Systems and methods for light manipulation - Patents.com

Abstract

The present disclosure provides systems, devices, and methods for imaging and imaging a subject. The systems and devices may include one or more optical filtering devices. The optical filtering device may include a chamber having an input and an output. The chamber may be configured to receive a light beam. The chamber may include a plurality of reflectors configured to direct the received light in an optical path from the input to the output by reflection of the light between one or more of the plurality of reflectors. The chamber may be configured to reject out-of-focus light along the optical path.

Description

[Technical field]

[0001] Statement of Government Interest This invention was made with United States Government support under Small Business Innovation Research (SBIR) Grant No. 2R44CA221591-02A1 awarded by the Department of Health and Human Services, National Cancer Institute. The United States Government has certain rights in the invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 285,422, filed December 2, 2021, which is incorporated herein in its entirety. [Background technology]

[0003] Confocal microscopic imaging uses excitation light directed at the tissue and collects the resulting reflected, transmitted or fluorescent light. Confocal microscopic imaging can create high resolution images by passing the collected light through a pinhole (or single mode fiber) to eliminate out-of-focus light. A focusing lens configuration can be used to focus the collected light to a plane or spot that optically coincides with the objective focus to the imaging plane within the tissue. The pinhole is used as a spatial filter where background or out-of-focus light is physically eliminated or blocked while the focused light passes through the pinhole. Precise alignment of the focused light is required on the x, y and z axes where the z axis is in the direction of the focused light beam. The pinhole and spot size depend on the resolution selected. The pinhole and pinhole alignment are approximately the same scale as the resolution. In tissue imaging using reflectance confocal microscopy, the focused spot of light can be 1-10 microns and can be directed through a pinhole that is approximately 1-10 microns. Thus, reflectance confocal microscopy imaging can use a small spot of focused light that is centered or aligned with a very small aperture. Achieving small magnitude focusing element tolerances can be difficult, especially in small handheld devices that are prone to losing alignment with any instability and / or thermal expansion of the imaging structure during use. Summary of the Invention [Means for solving the problem]

[0004] Provided herein are improved systems and methods for confocal microscopy imaging. Further provided herein are methods and apparatus in portable or handheld optical devices. Further provided herein are methods and apparatus for imaging tissue.

[0005] In one aspect, the disclosure provides a device comprising: a probe configured to (i) direct a light beam from a light source to an imaging target and (ii) collect light from the imaging target when the light beam contacts the imaging target; and an optical filtering device in optical communication with the probe, the optical filtering device comprising: (a) a chamber having (i) an input configured to receive light collected by the probe and (ii) an output configured to transmit the light away from the chamber; and (b) a plurality of reflectors disposed within the chamber, the plurality of reflectors configured to direct the received light in an optical path from the input to the output by reflection of light between reflectors of the plurality of reflectors, the chamber configured to eliminate out-of-focus light along the optical path.

[0006] In one embodiment, the cross-sectional diameter of the output is greater than or equal to about 0.1 times the cross-sectional diameter of the input.

[0007] In one embodiment, the chamber has a longest internal linear dimension and the light path has a path length that is at least three times the length of the longest linear dimension.

[0008] In one embodiment, the optical path has a path length of 0.25 meters, 0.5 meters, or 1 meter or more.

[0009] In one embodiment, at least one of the plurality of reflectors is a retroreflector.

[0010] In one embodiment, at least one of the plurality of reflectors is a mirror.

[0011] In one embodiment, one of the plurality of reflectors is a mirror and another of the plurality of reflectors is a retroreflector.

[0012] In one embodiment, the output is configured to direct a focused portion of the light to a collector.

[0013] In one embodiment, the device comprises a collector.

[0014] In one embodiment, an optical filtering device is coupled to an optical fiber, the optical fiber being configured to deliver light to a collector.

[0015] In one embodiment, the multiple reflectors include a first reflector, the multiple reflectors arranged to direct focused portions of light on an optical path between the reflectors, the optical path returning to the first reflector multiple times before reaching the output.

[0016] In one embodiment, the plurality of reflectors comprises a first reflector at a first position within the chamber and a second reflector at a second position within the chamber, the first reflector configured to direct light to the second reflector.

[0017] In one embodiment, the optical path has a path length, the first reflector and the second reflector are separated by a distance, and the path length is at least five times the distance.

[0018] In one embodiment, the path length traversed by the light is between 5 and 30 times the distance.

[0019] In one embodiment, the path length is about 0.5 meters or greater.

[0020] In one embodiment, the distance is about 15 centimeters or less.

[0021] In one embodiment, the first reflector and the second reflector are spaced apart along an axis parallel to the length of the chamber.

[0022] In one embodiment, the device further comprises a scanning unit disposed between the light source and the probe, the scanning unit configured to scan the light beam across the imaging object.

[0023] In one embodiment, the device further comprises a focusing unit disposed between the light source and the probe, the focusing unit configured to scan a focal point of the light beam within the imaged object.

[0024] In one embodiment, the probe is configured to contact or penetrate the imaging subject.

[0025] In one embodiment, the probe includes an objective lens configured to collimate the light.

[0026] In one embodiment, the device further comprises an alignment unit disposed between the probe and the optical filtering device, the alignment unit configured to (i) guide the light to the optical filtering device and (ii) adjust the angle of incidence of the light into the chamber.

[0027] In one embodiment, the device further comprises a beam splitter disposed between the probe and the optical filtering device, the beam splitter configured to (i) split the light to generate split light, and (ii) direct at least a portion of the split light to the optical filtering unit.

[0028] In one embodiment, the device is configured for confocal imaging.

[0029] In one embodiment, the device is configured for tandem confocal and multiphoton imaging.

[0030] In one embodiment, the device is portable.

[0031] In another aspect, the present disclosure provides a device comprising: a chamber configured to receive a light beam; and a plurality of reflectors comprising a first reflector and a second reflector, wherein the first reflector is disposed within the chamber and the second reflector is disposed within the chamber, the first reflector configured to direct the light beam from the first reflector to the second reflector, and the second reflector configured to direct the light beam from the second reflector to the first reflector at least once before the first reflector, the second reflector, or another reflector of the plurality of reflectors directs the light beam out of the chamber.

[0032] In one embodiment, the chamber is configured to filter out unfocused light from the light beam.

[0033] In one embodiment, the first reflector or the second reflector is a retroreflector.

[0034] In one embodiment, the retroreflector is configured for total internal reflection.

[0035] In one embodiment, at least one of the first reflector or the second reflector is a mirror.

[0036] In one embodiment, the mirror comprises a dielectric coating.

[0037] In one embodiment, the dielectric coating is configured to direct a portion of the light beam having a wavelength between about 700 nanometers and 900 nanometers.

[0038] In one embodiment, the chamber further comprises an input and an output, the input configured to receive a signal and the output configured to direct the signal to a collection unit.

[0039] In one embodiment, the collection unit is disposed adjacent to the first location or the second location, the collection unit being configured to collect the light beam.

[0040] In one embodiment, the first reflector and the second reflector are spaced apart along an axis parallel to the length of the chamber.

[0041] In one embodiment, a distance within the chamber separates the first reflector and the second reflector, and a path length traversed by the light beam within the chamber is at least three times the distance separating the first reflector and the second reflector.

[0042] In one embodiment, the path length traversed by the light beam is at least five times the distance separating the first reflector and the second reflector.

[0043] In one embodiment, the path length is between 5 and 30 times the distance separating the first reflector and the second reflector.

[0044] In one embodiment, the distance between the first reflector and the second reflector is about 15 centimeters or less.

[0045] In one embodiment, the distance between the first reflector and the second reflector is about 10 centimeters or less.

[0046] In one embodiment, the path length is about 0.5 meters or greater.

[0047] In one embodiment, the path length is about 1 meter or greater.

[0048] In one embodiment, the first reflector or the second reflector has a reflective surface with a dimension of about 0.5 centimeters to 2 centimeters.

[0049] In one embodiment, the device further comprises a probe configured to (i) provide a light beam from a light source to the imaging target and (ii) collect light from the imaging target when the light beam contacts the imaging target, the probe being in optical communication with the chamber.

[0050] In one embodiment, the first reflector or the second reflector comprises a plurality of reflective elements.

[0051] In one aspect, the present disclosure provides a system for filtering light, the system comprising: a light source for generating a light beam; a light filtering device in optical communication with the light source, the light filtering device comprising: (i) a chamber configured to receive the light beam from the light source and having an input configured to receive light from a probe and an output configured to transmit the light away from the chamber; and (ii) a plurality of reflectors disposed within the chamber, the plurality of reflectors configured to direct the received light in an optical path from the input to the output by reflection of light between reflectors of the plurality of reflectors, the chamber configured to eliminate out-of-focus light along the optical path; and one or more computer processors operatively coupled to the light source and the light filtering device, the one or more computer processors individually or collectively programmed to process the light beam transmitted away from the chamber at the output to generate an image.

[0052] In one embodiment, the system further comprises a beam deexpander unit located between the light beam and the optical filtering device.

[0053] In one embodiment, the beam deexpander unit comprises a beam deexpander unit chamber comprising a first lens and a second lens, the first lens being positioned at a first end of the beam deexpander unit chamber and the second lens being positioned at a second end of the beam deexpander unit chamber, the first end of the beam deexpander unit chamber being on opposite sides of the beam deexpander unit chamber.

[0054] In one embodiment, the chamber of the beam deexpander unit comprises a deformable wall.

[0055] In one embodiment, the beam deexpander unit comprises an adjustment structure.

[0056] In one embodiment, the adjustment structure is configured to adjust the distance between the first lens and the second lens.

[0057] In one embodiment, the system further comprises an imaging device comprising an optical filtering device.

[0058] In one embodiment, the cross-sectional diameter of the output is greater than or equal to about 0.1 times the cross-sectional diameter of the input.

[0059] In one embodiment, the chamber has a longest internal linear dimension and the path has a path length at least three times as long as the longest linear dimension.

[0060] In one embodiment, the imaging device comprises a probe disposed in an optical path between a light source and an optical filtering device, the probe configured to (i) direct a light beam to the imaging object, (ii) collect light from the imaging object when the light beam contacts the imaging object, and (iii) direct a signal to the optical filtering device.

[0061] In one embodiment, the probe is configured to contact or penetrate the imaging subject.

[0062] In one embodiment, the probe includes an objective lens configured to collimate the signal.

[0063] In one embodiment, the imaging device is a handheld device.

[0064] In an embodiment, the imaging device further comprises one or more members selected from the group consisting of a focusing unit, a scanning unit, an alignment unit, an objective lens, a beam splitter, a frequency multiplier, and any combination thereof.

[0065] In one embodiment, the system further comprises a focusing unit disposed between the light source and the light filtering device, the focusing unit configured to vary the focus of the light beam.

[0066] In one embodiment, the system further comprises a scanning unit disposed between the light source and the light filtering device, the scanning unit configured to scan the light beam in at least one dimension.

[0067] In one embodiment, the system further comprises an alignment unit disposed in the optical path between the light source and the optical filtering device, the alignment unit configured to (i) guide the light beam to the optical filtering device and (ii) adjust the angle of incidence of the light beam into the chamber.

[0068] In one embodiment, the alignment unit comprises a mirror.

[0069] In one embodiment, the system further comprises a beam splitter disposed between the light source and the optical filtering device, the beam splitter configured to (i) split the light beam to generate split light beams, and (ii) direct at least a portion of the split light beam to the optical filtering unit.

[0070] In one embodiment, the multiple reflectors include a first reflector arranged to direct focused portions of light on a path between the reflectors that returns to the first reflector multiple times before reaching the output.

[0071] In one embodiment, the plurality of reflectors comprises (c) a first reflector disposed at a first location within the chamber and configured to direct light to a second location within the chamber, and (c) a second reflector disposed at the second location and configured to direct light to the first reflector.

[0072] In one embodiment, a distance within the chamber separates the first reflector and the second reflector, and a path length that the light traverses between the first position and the second position is at least three times the distance separating the first reflector and the second reflector.

[0073] In one embodiment, the path length traversed by the light beam is at least five times the distance separating the first reflector and the second reflector.

[0074] In one embodiment, the path length traversed by the light beam is approximately 5 to 30 times the distance separating the first reflector and the second reflector.

[0075] In one embodiment, the distance between the first reflector and the second reflector is about 15 centimeters or less.

[0076] In one embodiment, the path length is about 0.5 meters or greater.

[0077] In one embodiment, at least one of the first reflector or the second reflector is a mirror.

[0078] In one embodiment, the first reflector, the second reflector, or both the first reflector and the second reflector comprise a retroreflector.

[0079] In one embodiment, the system further comprises a fiber bundle having a first end coupled to the output of the chamber and a second end coupled to a collection unit, the fiber bundle configured to collect the light beam following the light beam passing through the chamber and direct the light beam to the collection unit.

[0080] In one embodiment, the first end of the fiber bundle comprises a linear array of fibers.

