a center-point.

26. The method of claim 25, wherein the weighting mask is characterized by a radius and an anguLar orientation.

27. The method of claim 21, wherein determining a phase for the partition is according to a genetic algorithm.

28. The method of claim 21, for photo-bleaching a fluorescently- tagged target, wherein the light has an intensity, and further comprising:

providing a scanning intensity value, a bleaching intensity value, and a target acquisition threshold; performing an optimized scan of the target with the intensity at the scanning intensity value; and

if the back-propagated two-photon fluorescence (2PF) meets the

target acquisition threshold, then performing an optimized scan of the target with the intensity at the bleaching intensity value.

29. A computer product for optimizing remote imaging and focusing of light in an apparatus that includes: a multi-mode fiber (MMF) having a proximal end and a distal end, a spatial light modulator (SLM) at the proximal end, and a primary light detector at the proximal end which measures an intensity of a back-propagated two-photon fluorescence (2PF) from the distal end; the computer product comprising a non-transitory storage containing executable instructions, which instructions, when executed by a data processing device cause the data processing device to perform:

logically partitioning the SLM into a plurality of square partitions;

for at least one partition:

deterrnining a phase for the partition that optimizes the intensity of the back-propagated 2PF according to an optimization function; and

using a phase determined for the at least one partition in the SLM.