[0081] In one embodiment, the fiber bundle has a diameter of about 1 millimeter to about 5 millimeters.

[0082] In one embodiment, the system further comprises a confocal imaging arrangement, and the optical filtering device is part of the confocal imaging arrangement.

[0083] In one embodiment, the confocal imaging setup does not include an emission pinhole for light filtering.

[0084] In one embodiment, the system further comprises a multiphoton imaging arrangement.

[0085] In one embodiment, the system is configured for tandem confocal and multiphoton imaging.

[0086] In one embodiment, the system is configured to generate a three-dimensional image of the imaging object.

[0087] In one embodiment, the system is portable.

[0088] In one aspect, the disclosure provides a method for filtering light, the method including: (a) providing a light filtering device comprising: (i) a chamber having a plurality of reflectors arranged to reflect a light beam therebetween to form a light path; (b) directing the light beam into the chamber; and (c) directing a focused portion of the light beam in the light path from an input to an output between the reflectors while removing unfocused light from the light path.

[0089] In one embodiment, the method further comprises, prior to (b), transmitting a light beam from the light source to the imaging object and collecting light from the imaging object as the light beam contacts the imaging object.

[0090] In one embodiment, the method further comprises varying the focus of the light beam within the imaged object using a focusing unit disposed in the optical path between the light source and the imaged object.

[0091] In one embodiment, the method further includes scanning the light beam in at least one dimension across the imaged object using a scanning unit disposed in an optical path between the light source and the imaged object.

[0092] In one embodiment, the method further includes using a beam splitter positioned in an optical path between the imaging object and the optical filtering device to (i) split the light to generate split light, and (ii) direct at least a portion of the split light to the optical filtering device.

[0093] In one embodiment, the method further includes directing a light beam to the imaging object and collecting light generated from the imaging object using a probe positioned in an optical path between the light source and the imaging object.

[0094] In one embodiment, the method further includes processing the light after directing the focused portion of the light beam to an output of the chamber to generate an image of the imaged object.

[0095] In one embodiment, the image is a three-dimensional image.

[0096] In one embodiment, the method further comprises using the light for confocal imaging.

[0097] In one embodiment, the method further comprises, prior to (b), using an alignment unit: (i) aligning the light beam with the chamber; and (ii) adjusting the angle of incidence of the light beam to the chamber.

[0098] In one embodiment, a light filtering device is provided in the imaging device.

[0099] In one embodiment, the imaging device is handheld.

[0100] In one embodiment, the imaging device further comprises a confocal imaging arrangement comprising an optical filtering device.

[0101] In an embodiment, the imaging device further comprises a multiphoton imaging arrangement.

[0102] In one embodiment, the method further comprises using a confocal imaging arrangement and a multiphoton imaging arrangement for tandem confocal and multiphoton imaging.

[0103] In one embodiment, the imaging device includes an objective lens that collimates the light beam.

[0104] In one embodiment, the path length of the optical path that the light beam traverses within the chamber is at least 0.25M.

[0105] In one embodiment, the light beam is a collimated light beam.

[0106] In one aspect, the disclosure provides a method for imaging an object, the method including: (a) providing an optical filtering device having (i) a chamber and (ii) a probe in optical communication with the object; (b) providing a light beam to the object using the probe and collecting a resultant return light beam from the object; (c) directing the return light beam from the object to the chamber of the optical filtering device; (d) repeatedly directing the light beam from a first reflector to a second reflector in the chamber of the optical filtering device such that a path length that the light beam traverses between the first reflector and the second reflector is at least three times the distance separating the first reflector and the second reflector; and (e) processing the light beam to generate an image of the object.

[0107] In one embodiment, the method further comprises, prior to (b), transmitting a light beam from the light source through the probe to the imaging object and collecting light from the imaging object as the light beam contacts the imaging object.

[0108] In one embodiment, the method further comprises, prior to (c), directing the returning optical beam to a beam deexpander unit. In one embodiment, the method further comprises adjusting the beam deexpander after directing the returning optical beam to the beam deexpander unit.

[0109] In one embodiment, the beam deexpander unit comprises a beam deexpander unit chamber comprising a first lens and a second lens, the first lens being positioned at a first end of the beam deexpander unit chamber and the second lens being positioned at a second end of the beam deexpander unit chamber, the first end of the beam deexpander unit chamber being on opposite sides of the beam deexpander unit chamber.

[0110] In one embodiment, the chamber of the beam deexpander unit comprises a deformable wall.

[0111] In one embodiment, the beam deexpander unit further comprises an adjustment structure.

[0112] In one embodiment, the adjustment structure is configured to adjust the distance between the first lens and the second lens.

[0113] In one embodiment, the method further comprises, subsequent to (c), processing the light to generate an image of the imaging object.

[0114] In one embodiment, the method further comprises using the light for confocal imaging.

[0115] In one embodiment, the method further comprises using the light for multiphoton imaging.

[0116] In one embodiment, the method further comprises using the light for tandem confocal and multiphoton imaging.

[0117] In one embodiment, the path length traversed by the light beam is at least five times the distance separating the first location and the second location.

[0118] In one embodiment, the path length is between 5 and 30 times the distance separating the first reflector and the reflector.

[0119] In one embodiment, the path length is about 0.5 meters or greater.

[0120] In one embodiment, the distance is about 15 centimeters or less.

[0121] In one aspect, the present disclosure provides an imaging system comprising: a light source configured to provide a light beam to an imaging target; The present invention provides an imaging device comprising: a probe in optical communication with a light source, the probe configured to direct a light beam from the light source to an imaging object; an optical filtering device in optical communication with the probe, the optical filtering device comprising a chamber having a first reflector and a second reflector, the optical filtering device configured to (i) receive the light beam from the probe, and (ii) such that a path length traversed by the light beam between the first reflector and the second reflector is at least three times a distance separating the first reflector and the second reflector; and a collection unit in optical communication with the optical filtering device, configured to collect the light beam from the optical filtering device, the light beam being usable to generate an image of the imaging object.

[0122] In one embodiment, the method further comprises a beam deexpander unit positioned between the light beam and the optical filtering device.

[0123] In one embodiment, the beam deexpander unit comprises a beam deexpander unit chamber comprising a first lens and a second lens, the first lens being positioned at a first end of the beam deexpander unit chamber and the second lens being positioned at a second end of the beam deexpander unit chamber, the first end of the beam deexpander unit chamber being on opposite sides of the beam deexpander unit chamber.

[0124] In one embodiment, the chamber of the beam deexpander unit comprises a deformable wall.

[0125] In one embodiment, the beam deexpander unit comprises an adjustment structure.

[0126] In one embodiment, the adjustment structure is configured to adjust the distance between the first lens and the second lens.

[0127] In one embodiment, the path length traversed by the light beam is at least five times the distance separating the first reflector and the second reflector.

[0128] In one embodiment, the path length is between 5 and 30 times the distance separating the first reflector and the second reflector.

[0129] In one embodiment, the path length is about 0.5 meters or greater.

[0130] In one embodiment, the distance is about 15 centimeters or less.

[0131] In one aspect, the disclosure provides a system comprising: a probe configured to (i) provide a light beam from a light source to an imaging target and (ii) collect light from the imaging target when the light beam contacts the imaging target; and an optical filtering device in optical communication with the probe, the optical filtering device comprising: (i) a chamber having an input and an output, the chamber configured to receive light from the imaging target at the input; and (ii) a plurality of reflectors positioned within the chamber, including a first reflector, the reflectors of the plurality of reflectors being positioned to direct focused portions of light on a path between the reflectors, the path returning to the first reflector multiple times before reaching the output.

[0132] In one aspect, the present disclosure provides a system comprising: a probe configured to (i) provide a light beam from a light source to an imaging target and (ii) collect light from the imaging target when the light beam contacts the imaging target; and an optical filtering device in optical communication with the probe, the optical filtering device comprising: (a) a chamber having (i) an input configured to receive light from the probe and (ii) an output configured to transmit light away from the chamber; and (b) a plurality of reflectors disposed within the chamber and configured to direct light received from the input to an output by reflection of light between reflectors of the plurality of reflectors, wherein a path length that the light traverses from the input to the output by reflection through the reflectors is at least 0.25 meters.

[0133] In one aspect, the disclosure provides a device comprising a chamber providing an optical path from an input to an output of said chamber, (i) the optical path configured to direct light from the input to the output, and (ii) the chamber configured to reject out-of-focus light along the optical path, wherein a cross-sectional diameter of the output is about 0.1 times or greater than a cross-sectional diameter of the input.

[0134] In one aspect, the disclosure provides a device comprising a chamber providing an optical path from an input to an output of said chamber, (i) the optical path configured to direct light from the input to the output, and (ii) the chamber configured to reject out-of-focus light along the optical path, the chamber having a longest internal linear dimension, and the optical path having a path length that is at least three times the length of the longest linear dimension.

[0135] In one aspect, the present disclosure provides: a chamber providing an optical path from an input to an output of the chamber, (i) the optical path having a path length and configured to direct light from the input to the output, (ii) the chamber configured to reject out-of-focus light along the optical path, the chamber having a longest internal linear dimension, the path length being at least three times as long as the longest linear dimension; Provide the device.

[0136] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which merely exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature, and not as restrictive.

[0137] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with a disclosure contained in the specification, the specification is intended to supersede and / or override any such conflicting material.

[0138] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "FIGS." and "FIGS."), in which: [Brief description of the drawings]

[0139] [Figure 1A] 1 shows a schematic example of a confocal imaging setup, with a probe integrated with an optical filtering device; [Figure 1B] 1 shows an example of a confocal imaging setup, including a probe connected to an optical filtering device; [Figure 2A] 1A-1C are schematic diagrams illustrating exemplary optical filtering devices and the resulting paths of optical beams, and an example of an optical filtering device including a mirror and a retroreflector; [Figure 2B] 1A-1C are schematic diagrams illustrating exemplary optical filtering devices and the resulting paths of optical beams, and an example of an optical filtering device including two mirrors; [Figure 2C] 1A-1C are schematic diagrams illustrating exemplary optical filtering devices and the paths of the resulting optical beams, and an example of an optical filtering device including two retroreflectors; [Figure 3A] 1A and 1B are schematic diagrams of collector fiber optic bundles, showing an example of a linear arrangement of collector fiber optic bundles; [Figure 3B] 1 shows a schematic of optical fiber bundles in a collector, illustrating an example of optical fiber bundles arranged in a circular ring shape in a collector. [Figure 4A] 1 shows an example of a light path through an optical filtering device, and an example of a folded light path. [Figure 4B] 4B illustrates an example optical path through an optical filtering device, with an example effective optical length of the optical path of FIG. 4A. [Diagram 5]1 illustrates an example of an optical element including a focusing unit that can be used to scan an imaging object. [Figure 6] 1 illustrates another example of an optical element that includes a focusing unit that can be used to scan an imaging object. [Figure 7] 1 illustrates a schematic diagram of an example handheld device that includes an optical element for scanning an imaging object. [Figure 8] 1 shows an example of an optical path between a mirror and a retroreflector. [Figure 9] 1 shows examples of optical elements in multiple reflective configurations and schematic light paths. [Figure 10A] 1 shows an example of an image generated from a scanned in vivo depth profile, and an example of a two-photon image. [Figure 10B] 1 shows an example of an image generated from a scanned in vivo depth profile, and an example of a second harmonic generated image. [Figure 10C] 1 shows an example of an image generated from a scanned in vivo depth profile; FIG. 2 shows an example of a reflectance confocal microscope image; [Figure 10D] 1 shows an example of an image generated from a scanned in vivo depth profile, and an example of a three-channel average image. [Figure 11A] 1 illustrates a schematic diagram of an example of a support system. [Figure 11B] 1 illustrates a schematic diagram of an example of a support system. [Figure 12] 1 illustrates an example of a portable imaging system that includes a handheld device coupled to a support system. [Figure 13] 1 shows an example of a portable support system for imaging. [Figure 14] 1 illustrates an example of an imaging system that includes a handheld device used to image a subject. [Figure 15] 1 illustrates a computer system that is programmed or otherwise configured to carry out the methods provided herein. [Figure 16] 1 illustrates a schematic of an example of a light path through a beam deexpander. [Figure 17A]1 illustrates a schematic diagram of an example of a beam deexpander with a chamber; 2 illustrates a schematic diagram of a beam deexpander disposed within an imaging system; [Figure 17B] 1 shows a schematic of an example of a beam deexpander with a chamber, and a beam deexpander with an actuation element for deforming the chamber; [Figure 17C] 1 shows a schematic of an example of a beam deexpander with a chamber, the beam deexpander and the actuating elements being shown in an undeformed state of the chamber of the beam deexpander; [Figure 17D] 1 shows a schematic of an example of a beam deexpander with a chamber, the beam deexpander being shown with the chamber deformed; [Figure 18A] 1 shows a schematic of an example of a beam deexpander with a chamber, and a beam deexpander with an adjustment element for deforming the chamber. [Figure 18B] 1 shows a schematic of an example of a beam deexpander with a chamber, the beam deexpander being shown with the chamber undeformed; [Figure 18C] 1 shows a schematic of an example of a beam deexpander with a chamber, the beam deexpander being shown with the chamber deformed; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0140] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are given by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0141] The term "subject" as used herein generally refers to an animal, such as a mammal. The subject may be a human or a non-human mammal. The subject may be a plant. The subject may be suffering from a disease or may be suffering from or suspected of having a disease. The subject may not be suffering from or suspected of having a disease. The subject may be symptomatic. Alternatively, the subject may be asymptomatic. In some cases, the subject may be treated to alleviate symptoms of a disease or to cure the subject of a disease. The subject may be a patient receiving treatment by a health care provider, such as a physician.

[0142] The term "tissue characteristic" as used herein generally refers to the state of a tissue. Examples of tissue characteristics include, but are not limited to, disease, abnormality, normality, condition, tissue hydration state, tissue structural state, tissue health state, or beneficial state. A characteristic may be a disease state. A characteristic may be benign (e.g., information about healthy tissue). A tissue characteristic may include one or more features that may aid in the classification or diagnosis of tissue. A tissue characteristic may be eczema, dermatitis, psoriasis, lichen planus, bullous pemphigoid, vasculitis, granuloma annulare, verruca vulgaris, seborrheic keratosis, basal cell carcinoma, actinic keratosis, squamous cell carcinoma in situ (e.g., intraepidermal carcinoma), squamous cell carcinoma, cyst, lentigo, pigmented nevi, melanoma, dermatofibroma, crust, fungal infection, bacterial infection, burn, wound, etc., or any combination thereof.

[0143] The term "feature" as used herein generally refers to an aspect of a tissue or other body part that exhibits a given tissue characteristic or characteristics. Examples of features include: properties; physiology; anatomy; composition; histology; function; processing; size; geometry; regularity; irregularity; optical properties; chemical properties; mechanical properties or other properties; color; vasculature; appearance; structural elements; quality; age of the tissue of a subject; data corresponding to the tissue characteristic; spondylosis in acute eczema with associated lymphocytic exocytosis; acanthosis in chronic eczema; parakeratosis and / or perivascular lymphohistiocytic infiltrate; signs of excoriation and / or chafing in chronic cases (e.g., lichen simplex) (e.g., irregular acanthosis and dermal papillae); vertical orientation of collagen in the epidermis; hyperkeratosis (e.g., parakeratosis), orthokeratosis; neutrophils in the stratum corneum and squamous epithelium; hypogranulosis; epidermis thin over the papillary dermis; regular acanthosis, acanthorectic ridges; relatively few spondylosis; dilated capillaries in the papillary dermis; perivascular lymphohistiocytic infiltrate; orthokeratosis; hypergranulosis; irregular acanthosis with serrated reticulate ridges; colloid bodies in the lower epidermis and upper dermis; liquefied degeneration of the basal layer; lichenoid lymphohistiocytic infiltrate in the upper dermis (e.g., interface dermatitis) and / or epidermis; melanin incontinence; subepidermal blisters; new blebs Viable roof over the blister, necrosis over the older blister; variable perivascular infiltrate (e.g., lymphocytes, histiocytes, eosinophils); prebullous lesions may show spongiosis with eosinophil exocytosis (e.g., eosinophilic spondylosis); vessel wall damage (e.g., necrosis, hyalinization, fibrin); infiltration of inflammatory cells into vessel walls; erythrocyte extravasation; nuclear dust from neutrophil leukocytoclasticity; ischemic necrosis of the epidermis; normal epidermis; central focus of dermal collagen degeneration (e.g., necrosis), mucin accumulation; palisading of histiocytes; multinucleated giant cells; 1 of inflammatory cells between collagen bundles filling (e.g., "busy" dermis); hyperkeratosis, papillomatosis, acanthosis; basal-like keratinocytes; keratinocyte cysts; abundant melanin throughout the basal layer and / or epidermis; sharp borders at the base of epidermal hyperplasia; locations; aggregated nests of basal-like tumor cells (e.g., sometimes with a small amount of squamous differentiation); peripheral parasitism of nuclei at the periphery of the cellular nests; artifacts (e.g., fissures) around the cellular nests; variable inflammatory infiltrates and ulceration; hyperkeratosis and / or ulceration; columns of keratosis that may overlie atypical keratinocytes that may be separated by areas of keratosis;Basal atypical keratinocytes with varying degrees of overlapping loss of maturation, hyperpigmentation, pleomorphism, increased and abnormal mitoses, and abnormal keratinization - full thickness changes may be called "Bowen-like actinic keratosis"; variable superficial perivascular or lichenoid chronic inflammatory infiltrate; solar elastosis; hyperkeratosis, parakeratosis; acanthosis; full thickness epidermal involvement by atypical keratinocytes with pale vacuolated or multinucleated cells; in some lesions, Paget-like spread at the margins; proliferation of atypical keratinocytes; infiltrates of the dermis; variable degrees of keratinization, optionally squamous or keratinous pearls; squamous epidermal hyperkeratosis; cysts lined by epidermis, occasionally flat, with a granular layer; lamellar keratin within the cyst; elongated reticular ridges of hyperpigmentation; increased melanocytes; squamous epithelial lining, but without a granular layer; dense keratin content; frequent calcifications; various epidermal changes (e.g., lichen simplex); melanocyte / nevus cell nests at the dermoepidermal junction (e.g., parakeratosis) and / or in the dermis (e.g., interface dermatitis); nevus cells in the epidermis confined to the basal layer, optionally at the tip of the reticular ridge; generally rounded, showing a decrease in size of both cells and cell nests with increasing depth into the dermis nevus cells (e.g., lymphocytes, histiocytes, eosinophils); inflammation, which varies based on traumatic state; asymmetric proliferation of melanocytes; atypical melanocytes infiltrating upward through the epidermis and downward into the dermis; variable cellular atypia (e.g., eosinophilic spondylosis); epidermal hyperplasia (may mimic basal cell carcinoma); hyperpigmented basal layer; peripheral but poorly circumscribed proliferation of spindle fibroblasts; histiocytes and few giant cells; variable amounts of collagen; focal epidermal hyperplasia with hyperkeratosis, dyskeratosis, and papillomatosis (not flat warts); tridentate keratinization; playrocytes (e.g., necrotic, hyalinized keratinocytes with pointed protuberances resembling whorled peaks, extravasated erythrocytes or hemosiderin; the granular layer is thickened with prominent keratohyalin granules and keratinocytes and shows perinuclear clearing (e.g., "busy" dermis); lymphocytic infiltrates in the upper dermis; involuted lesions with chronic inflammatory infiltrates in the dermis and epidermis with degenerative epithelial changes;These include, but are not limited to, numerous coarse basophilic intracytoplasmic keratohyalin granules resembling molluscan bodies, or any combination thereof;

[0144] The term "disease" as used herein generally refers to an abnormal condition affecting some or all of a subject or a disturbance of a biological function or structure, such as an organ. A disease may be caused by an agent from an external source, such as an infection, or may be caused by an internal malfunction, such as an autoimmune disease. A disease may refer to any condition that causes pain, dysfunction, suffering, social problems, and / or death to the affected subject. A disease may be an acute or chronic condition. A disease may refer to an infectious disease that may result from the presence of a pathogenic microorganism, including viruses, bacteria, fungi, protozoa, multicellular organisms, and abnormal proteins such as prions. A disease may refer to a non-infectious disease, including, but not limited to, cancer and genetic diseases. In some cases, the disease can be cured. In some cases, the disease cannot be cured. In some cases, the disease is an epithelial cancer. An epithelial cancer is a skin cancer, including, but not limited to, non-melanoma skin cancer, such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), and melanoma skin cancer.

[0145] The terms "epithelial tissue" and "epithelium" as used herein generally refer to tissue that lines the cavities and surfaces of blood vessels and organs throughout the body. Epithelial tissue generally comprises epithelial cells that come in three shapes: flat, columnar, and cuboidal. Epithelial cells can be arranged in a single layer of cells as a simple epithelium containing either flat, columnar, or cuboidal cells, or in layers of two or more cells with a deeper layer structure (stratified) containing either flat, columnar, and / or cuboidal cells.

[0146] The term "cancer" as used herein generally refers to a proliferative disorder caused or characterized by the proliferation of cells that may have lost susceptibility to normal growth control. Cancers of the same tissue type usually originate from the same tissue and can be classified into different subtypes based on their biological characteristics. Non-limiting examples of cancer categories are carcinoma (epithelial cell derived), sarcoma (connective tissue or mesoderm derived), leukemia (blood-forming tissue derived) and lymphoma (lymphatic tissue derived). Cancer can involve any organ or tissue of the body. Examples of cancer include melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma, glioma, Hodgkin's lymphoma, and chronic lymphocytic leukemia. Examples of organs and tissues that may be affected by various cancers include the pancreas, breast, thyroid, ovaries, uterus, testes, prostate, pituitary, adrenal glands, kidneys, stomach, esophagus, rectum, small intestine, colon, liver, gallbladder, head and neck, tongue, mouth, eyes and orbits, bones, joints, brain, nervous system, skin, blood, nasopharyngeal tissue, lungs, larynx, urinary tract, cervix, vagina, exocrine glands, and endocrine glands. In some cases, the cancer may be multicentric. In some cases, the cancer may be of unknown primary (CUP).

[0147] The term "lesion" as used herein generally refers to an area(s) of disease and / or suspected disease, a wound, incision, or surgical margin. Wounds can include, but are not limited to, abrasions, abrasions, cuts, lacerations, breaks, punctures, slices, and / or any injury that results in sufficient bleeding and / or skin trauma to allow entry of a foreign body. Incisions can include, but are not limited to, incisions made by medical professionals, such as doctors, nurses, assistants and / or nurse practitioners, and dental professionals during procedures such as surgical procedures.

[0148] The term "light" as used herein generally refers to electromagnetic radiation. Light may range in wavelength from infrared (e.g., about 700 nm to about 1 mm) to ultraviolet (e.g., about 10 nm to about 380 nm). Light may be visible light. Alternatively, light may be non-visible light. Light may include wavelengths of light in the visible and non-visible wavelengths of the electromagnetic spectrum.

[0149] As used herein, the term "ambient light" generally refers to the light surrounding an environment or subject, such as the light at a point-of-care location (e.g., a patient's home or office, an examination room, or a surgical suite) where the devices, methods, and systems of the present disclosure are used.

[0150] The term "optical axis" as used herein generally refers to a line about which there may be some degree of rotational symmetry in an optical system such as a camera lens or a microscope. The optical axis may be a line that passes through the center of curvature of a lens or spherical mirror and is parallel to the axis of symmetry. The optical axis herein may also be referred to as the Z-axis. In the case of a simple lens and mirror system, the optical axis may pass through the center of curvature of each surface and coincide with the axis of rotational symmetry. The optical axis may coincide with the mechanical axis of the system, as in the case of off-axis optical systems. In the case of an optical fiber, the optical axis (also called the fiber axis) may be along the center of the fiber core.

[0151] The term "position" as used herein generally refers to a position on a plane perpendicular to the optical axis, as opposed to "depth", which is parallel to the optical axis. For example, the position of the focal point may be the position of the focal point in the xy plane, whereas the "depth" position may be the position along the z-axis (optical axis). The position of the focal point may be varied throughout the xy plane. The focal point may also be varied simultaneously along the z-axis. The position may be the position of the focal point.

[0152] The term "location" can also refer to the location of the optical device (or housing), which can include the location in space of the probe; the location relative to the anatomical features of the subject; and the orientation or angle of the probe and / or its optics or optical axis. Location can refer to the location or orientation of the probe on or near the tissue or tissue boundary of the subject. Location can also refer to the location with respect to other characteristics or features identified in the tissue of the subject, or with respect to other data collected or observed from the tissue of the subject. Location of the optical device can also refer to the location and / or orientation of the probe or its optics with respect to tags, markers, or guides.

[0153] The term "focal point" or "focal spot" as used herein generally refers to a point of light on the axis of a lens or mirror of an optical element where parallel light rays converge. The focal point or focal spot may be within the tissue sample being imaged, from which a return signal is generated and can be processed to form a depth profile.

[0154] The term "focal plane" as used herein generally refers to a plane formed by a focal point directed along a scan path. The focal plane can be where the focal point moves in the X and / or Y directions with movement in the Z direction, where the Z axis is generally the optical axis. The scan path can also be considered as a focal path that includes at least two focal points that define a path that is non-parallel to the optical axis. For example, the focal path can include multiple focal points shaped as a spiral. A focal path as used herein may or may not be a plane, and may be a plane when projected onto the XZ or YZ plane. The focal plane may be an inclined plane. The inclined plane may be a plane oriented at an angle to the optical axis of the optical element (e.g., lens or mirror). The angle may be from about 0° to about 90°. The inclined plane may be a plane having a non-zero Z axis component.

[0155] The term "depth profile" as used herein generally refers to information or optical data derived from generated signals resulting from scanning a tissue sample. Scanning a tissue sample can involve an imaging focus extending in a direction parallel to the optical axis or z-axis, and various positions on the xy axis. The tissue sample can be, for example, an in vivo skin tissue, where the depth profile can extend across layers of the skin, such as the dermis, epidermis, and subcutaneous layers. The depth profile of the tissue sample can include data that, when projected onto the XZ or YZ plane, forms a vertical plane profile that can be converted into a projected vertical cross-sectional image. The vertical cross-sectional image of the tissue sample derived from the depth profile can be vertical or nearly vertical. In some cases, the depth profile provides a varying vertical focus coordinate, while the horizontal focus coordinate may or may not vary. The depth profile can be in the form of at least one plane that is at an angle to the optical plane (on the optical axis). For example, the depth profile can be parallel to the optical plane or at an angle less than 90 degrees and greater than 0 degrees to the optical plane. The depth profile can be generated using an optical device contacting the tissue at an angle. For example, the optical device can include a probe in contact with the tissue. The probe can include one or more objective lenses. The one or more objective lenses may be in contact with the tissue. For example, the depth profile may not be perpendicular to the optical axis, but rather offset by as much as the angle at which the optical device is in contact with the tissue. The depth profile can provide information at various depths of the sample, for example, various depths of skin tissue. The depth profile can be provided in real time. The depth profile may or may not correspond to a planar slice of the tissue. The depth profile can correspond to a slice of the tissue on an oblique surface. The depth profile can correspond to a tissue region that is not exactly a planar slice (e.g., the slice can have components in all three dimensions). The depth profile can be a virtual slice or cross section of the tissue. The depth profile can be optical data scanned from an in vivo tissue.The data used to form the projected cross-sectional image may be derived from multiple focal points distributed along a common shape or pattern. The multiple distributed points may be in the form of a scanned tilted plane, multiple scanned tilted planes, or a non-planar scanning pattern or shape (e.g., a spiral pattern, a wave pattern, or other predetermined or random or pseudo-random pattern of focal points). The location of the focal points used to form the depth profile may be changed or changeable to track objects or regions of the subject within the tissue that are detected or identified during the scan or associated data processing. The depth profile may be formed from one or more separate return signals or signals corresponding to anatomical features or characteristics that may form separate layers of the depth profile. The generated signals used to form the depth profile may be generated from an excitation light beam. The generated signals used to form the depth profile may be synchronized in time and position. The depth profile may include multiple depth profiles, each corresponding to a particular signal or subset of signals corresponding to (one or more) anatomical features or characteristics. The depth profile may form a composite depth profile generated using the time- and position-synchronized signals. A depth profile herein may be an in vivo depth profile from which optical data of in vivo tissue is obtained. A depth profile may be a composite of multiple depth profiles or layers of optical data generated from different generated signals synchronized in time and position. A depth profile may be a depth profile generated from a subset of generated signals synchronized in time and position with other subsets of generated signals. A depth profile may include one or more layers of optical data, each layer corresponding to a different subset of signals. A depth profile or depth profile optical data may also include data from processing information that corresponds to the depth profile, optical device, optical device position, other sensors, or information identified and time-corresponding to the depth profile or other related information.Additionally, other data corresponding to subject information, such as, for example, medical data, physical conditions, or other data or characteristics, may also be included along with the optical data in the depth profile. The depth profile may be an annotated depth profile having annotations or markings.

[0156] The term "medical data" as used herein generally refers to medical data of a subject including at least one medical data selected from the group consisting of health status, medical history, test results, current and past occupations, age, sex, race, skin type, Fitzpatrick skin type, other metrics related to skin health and appearance, subject nationality, environmental exposures, mental health, and medications. The subject's physical condition can be obtained via one or more medical instruments. The one or more medical instruments may include, but are not limited to, a stethoscope, a suction device, a thermometer, a tongue depressor, a blood transfusion kit, a calibrating fork, a respirator, a clock, a stopwatch, a weighing scale, an alligator forceps, a bedpan, a cannula, a cardioverter, a defibrillator, a catheter, a dialysis machine, an electrocardiograph machine, an enema machine, an endoscope, a gas cylinder, a gauze sponge, a hypodermic needle, a syringe, an infection control device, an instrument sterilizer, a kidney dish, a measuring tape, a medical halogen penlight, a nasogastric tube, a nebulizer, an ophthalmoscope, an otoscope, an oxygen mask and tube, a pipette, a dropper, a proscope, a reflex hammer, a blood pressure monitor, a spectrometer, a dermatoscope, and a camera. In some embodiments, the physical condition comprises a vital sign of the subject. The vital signs may be a measurement of a basic bodily function of the patient. The vital signs may include a temperature, a pulse rate, a respiratory rate, and a blood pressure. In some embodiments, the methods described herein further include receiving or using the medical data of the subject.

[0157] The term "projected cross-sectional image" as used herein generally refers to an image constructed from depth profile information projected onto an XZ or YZ plane to form an image plane. In this situation, there may be no distortion of the depth of the structure relative to the surface of the tissue. The projected cross-sectional image may be defined by the portion of the tissue being scanned. The projected cross-sectional image may extend in a direction perpendicular to the surface of the skin tissue. The data used to form the projected cross-sectional image may be derived from one or more tilted planes scanned, and / or non-planar scanning patterns, shapes (e.g., spiral, wave, etc.) or predetermined or random patterns of focus.

[0158] The term "fluorescence" as used herein generally refers to radiation that can be emitted as a result of absorption of incident electromagnetic radiation of one or more wavelengths (e.g., a single wavelength or two different wavelengths). In some cases, fluorescence can result from emission of light from an exogenously provided tag or marker. In some cases, fluorescence can result as an intrinsic response of one or more endogenous molecules to excitation by electromagnetic radiation.

[0159] As used herein, the term "autofluorescence" generally refers to fluorescence from one or more endogenous molecules due to excitation by electromagnetic radiation.

[0160] The term "multiphoton excitation" as used herein generally refers to excitation of a fluorophore with two or more photons, resulting in emission of a fluorescent photon. In some cases, the emitted photons are at higher energy than the excitatory photon. In some cases, multiple multiphoton excitations can be generated in a tissue. Multiple multiphoton excitations can generate multiple multiphoton signals. For example, a cell nucleus can undergo two-photon excitation. As another example, a cell wall can undergo three-photon excitation. At least a subset of the multiple signals can be different. The different signals can have different wavelengths that can be used in the methods described herein. For example, the different signals (e.g., two-photon or three-photon signals) can be used to form a map that can indicate different elements of the tissue. In some cases, the map is used to train a machine learning based diagnostic algorithm.

[0161] As used herein, the terms "second harmonic generation" and "SHG" generally refer to a nonlinear optical process in which photons interacting with a nonlinear material are effectively "combined" to form new photons that have approximately twice the energy of the initial photons, and therefore approximately twice the frequency and approximately half (1 / 2) the wavelength.

[0162] As used herein, the terms "third harmonic generation" and "THG" generally refer to a nonlinear optical process in which photons interacting with a nonlinear material effectively "combine" to form new photons that have approximately three times the energy of the initial photon, and therefore approximately three times the frequency and approximately one-third (1 / 3) the wavelength.

[0163] The term "reflectance confocal microscopy" or "RCM" as used herein generally refers to a process of collecting and / or processing reflected light from a sample (e.g., tissue or any component thereof). The process may be a non-invasive process in which a light beam is directed at a sample and return light from a focal point within the sample ("RCM signal") may be collected and / or analyzed. The process may be in vivo or ex vivo. RCM signals may be traced back the direction of the light beam that generated them. RCM signals may be polarized or unpolarized. RCM signals may be combined with pinholes, single mode fibers, multimode fibers, crossed excitation and collection light paths, or other confocal arrangements that restrict the collected light to that portion that originates from the focal point. A confocal microscope device as defined herein may be a device that focuses a limited focal point that produces light at a finite or substantially infinite distance through an aperture and / or optical fiber or fiber bundle for collection. In some embodiments, the light from the focal point may be collimated at a substantially infinite distance and passed through an aperture / fiber of approximately equal or similar diameter to the collimated beam. RCM generally refers to collecting reflected light, but can also refer to confocal arrangements capable of collecting transmitted or fluorescent light.

[0164] As used herein, the term "polarized" generally refers to light having waves that oscillate in one plane. Unpolarized light can generally refer to light having waves that oscillate in two or more planes.

[0165] The term "excitation light beam" as used herein generally refers to a focused light beam directed at a tissue to generate a generated signal. The excitation light beam can be a single light beam. The excitation light beam can be a pulsed single light beam. The excitation beam of light can be multiple light beams. The multiple light beams can be synchronized in time and position as described herein. The excitation beam of light can be a pulsed beam or a continuous beam or a combination of one or more pulsed and / or continuous beams that are simultaneously delivered to a focal point of the tissue to be imaged. The excitation light beam can be selected according to a given type of return signal or generated signal, as described herein. The excitation beam of light as used herein includes the illumination light used to generate a reflected or transmitted signal, e.g., a confocal microscopy signal.

[0166] The term "generated signal" as used herein generally refers to a signal returned from tissue to tissue originating from the direction of the focused light, e.g., excitation light, including but not limited to reflected, absorbed, scattered, or refracted light. The generated signal may include but is not limited to an endogenous signal originating from the tissue itself or a signal from an exogenously provided tag or marker. The generated signal may occur in either in vivo or ex vivo tissue. The generated signal may be characterized as either a single-photon generated signal or a multi-photon generated signal, as determined by the number of excitation photons contributing to the signal generation event. Single-photon generated signals may include but are not limited to reflectance confocal microscopy ("RCM") signals, single-photon fluorescence, and single-photon autofluorescence. Single-photon generated signals, such as RCM, may originate from either a continuous light source, a pulsed light source, or a combination of light sources, which may be either pulsed or continuous. Single-photon generated signals may be superimposed. Single-photon generated signals may be deconvolved. The multiphoton generated signal may be generated by at least 2, 3, 4, 5 or more photons. The multiphoton generated signal may include, but is not limited to, second harmonic generation, two-photon autofluorescence, two-photon fluorescence, third harmonic generation, three-photon autofluorescence, three-photon fluorescence, multiphoton autofluorescence, multiphoton fluorescence, and coherent anti-Stokes Raman spectroscopy. The multiphoton generated signal may come from either a single pulsed light source or a combination of pulsed light sources, such as in the case of coherent anti-Stokes Raman spectroscopy. The multiphoton generated signals may be superimposed. The multiphoton generated signals may be deconvolved. Other generated signals include, but are not limited to, optical coherence tomography (OCT), single or multiphoton fluorescence / autofluorescence lifetime imaging, polarized light microscopy signals, additional confocal microscopy signals, and ultrasound diagnostic signals. Single-photon and multi-photon generated signals can be combined with polarized light microscopy by selectively detecting components of the generated signals that are either linearly polarized, circularly polarized, unpolarized, or any combination thereof. Polarized light microscopy can further include blocking all or a portion of the generated signals that have a polarization direction parallel or perpendicular to the polarization direction of the light used to generate the signals, or any intermediate polarization direction.The generated signals described herein can be combined with confocal techniques utilizing pinholes, single mode fibers, multimode fibers, crossed excitation and collection light paths, or other confocal arrangements that restrict the light detected from the generated signal to that portion of the generated signal originating from the focal point. For example, a pinhole can be placed in a Raman spectroscopy device to generate a confocal Raman signal. Raman spectroscopy signals can generate different signals based at least in part on different vibrational states present in the sample or tissue. Optical coherence tomography signals can use light containing multiple phases to image tissue. Optical coherence tomography can be similar to optical ultrasound. Ultrasound can generate signals based at least in part on the reflection of sound waves from features (e.g., tissue) in the sample.

[0167] The term "imaging agent" as used herein generally refers to any agent, including but not limited to fluorophores, metal nanoparticles, nanoshell composites, and semiconductor nanocrystals, that can be applied to a sample to enhance the contrast of an image of the sample obtained using optical imaging techniques. Fluorophores can be antibody-targeted fluorophores, peptide-targeted fluorophores, and metabolically active fluorescent probes. Metal nanoparticles can include metals such as gold and silver that can scatter light. Nanoshell composites can include nanoparticles that include a dielectric core and a metal shell. Semiconductor nanocrystals can include quantum dots, such as quantum dots containing cadmium selenide or cadmium sulfide. Other contrast agents can also be used herein, for example, by applying acetic acid to the tissue.

[0168] As used herein, the terms "in real time" and "real-time" generally refer to immediate, rapid, without operator intervention, automatic, and / or programmed. Real time may include, but is not limited to, measurements in femtoseconds, picoseconds, nanoseconds, milliseconds, seconds, as well as measurements at longer and shorter time intervals.

[0169] The term "tissue" as used herein generally refers to any tissue or tissue contents. Tissue may be a healthy, benign, or disease-free sample. Tissue may be a sample removed from a subject, such as a tissue biopsy, tissue resection, aspirate (such as a fine needle aspirate), tissue washing, cytology specimen, bodily fluid, or any combination thereof. The tissue from which an image may be obtained may be any tissue or tissue contents of a subject, including, but not limited to, connective tissue, epithelial tissue, organ tissue, muscle tissue, ligament, tendon, skin tissue, breast tissue, bladder, kidney tissue, liver tissue, colon tissue, thyroid tissue, cervical tissue, prostate tissue, lung tissue, heart tissue, cardiac tissue, muscle tissue, pancreatic tissue, anal tissue, bile duct tissue, bone tissue, bone marrow, uterine tissue, ovarian tissue, endometrial tissue, vaginal tissue, vulvar tissue, stomach tissue, eye tissue, nasal tissue, paranasal sinus tissue, penile tissue, salivary gland tissue, intestinal tissue, gallbladder tissue, gastrointestinal tissue, bladder tissue, brain tissue, spinal cord tissue, neurons, cells representing the blood-brain barrier, blood, hair, nails, keratin, collagen, or any combination thereof.

[0170] As used herein, the term "imaged subject" generally refers to an object, material, specimen, sample or tissue, including, but not limited to, in vivo or ex vivo tissue.

[0171] The term "reflector" as used herein generally refers to an element that can be configured to reflect at least a portion of a light beam, including but not limited to an individual structural component, unit, or element. A reflector may be a material covering a surface configured to reflect at least a portion of a light beam. A reflector may be a retroreflector, a mirror, or other optical element configured to reflect at least a portion of a light beam.

[0172] The term "collector" as used herein generally refers to an element that can be configured to collect at least a portion of a light beam or other signal. A collector can refer to a sensor, including but not limited to an optical sensor, that can collect a portion of an optical signal.

[0173] The term "numerical aperture" as used herein generally refers to a dimensionless number that characterizes the range of angles at which a system can accept or emit light. Numerical aperture may be used in microscopy to describe the light acceptance cone of an objective lens (and therefore its light gathering ability and resolution).

[0174] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0175] Whenever the terms "not greater than," "less than," or "equal to or less than" precede the first number in a series of two or more numbers, the term "not greater than," "less than," or "equal to or less than" applies to each number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, and 1 or less.

[0176] Systems and devices for optical filtering and imaging - Patents.com In one aspect, the present disclosure provides a device for light filtering and / or imaging. The device may comprise a probe. The probe may be configured to direct a light beam from a light source to an imaging target. The probe may be configured to direct the light beam so that the light beam contacts the imaging target. The probe may be configured to collect light from the imaging target on the light beam contacting the imaging target. The device may further comprise a light filtering device. The light filtering device may be in optical communication with the probe (e.g., in the optical path). The light filtering device may comprise a chamber and a plurality of reflectors. The chamber may comprise an input and an output. The input may be configured to receive light collected by the probe. The output may be configured to transmit the light away from the chamber. The plurality of reflectors may be disposed within the chamber. The plurality of reflectors may be configured to direct the received light. For example, the plurality of reflectors may be configured to direct the light (received from the probe) in an optical path from an input of the chamber to an output of the chamber. The light may be directed by reflection between the reflectors of the plurality of reflectors. The chamber may be configured to eliminate out-of-focus light along the optical path between the input and output by reflection of the light between multiple reflectors.

[0177] In one aspect, the present disclosure provides a device for optical filtering and imaging. In one aspect, the present disclosure provides a device for optical filtering for confocal imaging. The optical filtering device can include a chamber having a plurality of reflectors. The chamber can be configured or may receive a light beam. The chamber can be configured to filter out unfocused light from the light beam. Alternatively or additionally, the chamber can include an optical filtering device. Each reflector of the plurality of reflectors can be located at a position within the chamber. Each reflector can be uniquely located from all other reflectors, or one or more reflectors can be substantially at the same position. A light beam directed into the reflecting chamber can follow a path defined by path segments between the reflectors, whereby the light beam travels from one of the reflectors to the other to define an optical path from an input into the chamber to an output from the chamber. The optical filtering device or chamber can be configured to eliminate out-of-focus light along the optical path. A light beam directed into the reflecting chamber can follow a path defined by path segments between the units, whereby the light beam travels from one of the reflectors to another in a path that returns to the first reflector at least once before reaching the output of the chamber. The path can include two or more path segments, where a path segment is a portion of the light path, that portion being between two reflectors of the plurality of reflectors, between the input of the chamber and a reflector, or between a reflector and the output of the chamber.

[0178] In another aspect, the present disclosure provides a device for optical filtering and imaging. According to some embodiments, the device for optical filtering and imaging may be a confocal imaging device. The imaging device may include a light source, a probe, an optical filtering device, and a collector. The light source may be configured or may provide a light beam to an imaging target. The probe may be in optical communication with the light source (e.g., in the optical path). The probe may be configured or may direct the light beam from the light source to the imaging target. The optical filtering device may be in optical communication with the probe. The optical filtering device may include a chamber having a plurality of reflectors. The optical filtering device may be configured or may receive the light beam from the probe. The optical filtering device may further be configured to have a path length where the light beam travels from the aperture through the reflectors to the output. The collector may be in optical communication with the optical filtering device (e.g., in the optical path). The collector may be configured or may collect the light beam from the optical filtering device. The collector may be coupled to or disposed at the output of the optical filtering device. The collector may include or be coupled to a sensor and / or an image processor. The optical beam may be usable to generate an image of the imaging subject.

[0179] In another aspect, the present disclosure provides a system for light filtering and imaging. The system may include a light source, a light filtering device, and one or more computer processors. The light source may be configured to generate a light beam. The light filtering device may be in optical communication with the light source. The light filtering device may include a chamber and a plurality of reflectors. The chamber may be configured to receive or may receive a light beam from the light source. The first reflector may be disposed within the chamber. The first reflector may be configured to or may direct at least a portion of the light beam from the first reflector within the chamber to a second reflector within the chamber. The second reflector may be configured to or may direct another portion of the light beam from a second location within the chamber to the first reflector within the chamber. A chamber comprising a plurality of reflectors (e.g., a first reflector and a second reflector) may have a longest interior dimension. The longest interior dimension may be the length of the interior of the chamber from one point to a second point. The longest internal dimension may be longer than any other length between any two other points in the chamber. The light beam may be directed into an optical path between the input of the chamber and the output of the chamber by reflection between one or more of the multiple reflectors in the chamber. The length of the optical path may be the sum of the lengths of the segments of the optical path. In some cases, the length of the optical path may be greater than the longest internal dimension of the chamber. The length of the optical path may be about 0.01 meters, 0.05 meters, 0.08 meters, 0.1 meters, 0.14 meters, 0.17 meters, 0.20 meters, 0.25 meters, 0.30 meters, 0.35 meters, 0.40 meters, 0.50 meters, 0.60 meters, 0.75 meters, 0.90 meters, 1 meter, 1.25 meters, 1.5 meters, 1.75 meters, 2 meters, 2.5 meters, 3 meters, 5 meters, 10 meters, or more.The length of the optical path may be less than or equal to about 15 meters, 11 meters, 6 meters, 4 meters, 2 meters, 1 meter, 0.80 meters, 0.65 meters, 0.55 meters, 0.45 meters, 0.5 meters, 0.25 meters, 0.20 meters, 0.18 meters, 0.16 meters, 0.13 meters, 0.10 meters, 0.07 meters, 0.04 meters, 0.02 meters, or less. The length of the optical path may be between any two of the above lengths, for example, between about 0.25 meters and 1 meter.

[0180] In some cases, the second reflector may return the light beam to the first reflector at least once before the light beam is directed out of the chamber. One or more computer processors may be operatively coupled to the light source and the light filtering device. The one or more computer processors may be individually or collectively programmed to process the light beam to generate an image.

[0181] The devices described herein can be used in any type of microscopy, including those in which confocal imaging is used. The devices described herein can be used in any laser scanning microscope, with or without a needle-tip probe or other probe, and / or with or without a pulsed laser. The devices described herein can also be used for any type of tissue imaging (e.g., as tissue is defined herein). Additionally, the reflector of the devices described herein can be a reflective surface or material. Additionally, the reflector can be an individual structural component or subunit, and / or can be a material that covers the interior surface of a chamber.

[0182] An exemplary device for confocal optical imaging is shown in FIG. 1A and FIG. 1B. The optical device may include a light source 101 providing a light beam to a beam splitter 102. The beam splitter 102 may direct a portion of the light beam to a scanning unit 103. The scanning unit 103 may scan the light beam in two dimensions (e.g., scanning in the x- and y-directions) across an imaging object 105. The scanning unit 103 may provide a depth scan (e.g., scanning in the z-direction) of the imaging object 105. The scanning unit 103 may scan in any combination of the x, y, and z directions. The scanning unit may be configured to scan a three-dimensional image, a two-dimensional image, a slice of an image in a direction (e.g., a two-dimensional slice in the xy plane repeated in the z-direction), or other types of imaging using three-dimensional or two-dimensional scanning. The scanning unit 103 may provide multiple scanning layers to form a composite image. The scanning unit 103 may provide a multi-dimensional image, for example, a two-dimensional or three-dimensional image and / or a time-dependent image. The scanning unit 103 can provide a light beam to the probe 104 before the probe 104 provides the light beam to the imaging target 105. The probe 104 can include an objective lens. Contacting the imaging target 105 with the light beam can generate a light signal. The generated light signal can be indicative of one or more characteristics of the imaging target. The light signal can be used to image the imaging target 105. The probe 104 can operate, in part, as a collector that collects light from a subject or sample. The generated light signal can be collected by the probe 104 from the imaging target 105. The probe 104 can direct the generated light signal to the beam splitter 102. The beam splitter 102 can provide at least a portion of the light signal to the optical filtering device 106. The optical filtering device 106 can generate a path length or an effective path length of the light signal that is greater than a dimension of the optical filtering device 106.According to some embodiments, the optical filtering device 106 can include one or more reflectors (e.g., mirrors, retroreflectors, or coated surfaces) that allow the optical signal (e.g., a beam of light) to reflect between the reflectors to form an optical path having a path length. By repeatedly reflecting the signal between the reflectors, the path length of the optical signal can be increased multiple times to filter out unfocused or uncollimated light. As shown in FIG. 1A, the optical filtering device 106 can include a chamber, as described in more detail with reference to FIGS. 2A and 2B. The optical filtering device 106 can generate an optical path, path length, or effective path length of the optical signal, which is greater than the maximum linear dimension within the chamber. As shown in FIG. 1A, the optical filtering device 106 can be coupled to or integrated with a collector 107. The collector 107 can collect light from the optical filtering device 106 at an end of the optical path. Alternatively or additionally, the optical filtering device may be coupled to a fiber (eg, optical fiber bundle) 108 that is included as part of or coupled to collector 107, as shown in FIG. 1B.

[0183] Examples of optical or imaging devices described with respect to Figures 1A and 1B include those that can be used in any type of microscopy, including where imaging is used. The devices described with respect to Figures 1A and 1B herein can be used in any laser scanning microscope, with or without a needle-tip probe or other probe, and / or with or without a pulsed laser. The devices described with respect to Figures 1A and 1B herein can also be used for any type of tissue imaging. The devices described with respect to Figures 1A and 1B herein can be used for confocal imaging. Examples of optical or imaging devices described with respect to Figures 2A-15 herein.

[0184] According to an embodiment of the present invention, an optical filtering device is provided that includes a chamber (e.g., a reflection chamber) to remove out-of-focus light from a collected reflected, transmitted, fluorescent, or autofluorescent optical signal. In some embodiments, the collected optical signal is a confocal microscope signal. In some embodiments, the collected optical signal is generated from a single-photon excitation optical signal that is focused on an imaging target (e.g., tissue) to be imaged. In some embodiments, the reflection chamber is configured to be used with an optical imaging device. In some embodiments, the optical imaging device is a handheld optical device. In some embodiments, the optical device is portable. In some embodiments, the optical device is a handheld device. As the collected light passes through the chamber (e.g., the reflection chamber) and reflects between the reflectors, the out-of-focus light is dispersed. The focused light can then pass through an output of the chamber and enter a collector. The output of the chamber can include an output opening of the chamber. The output opening can be an opening at the end of the reflection path. The output aperture may be an interface with or input to a collection optic, fiber (e.g., an array of collection fibers or a fiber optic bundle) at the end of the reflection path, and the output aperture may be an interface with or input to a sensor at the end of the reflection path. In some embodiments, the output aperture or collection fiber may have a minimum diameter approximately equal to the diameter of a focused collimated beam of light. In some embodiments, the minimum output aperture or fiber cross-sectional diameter or dimension may be about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, or 0.1 mm in diameter. In some embodiments, the minimum output aperture or fiber cross-sectional dimension may be about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, or 0.1 mm in diameter. In some embodiments, the cross-section of the output (and output aperture or input collection fiber) may be a slit or have a slit shape. According to some embodiments, the collected light may not be focused after collection onto the imaging device.According to some embodiments, the collected light may not be further focused in the chamber to a selected level of resolution. For example, for a resolution of about 1 to 10 microns, the collected light beam and / or collection fiber or fiber bundle may have a cross-sectional dimension (e.g., diameter) of 1.5 millimeters (mm) and / or a minimum cross-sectional diameter of about 0.2 mm. In some embodiments, the output aperture or collection fiber may have a cross-sectional dimension (e.g., diameter) similar in scale to a collimated beam of focused light. In some embodiments, the output aperture or fiber may have a minimum cross-sectional dimension (e.g., diameter) of about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, 0.1 mm or less. The output aperture or fiber may have a minimum cross-sectional dimension (e.g., diameter) of about 0.1 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or more.The output aperture or fiber may be from about 0.1mm to about 0.5mm, 0.1mm to 1mm, 0.1mm to 1.5mm, 0.1mm to 2mm, 0.1mm to 3mm, 0.1mm to 4mm, 0.1mm to 5mm, 0.1mm to 6mm, 0.1mm to 7mm, 0.1mm to 8mm, 0.1mm to 9mm, 0.1mm to 10mm, 0.5mm to 1mm, 0.5mm to 1.5mm, 0.5mm to 2mm, 0.5mm to 3mm, 0.5m m~4mm,0.5mm~5mm,0.5mm~6mm,0.5mm~7mm,0.5mm~8mm,0.5mm~9mm,0.5mm~10mm,1mm~1.5mm,1mm~2mm,1mm~3mm, 1mm~4mm,1mm~5mm,1mm~6mm,1mm~7mm,1mm~8mm,1mm~9mm,1mm~10mm,1.5mm~2mm,1.5mm~3mm,1.5mm~4mm,1.5mm~5 mm,1.5mm~6mm,1.5mm~7mm,1.5mm~8mm,1.5mm~9mm,1.5mm~10mm,2mm~3mm,2mm~4mm,2mm~5mm,2mm~6mm,2mm~7mm ,2mm~8mm,2mm~9mm,2mm~10mm,3mm~4mm,3mm~5mm,3mm~6mm,3mm~7mm,3mm~8mm,3mm~9mm,3mm~10mm,4mm~5mm,4mm It may have a minimum cross-sectional dimension (e.g., diameter) of 6mm, 4mm to 7mm, 4mm to 8mm, 4mm to 9mm, 4mm to 10mm, 5mm to 6mm, 5mm to 7mm, 5mm to 8mm, 5mm to 9mm, 5mm to 10mm, 6mm to 7mm, 6mm to 8mm, 6mm to 9mm, 6mm to 10mm, 7mm to 8mm, 7mm to 9mm, 7mm to 10mm, 8mm to 9mm, 8mm to 10mm, or 9mm to 10mm.

[0185] In some embodiments, the collimated light beam can have a cross-sectional dimension (e.g., diameter) of about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, 0.1 mm or less. The collimated light beam can have a cross-sectional dimension (e.g., diameter) of about 0.1 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or more. Fibers are available in sizes from about 0.1mm to about 0.5mm, 0.1mm to 1mm, 0.1mm to 1.5mm, 0.1mm to 2mm, 0.1mm to 3mm, 0.1mm to 4mm, 0.1mm to 5mm, 0.1mm to 6mm, 0.1mm to 7mm, 0.1mm to 8mm, 0.1mm to 9mm, 0.1mm to 10mm, 0.5mm to 1mm, 0.5mm to 1.5mm, 0.5mm to 2mm, 0.5mm to 3mm, 0.5mm to 4mm, 0.5mm~5mm,0.5mm~6mm,0.5mm~7mm,0.5mm~8mm,0.5mm~9mm,0.5mm~10mm,1mm~1.5mm,1mm~2mm,1mm~3mm,1mm~4 mm,1mm~5mm,1mm~6mm,1mm~7mm,1mm~8mm,1mm~9mm,1mm~10mm,1.5mm~2mm,1.5mm~3mm,1.5mm~4mm,1.5mm~5mm,1 .5mm~6mm,1.5mm~7mm,1.5mm~8mm,1.5mm~9mm,1.5mm~10mm,2mm~3mm,2mm~4mm,2mm~5mm,2mm~6mm,2mm~7mm,2m m~8mm,2mm~9mm,2mm~10mm,3mm~4mm,3mm~5mm,3mm~6mm,3mm~7mm,3mm~8mm,3mm~9mm,3mm~10mm,4mm~5mm,4mm~6 The cross-sectional dimensions (e.g., diameter) may be from 1 mm, 4 mm to 7 mm, 4 mm to 8 mm, 4 mm to 9 mm, 4 mm to 10 mm, 5 mm to 6 mm, 5 mm to 7 mm, 5 mm to 8 mm, 5 mm to 9 mm, 5 mm to 10 mm, 6 mm to 7 mm, 6 mm to 8 mm, 6 mm to 9 mm, 6 mm to 10 mm, 7 mm to 8 mm, 7 mm to 9 mm, 7 mm to 10 mm, 8 mm to 9 mm, 8 mm to 10 mm, or 9 mm to 10 mm.

[0186] In some embodiments, the ratio of the minimum cross-sectional diameter of the output aperture to the cross-sectional diameter of the beam of collimated light collected at the output is 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:4, 1:3, 1:2, or 1:1 or more. In some embodiments, the ratio of the minimum cross-sectional diameter of the collection fiber to the cross-sectional diameter of the collimated beam of light is greater than or equal to about 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:4, 1:3, 1:2, or 1:1. In some embodiments, the ratio of the minimum cross-sectional diameter of the output aperture collection fiber to the cross-sectional diameter of the collimated beam of light is between any two of the above values, such as between about 1:3 and about 1:2.

[0187] FIG. 2A illustrates an example of a filtering device 200 configured to remove out-of-focus light from a collected light sample 250. The collected light sample 250 can include, for example, reflected light, transmitted light, fluorescent light, or any combination thereof resulting from an illumination light beam focused at a plane or location within an imaging subject (e.g., a tissue sample in vivo or ex vivo). The filtering device 200 can include a chamber 210, an input 220 through which the collected light beam is directed into the chamber 210, and an output 240 configured to receive the focused light passing along a path 230 through the chamber 210. The output 240 can include an output aperture. The output 240 can be, include, or be configured to direct light to a collector. Alternatively or additionally, the reflective chamber can include one or more waveguides that can allow light to enter or exit the chamber via the output. The input and / or output of the chamber can be coupled to or include a waveguide.

[0188] An alignment mirror 290 can be used to direct the light sample 250 through the input 220 into the reflecting chamber 210. The alignment mirror 290 can move the direction of the light and control the angle of the light entering the reflecting chamber. The mirror 290 can change the position of the focal point of the light beam on the xy plane, or simply on a single x or y axis. Alignment of the light entering the chamber may not require any additional focal points or may not require alignment of the focal point on the z axis.

[0189] The chamber 210 may include multiple reflectors 270, 280. In one example, the chamber 210 may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more reflectors. The reflectors may be arranged in any configuration. The reflectors may have any arrangement within the chamber where light is reflected to form a light path. For example, two or more reflectors may be arranged in a side-by-side arrangement relative to other reflectors, a circular arrangement, or other shapes.

[0190] Examples of reflectors include, but are not limited to, mirrors, retroreflectors, and coated surfaces. Reflectors can have a reflective surface of any shape. For example, reflectors can have a reflective surface that is circular, square, rectangular, elliptical, or any other shape. Reflective surfaces can have dimensions (e.g., width, height, diameter, etc.) of about 5 cm, 4 cm, 3 cm, 2 cm, 1.5 cm, 1 cm, 0.5 cm or less. The reflective surface may have dimensions (e.g., width, height, diameter, etc.) of about 0.5 cm to 1 cm, 0.5 cm to 1.5 cm, 0.5 cm to 2 cm, 0.5 cm to 3 cm, 0.5 cm to 4 cm, 0.5 cm to 5 cm, 1 cm to 1.5 cm, 1 cm to 2 cm, 1 cm to 3 cm, 1 cm to 4 cm, 1 cm to 5 cm, 1.5 cm to 2 cm, 1.5 cm to 3 cm, 1.5 cm to 4 cm, 1.5 cm to 5 cm, 2 cm to 3 cm, 2 cm to 4 cm, 2 cm to 5 cm, 3 cm to 4 cm, 3 cm to 5 cm, or 3 cm to 5 cm. In one example, the reflective surface may have dimensions (e.g., width, height, diameter, etc.) of about 2 cm or less. In another example, the reflective surface may have dimensions (e.g., width, height, diameter, etc.) of about 0.5 cm to 2 cm.

[0191] FIG. 2A shows an arrangement of opposing interface parallel reflectors 270, 280. However, as previously mentioned, more than two reflectors can be used in the interface reflector arrangement, or the reflector arrangement may be in series, parallel or non-parallel orientations, or a combination of orientations. Some examples of multiple reflector or multiple chamber arrangements are shown in FIG. 9. As shown in FIGS. 2A-2C, the reflectors 270, 280 may include both a mirror 270 and a retroreflector 280 (see, e.g., FIG. 2A), a mirror 270 (see, e.g., FIG. 2B), or a retroreflector (see, e.g., FIG. 2C). Additionally or alternatively, a reflectively coated surface within the chamber may be used as a reflector. The collected light enters the reflecting chamber 210 through the input 220, and the focused or collimated light is reflected multiple times between at least two reflectors 270, 280. As the light travels between the reflectors 270, 280 in the chamber, the defocused, uncollimated, diverging, or converging light deviates from the collimated trajectory or disperses over an enlarged cross section when it reaches the location of the output 240 of the chamber and the input of the optical fiber 241. The optical fiber 241 collects the primarily focused collimated light. The output 240 may include an output aperture. The output 240 is shown in Figures 2A-2C as an input to or interface with a collector 242. The collector 242 may include a sensor. The optical fiber 241 of the collector 242 may be positioned at the output 240 to collect the collimated light at the end of the optical path where the output 240 of the chamber is the input of the fiber 241. However, the output aperture may be positioned before the optical fiber 241 with respect to the optical path 230 of the collimated beam. The output aperture may or may not have an aperture size larger or smaller than the input of the optical fiber 241 or the input of the collector 242 .

[0192] According to some embodiments, the first and second reflectors may be positioned along an axis parallel to the length of the chamber, as shown, for example, in Figures 2A-2C.

[0193] 3A-3B show examples of collection fibers 241 (of collector 242) or fiber optic bundles configured to collect light at the end of light path 230 at the output of reflector chamber 210. FIG.

[0194] In some embodiments, the collection fiber 241 is a bundle of fibers oriented linearly (e.g., slit-like) at the output 240 of the reflector chamber, as shown in FIG. 3A. In some embodiments, the collection fiber 241 is a bundle or round shape 243 at the receiver / detector side of the collection fiber, as shown in FIG. 3B. The fiber bundle 241 can be relatively larger than those used in confocal microscopy imaging for similar sized features to be imaged. For example, the fiber or linear fiber bundle may be about 1.5 mm in diameter or along its length dimension, rather than 5 micrometers. The individual fibers of the bundle may be smaller, for example about 0.2 mm in diameter.

[0195] There may be no z-component alignment used or required to focus the light on the collector since the defocused light is filtered out along the light path rather than being focused into a much smaller pinhole exit. The output may have an aperture with a cross section in the x and y plane that is relatively the same or relatively similar to the cross section in the x and y plane of the light beam passing from the input to the output of the chamber. In one example, the confocal microscope device does not include an emission pinhole for light filtering. Figures 4A-4B show a schematic light path from the input 220 of the chamber 210 to the output 240 of the chamber 210. The light path may be reflected between reflectors 270, 280. As shown in Figure 4A, the path may be folded each time the collected collimated light is reflected from a reflector to form multiple path segments 245. The path segments 245 from the input to the output after being reflected between reflectors form a light path configured to have a length along which the defocused light is removed.

[0196] The length of the path segment 245 may be similar in size to the distance between the reflectors 270, 280. At least two path segments 245 may be formed during reflection from the reflectors. The path segments may be equal to or slightly greater than the distance between the reflectors. According to some embodiments, the number of path segments 245 may be 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, or more. In one example, the number of path segments is about 2-3, 2-4, 2-5, 2-6, 2-8, 2-10, 2-12, 2-15, 2-20, 2-25, 2-30, 2-40, 2-50, 3-4, 3-5, 3-6, 3-8, 3-10, 3-12, 3-15, 3-20, 3-25, 3-30, 3-40, 3-50, 4-5, 4-6, 4-8, 4-10, 4-12, 4-15, 4-20, 4-25, 4-30, 4-40, 4-50, 5-6, 5-8, 5-10, 5-12, 5-15, 5-20, 5-25, 5-30, 5-40, 5-50, 6-8, 6-10, 6-12, It may be 6-15, 6-20, 6-25, 6-30, 6-40, 6-50, 8-10, 8-12, 8-15, 8-20, 8-25, 8-30, 8-40, 8-50, 10-12, 10-15, 10-20, 10-25, 10-30, 10-40, 10-50, 12-15, 12-20, 12-25, 12-30, 12-40, 12-50, 15-20, 15-25, 15-30, 15-40, 15-50, 20-25, 20-30, 20-40, 20-50, 25-30, 25-40, 25-50, 30-40, 30-50, or 40-50. In one example, the number of path segments 245 may be 3 or more. In another example, the number of path segments may be 5 or more. In another example, the number of path segments 245 may be about 5 to 30. According to some embodiments, the number of path segments 245 is between 5 and 30. According to some embodiments, the number of path segments 245 is between 3 and 50. According to some examples, light enters the chamber 210 at an incidence angle 250. The incidence angle 250 is approximately the same as the exit angle 255. FIG. 4B shows an effective length L 256 of the path (e.g., path length). As the angle 255 approaches zero, the effective length of the hypotenuse approaches the length of the adjacent side, which is shown as the effective length L 256.In some cases, the effective length can represent the length of the optical path. Thus, the effective length L 256 is an approximation based on small angles 255. The light reflects between reflectors in the chamber such that the focused light travels along the path until it reaches the output 240. As the collimated light travels along the path, the defocused light may diverge and scatter from the path.

[0197] The reflective chamber can include a first reflector and a second reflector. The first reflector and the second reflector can be a distance apart. The effective length L or path length can be at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, or greater than the maximum inner linear dimension of the chamber or the distance between the first and second reflectors. The effective length L or path length is about 2-3, 2-4, 2-5, 2-6, 2-8, 2-10, 2-12, 2-15, 2-20, 2-25, 2-30, 2-40, 2-50, 3-4, 3-5, 3-6, 3-8, 3-10, 3-12, 3-15, 3-20, 3-25, 3-30, 3-40, 3-50, 4-5, 4-6, 4-8, 4-10, 4-12, 4-15, 4-20, 4-25, 4-30, 4-40, 4-50, 5-6, 5-8, 5-10, 5-12, 5-15, 5-20, 5-25, 5-30, 5-40, 5- 50,6~8,6~10,6~12,6~15,6~20,6~25,6~30,6~40,6~50,8~10,8~12,8~15,8~20,8~25,8~30,8~40,8~50,10~12,10~15,10~20,10~25,10~30,10~40,10~50,12~ The length may be 15, 12-20, 12-25, 12-30, 12-40, 12-50, 15-20, 15-25, 15-30, 15-40, 15-50, 20-25, 20-30, 20-40, 20-50, 25-30, 25-40, 25-50, 30-40, 30-50, or 40-50. In one example, the path length is at least three times the maximum inner linear dimension of the chamber. In another example, the path length is at least three times the distance between the first reflector and the second reflector. In another example, the path length is at least five times the maximum inner linear dimension of the chamber. In another example, the path length is at least five times the distance between the first reflector and the second reflector. In another example, the path length is about five times to about thirty times the maximum inner linear dimension of the chamber. In another example, the path length is about 5 times to about 30 times the distance between the first reflector and the second reflector.

[0198] The larger the effective length L, the more the defocused light can diverge or dissipate from the collimated light beam along the path. The effective length L of the collimated light beam in the chamber corresponds to the angle of incidence 250 (α) and the height H of the chamber. The shallower the angle α of the incident light or the larger the height H, the more reflections there will be and the larger the effective length L. The relationship between the effective length L, the height H, and the angle α can be expressed by L=H / α. (See, for example, FIG. 4B). Thus, the angle α or the height H (e.g., the diameter of the reflector) can be selected to provide a selected effective length L. According to some embodiments, the effective length L or path length is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 3, 4, 5 meters (m) or more. According to some embodiments, the effective length L or path length is less than about 5, 4, 3, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 meters.The effective length L or path length may be about 0.1 m to 0.2 m, 0.1 m to 0.3 m, 0.1 m to 0.4 m, 0.1 m to 0.5 m, 0.1 m to 0.6 m, 0.1 m to 0.7 m, 0.1 m to 0.8 m, 0.1 m to 0.9 m, 0.1 m to 1 m, 0.1 m to 1.5 m, 0.1 m to 2 m, 0.1 m to 3 m, 0.1 m to 4 m, 0.1 m to 5 m, 0.2 m to 0.3 m, 0.2 m to 0.4 m, 0.2 m to 0.5 m, 0.2 m to 0.6 m, 0.2 m to 0.7 m, 0.2 m to 0.8 m, 0.2 m to 0.9 m, 0.2 m to 1 m, 0.2 m to 1.5 m, 0.2 m to 2 m, 0.2 m to 3 m, 0.2 m to 4 m, 0.2 m to 5 m, 0.3 m to 0.4 m, 0.3 m to 0.5 m, 0.3 m to 0.6 m, 0.3 m to 0.7 m, 0.3 m to 0.8 m, 0.3 m to 0.9 m, 0.3 m to 1 m, 0.3 m to 1.5 m, 0.3 m to 2 m, 0.3 m to 3 m, 0.3 m to 4 m, 0.3 m to 5 m, 0.4 m to 0.5 m, 0.4 m to 0.6 m, 0.4 m to 0.7 m, 0.4 m to 0.8 m, 0.4 m to 0.9 m, 0.4 m to 1 m, 0.4 m to 1.5 m, 0.4 m to 2 m, 0.4 m to 3 m, 0.4 m to 4 m, 0.4 m to 5 m, 0.5 m to 0.6 m, 0.5 m to 0.7 m, 0.5 m to 0.8 m, 0.5 m to 0.9 m, 0.5 m to 1 m, 0.5 m to 1.5 m, 0.5 m to 2 m, 0.5 m to 3 m, 0.5 m to 4 m, 0.5 m to 5 m, 0.6 m to 0.7 m, 0.6 m to 0.8 m, 0.6 m to 0.9 m, 0.6 m to 1 m, 0.6 m to 1.5 m, 0.6 m to 2 m, 0.6 m to 3 m, 0.6 m to 4 m, 0.6 m to 5 m, 0.7 m to 0.8 m, 0.7 m to 0.9 m, 0.7 m to 1 m, 0.7 m to 1.5 m, 0.7 m to 2 m, 0.7 m to 3 m, 0.7 m to 4 m, 0.7 m to 5 m, 0.8 m to 0.9 m, 0.8 m to 1 m, 0.8 m to 1.5 m, 0.8 m to 2 m, 0.8 m to 3 m, 0.8 m to 4 m, 0.8 m to 5 m, 0.9 m to 1 m, 0.9 m to 1.5 m, 0.9 m to 2 m, 0.9 m to 3 m, 0.9 m to 4 m, 0.9 m to 5 m, 1 m to 1.5 m, 1 m to 2 m, 1 m to 3 m, 1 m to 4 m, 1 m to 5 m, 1.5 m to 2 m, 1.5 m to 3 m, 1.5 m to 4 m, 1.5 m to 5 m, 2 m to 3 m, 2 m to 4 m, 2 m to 5 m, 3 m to 4 m, or 4 m to 5 m. According to some embodiments, the effective length L or path length is about 0.3 m to 1.5 m. In one example, the effective length L or path length is greater than about 0.5 m.In one example, the effective length L or path length is about 1 m or greater. In another example, the effective length L or path length is about 1.5 m or greater.

[0199] According to some embodiments, the filtering device 200 and the reflecting chamber 210 are configured or sized to fit within a handheld device. In addition to selecting the height H, the angle α, and the selected effective length L, the width W of the chamber can also be selected. The effective length L or path length depends on the angle α and the height H of the chamber, but may be independent of the width. Thus, by shortening the width, the width of the chamber can be compressed to make the chamber fit within the handheld device without changing the effective length L. However, some light (e.g., power) may be lost with each reflection, thus limiting the degree of chamber compression in the width direction to optimize the image resolution. According to some embodiments, the width of the chamber may be about 10 to 200 mm. In one example, the distance separating the reflectors (e.g., the distance between the first and second positions within the chamber) may be about 1 centimeter (cm), 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 12 cm, 15 cm, 20 cm, or more. In another example, the distance separating the reflectors (e.g., the distance between a first location and a second location within the chamber) may be about 20 cm, 15 cm, 12 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, 1 cm or less.In another example, the maximum inner linear dimension of the chamber is about 1 cm to 2 cm, 1 cm to 3 cm, 1 cm to 4 cm, 1 cm to 5 cm, 1 cm to 6 cm, 1 cm to 7 cm, 1 cm to 8 cm, 1 cm to 9 cm, 1 cm to 10 cm, 1 cm to 12 cm, 1 cm to 15 cm, 1 cm to 20 cm, 2 cm to 3 cm, 2 cm to 4 cm, 2 cm to 5 cm, 2 cm to 6 cm, 2 cm to 7 cm, 2 cm to 8 cm, 2 cm to 9 cm, cm,2cm~10cm,2cm~12cm,2cm~15cm,2cm~20cm,3cm~4cm,3cm~5cm,3cm~6cm,3cm~7cm,3cm~8cm,3cm~9cm,3 cm~10cm,3cm~12cm,3cm~15cm,3cm~20cm,4cm~5cm,4cm~6cm,4cm~7cm,4cm~8cm,4cm~9cm,4cm~10cm,4cm~ 12cm, 4cm~15cm, 4cm~20cm, 5cm~6cm, 5cm~7cm, 5cm~8cm, 5cm~9cm, 5cm~10cm, 5cm~12cm, 5cm~15cm, 5cm~20 cm,6cm~7cm,6cm~8cm,6cm~9cm,6cm~10cm,6cm~12cm,6cm~15cm,6cm~20cm,7cm~8cm,7cm~9cm,7cm~10cm, It may be 7cm-12cm, 7cm-15cm, 7cm-20cm, 8cm-9cm, 8cm-10cm, 8cm-12cm, 8cm-15cm, 8cm-20cm, 9cm-10cm, 9cm-12cm, 9cm-15cm, 9cm-20cm, 10cm-12cm, 10cm-15cm, 10cm-20cm, 12cm-15cm, 12cm-20cm, or 15cm-20cm. In one example, the maximum inner linear dimension of the chamber is 15cm or less. In another example, the maximum inner linear dimension of the chamber is 10cm or less.

[0200] As shown in FIG. 2A, the reflectors can be parallel and interfaced. Alternatively or additionally, the reflectors can be set to off. According to some embodiments, at least one of the reflectors can comprise a retroreflector having properties that reflect light in a path parallel to the path the light was received at the retroreflector. According to some embodiments, at least one of the reflectors can comprise a mirror. FIG. 2A shows a reflector 270, 280 that combines a retroreflector 280 and a mirror 270 for use in the reflection chamber 210. Furthermore, FIG. 2A is a schematic diagram of an exemplary light path 230 that uses a retroreflector 280 and a mirror 270 as reflectors 270, 280, respectively. Additional examples of the light paths of FIG. 2A are further shown in FIG. 8. The optical properties of the retroreflector 280 allow light to return in a direction parallel to the incident light, which can correct misalignment between opposing reflectors by returning light in a parallel orientation to the incident light. FIG. 2B shows a schematic of a reflective chamber 210 using a mirror 270 as the reflector 270, forming the optical path shown diagrammatically. FIG. 2C shows a schematic of a reflective chamber 210 using opposing parallel retroreflectors 280 as the reflectors 280, forming the optical path shown diagrammatically. As shown in FIG. 2A, the retroreflector 280 in combination with the mirror 270 nearly doubles the effective length L or path length compared to the two retroreflector element path shown in FIG. 2C. The combination of a mirror and a retroreflector results in fewer reflections for a given path length L compared to the two retroreflector element path shown in FIG. 2C.

[0201] The mirror reflector 270 in the reflective chamber 210 may be flat or may have a dielectric coating that preferentially reflects a selected wavelength or range of wavelengths. According to one example, the reflective coating may preferentially reflect wavelengths in the range of about 700 nm to 900 nm. The coating may provide some filtering effect to block some ambient light, especially with multiple reflections in the path. The retroreflector may be, for example, a corner cube retroreflector. The retroreflector may be configured for total internal reflection of light. The retroreflector may provide reflective properties such as low attenuation or absorption, or high reflectivity, and may be coated with gold plating or other coatings that may provide some preferential wavelength reflectivity (e.g., about 700 nm to 900 nm).

[0202] As shown in Figure 9, a light filtering device having multiple reflective elements is shown. Multiple interface reflector configurations (270a, 280a) and (270b, 280b) are shown in series. Collected light 250 follows a path. Some embodiments may include non-parallel orientations or combinations of reflector orientations.

[0203] According to some embodiments, the probe may comprise an optical filtering device or chamber as described herein. According to some embodiments, the optical filtering device or chamber may be configured to fit or be dimensioned to fit within a handheld optical device that comprises the probe.

[0204] FIG. 5 illustrates an example of focusing and scanning optics of an optical device (e.g., a handheld device) using the optical filtering device 200 as described with reference to FIGS. 1A-4B. The optical filtering device 200 may include one or more focusing 260 and scanning optics 265. According to some embodiments, the focusing unit 260 of the optical device may be used to scan and create a depth profile of an imaging object (e.g., tissue). The optical device may further include additional optical elements such as a light source 285 (e.g., a laser), a wave plate 286 (e.g., a half-wave plate), a beam splitter 287 (e.g., polarized or non-polarized), an alignment window 288, one or more relay lenses 289, a dichroic mirror 290, a probe 291, an alignment unit 292, or any combination thereof. FIGS. 5 and 6 illustrate an example of a focusing unit 260 of an optical device configured to simultaneously adjust the depth and position of the focus of the excitation light. FIG. 6 illustrates one or more of the focusing optics 260 and scanning optics 265 of FIG. 5 with additional collection optics 293 for collecting additional signals, such as multiphoton and SHG signals, that can be used to stack images or depth profiles of the imaged tissue. FIG. 7 illustrates an example of the focusing 260 and scanning 265 components or units of the optical device of FIG. 5 or FIG. 6 positioned within a handle 700 of a handheld optical device. In one example, the optical device can be configured for confocal imaging. In another example, the optical device can be configured for multiphoton imaging. In another example, the optical device can be configured for tandem confocal and multiphoton imaging.

[0205] 5, 6 and 7, the confocal imaging configuration described herein may be an optical device including an optical fiber 285 configured to transmit light from a laser to an optical device. The optical fiber 285 may be a single mode fiber, a multimode fiber, or a bundle of fibers. The optical fiber 285 may be a bundle of fibers configured to transmit light from multiple lasers or light sources, either pulsed or continuous beams, to the optical device. The optical fiber 285 may be coupled to a frequency multiplier 295 that converts the frequency to a predetermined excitation frequency (e.g., by multiplying the frequency by one or more factors). The frequency multiplier 295 may transmit the light from the optical fiber 285 to a half wave plate 286. The excitation light may be directed through the half wave plate 286 to shift the polarization direction of the light along the axis of the half wave plate 286. The light may be sent through a beam splitter 287 that directs a portion of the excitation light to a power monitor. Other sensors may be included in the probe and the power monitor. The sensors and monitors can provide additional information about the imaging device or the imaged subject (e.g., a sample) that can be included as data with the depth profile.

[0206] The illumination or excitation light can then be directed to an afocal z-axis scanner 260. The afocal z-axis scanner 260 can include a movable lens and an actuator (e.g., a voice coil) coupled to the movable lens. The afocal z-axis scanner (e.g., a focusing unit) can be disposed in an optical device between the light source and the probe. The afocal z-axis scanner 260 can converge or diverge the collimated light beam and move the focal point axially during imaging. By moving the focal point axially, a depth profile can be imaged. The illumination or excitation light can then be directed to a scanning unit 265 (e.g., a MEMS mirror). The scanning unit can be disposed in an optical device between the light source and the probe. The MEMS mirror 265 can enable scanning by moving the focal point in a horizontal or XY plane. According to some representative embodiments, the afocal Z scanner 260 and the MEMS mirror 265 are separately actuated by actuators driven by coordinated computer control such that their movements are synchronized to result in a synchronized movement of the focal point in the tissue. According to some representative embodiments, by moving both the movable lens and the MEMS mirror 265, the angle between the focal plane and the optical axis can be changed, allowing imaging of a depth profile through a plane (e.g., an inclined plane or focal plane as defined herein).

[0207] The MEMS mirror scanner 265 may be configured to direct at least a portion of the light through one or more relay lenses 289. The one or more relay lenses 289 may be configured to direct the light to a dichroic mirror 290. The dichroic mirror 290 may direct the excitation light into a probe 291, which may include one or more objective lenses. The probe 291 may be configured to direct the light to interact with an imaging object (e.g., tissue of a subject). The probe 291 may be configured to collect one or more signals generated by the light interacting with the imaging object, including but not limited to reflected light. One or more of the signals collected by the probe 291 may include reflection, transmission, fluorescence / autofluorescence signals, or any combination thereof, generated by the light interacting with the imaging object. A subset of the one or more signals collected by the probe 291 may include single photon or multiphoton generated signals.

[0208] The signal collected by the objective lens of the probe 291 can trace the reverse path of the light that generated the signal. In some embodiments, the returned signal can include an RCM or a fluorescence / autofluorescence single photon signal. The returned light can pass through a beam splitter 287, and at least a portion of the returned light is directed to the optical filtering device 200. The signal can contact an alignment unit 292 that can direct the light to the optical filtering device 200. The optical filtering device 200 can be coupled to the collector 242. The alignment unit 292 can include, for example, a mirror 270 that moves the light in the x-axis and y-axis and directs the light through the input 220 of the reflecting chamber 210. The collector 242 can include, for example, an optical fiber, such as a fiber bundle, as described with reference to FIGS. 3A and 3B, configured to collect the collimated light reflected on the optical path through the reflecting chamber 210.

[0209] The beam splitter 287 may be a polarization selective beam splitter. Thus, the off-axis returned, rotated or shifted polarization components can be directed by the beam splitter 287 to the filtering device 200. The rotated reflected light can represent reflections from birefringent objects, structures, features or molecules in the imaging subject (e.g., imaged tissue). For example, epidermal, connective tissue and dermal structures, cell types, pigments important for clinical imaging are birefringent and can rotate the polarity of the light. The specular reflected light may be dominant and brighter compared to the rotated light and retraces its original path back through the beam splitter 287. The polarizing beam splitter 287 can thus operate to select the light corresponding to the structure that rotated the light and direct it to the alignment unit 292 and then to the reflection chamber 210. The specular reflected light can be directed through a separate channel to a different reflection chamber or can dissipate as it returns through the beam splitter 287. The reflected signal can be further split via additional separate channels based on polarization or wavelength to different reflector chambers for processing.

[0210] The alignment unit 292 may be configured to move the light 250 signal separated by the beam splitter in the x-axis and y-axis to direct the light through the input 220 into the reflecting chamber 210 at a desired angle α. The optical filtering device 200 may be configured such that no further focusing of the light directed from the beam splitter 287 onto the optical fiber is used or no z-direction positioning of the optical focus is used. According to some embodiments herein, the x-axis and y-axis positioning or angle of the light entering the aperture 220 can be selected by observing the light or image from the output of the optical fiber 241. The optical fiber 241 may be a single mode fiber, a multimode fiber, or a bundle of fibers. The optical fiber 241 may be coupled to a photodetector for detecting the reflected signal.

[0211] 16-18, a beam de-expander unit can be added to the optical path before reaching the optical filtering device described herein in FIG. 1-9. The beam de-expander unit may comprise one or more lenses (e.g., a first lens and a second lens). The beam de-expander unit may comprise a chamber (e.g., a chamber of the beam de-expander unit). One or more walls of the chamber of the beam de-expander unit may be deformable walls (e.g., deformable inner or deformable outer). One or more walls of the chamber of the beam de-expander unit may not be deformable walls. The beam de-expander unit can include an adjustment structure. The adjustment structure may be configured to deform the deformable walls of the chamber of the de-expander. Thus, the removal of off-axis light can be further amplified when passing through the beam de-expander unit before entering the chamber of the optical filtering device described herein. In addition, the beam de-expander unit can reduce or narrow the beam diameter of the optical path.

[0212] According to some embodiments, the beam deexpander unit can include a chamber (e.g., a chamber of the beam deexpander unit). The beam deexpander (e.g., the beam deexpander unit) can include two lenses (e.g., a first lens and a second lens) disposed at opposite ends of the chamber of the beam deexpander unit.

[0213] The beam deexpander (e.g., beam deexpander unit) may be provided with adjustment structures to allow precise spacing between the lenses so that the light output from the beam deexpander (e.g., beam deexpander unit) is a focused, collimated beam of light.

[0214] In some embodiments, the adjustment structure (e.g., adjustment unit, adjustment structure unit, adjustment device, adjustment structure device, adjustment mechanism) can comprise one or more screws (e.g., a first screw and a second screw), a press, an actuator, a beam profiler, one or more jaws (e.g., a first jaw and a second jaw), one or more die elements (e.g., a first die element and a second die element), one or more block elements (e.g., a first block element and a second block element), a knob, a lever, or a combination thereof. In some embodiments, the adjustment structure can enable precise spacing between the first lens and the second lens using adjustment structure elements such as one or more screws (e.g., a first screw and a second screw), a press, an actuator, a beam profiler, one or more jaws (e.g., a first jaw and a second jaw), one or more die elements (e.g., a first die element and a second die element), one or more block elements (e.g., a first block element and a second block element), a knob, a lever, or a combination thereof. According to some embodiments, the adjustment structure may be configured to adjust the distance between the lenses.

[0215] In some embodiments, to adjust the distance between the lenses, the adjustment structural element is actuated to deform one or more walls of the chamber at one or more locations between the lenses. When actuated, the adjustment structural element engages and deforms the walls of the chamber, decreasing the length of the chamber, thereby decreasing the distance between the lenses. According to some embodiments, the deformation is uniform or symmetrical to provide a uniform change in the distance between the lenses. According to some embodiments, the actuator can actuate an element or mechanism that rotates a screw that moves the wall of the chamber outward (or inward). The screw can engage with a thread on the chamber wall. The screw can be disposed perpendicular to the optical path through the chamber. In other embodiments, to adjust the distance between the lenses, the actuator can drive a press toward the second jaw, which can drive a block element to symmetrically compress and deform the chamber of the beam deexpander wall inward. The deformation of the chamber of the beam deexpander wall can decrease the distance between the lenses. In some embodiments, the distance between the lenses can be about 1 millimeter (mm) to about 150 mm. In some embodiments, the distance between the lenses may be about 1 mm, 2 mm, 5 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 15 mm, 20 mm, 25 mm, 50 mm, 75 mm, 100 mm, 150 mm or more. In some embodiments, the distance between the lenses may be about 1 mm, 2 mm, 5 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 15 mm, 20 mm, 25 mm, 50 mm, 75 mm, 100 mm, or 150 mm or less. In some embodiments, the distance between the lenses may be between any two of the above values, for example, between about 5 mm and about 15 mm.

[0216] According to some embodiments, the chamber of the beam deexpander may be deformable. For example, one or more walls of the chamber of the beam deexpander may be deformable to decrease or increase the distance between opposing ends of the chamber (i.e., when lenses are disposed at opposing ends of the chamber). Alternatively or additionally, for example, one or more walls of the chamber of the beam deexpander may be deformable to increase or decrease the distance between a first wall and a second wall of one or more walls of the chamber of the beam deexpander. According to some embodiments, the chamber of the beam deexpander (e.g., the walls of the chamber) may be inwardly deformable. Alternatively or additionally, the chamber of the beam deexpander (e.g., the walls of the chamber) may be outwardly deformable.

[0217] The light beam enters and passes through a beam deexpander (e.g., a chamber of the beam deexpander). The collimated light may exit the beam deexpander and be directed to an optical filtering device comprising a reflective chamber (e.g., a chamber of the optical filtering device described with reference to Figures 1-9). The optical filtering device may comprise a collection fiber. The collection fiber may be configured to collect the light at an end of the chamber (e.g., the chamber of the optical filtering device).

[0218] FIG. 16 illustrates an example of a schematic of an optical beam path 1631 through a beam de-expander 1610 (e.g., a beam de-expander unit). The collected or returned light (which may include a collimated beam 1630) may follow the optical beam path 1631 by entering and passing through the beam de-expander 1610 before reaching the optical filtering device described herein in FIGS. 1-9. The beam de-expander 1610 may comprise a first lens 1611 and a second lens 1612. The first lens 1611 and the second lens 1612 may have a first focal length F1 and a second focal length F2, respectively. In some embodiments, the focal length F1 and the focal length F2 may be different. In some embodiments, the distance between the lenses 1611, 1612 may be the sum of the focal lengths F1, F2 of the lenses 1611, 1612, respectively.

[0219] In some embodiments, the focused light beam input to the deexpander 1610 may include returning light that includes focused light, as described herein with reference to FIGS.

[0220] The collected light, including the collimated light beam 1630, may be directed into the beam deexpander 1610 through an input 1615 of the beam deexpander (e.g., beam deexpander unit) 1610. The collected light may enter the input 1615 through a first lens 1611 of the beam deexpander 1610, whereby the collected light may be focused, but the divergence or convergence of the off-axis light is amplified. The second lens 1612 may have a shorter focal length than the first lens 1611 and is positioned such that the collimated beam 1630 entering the beam deexpander is collimated as it exits the output 1620 (e.g., beam deexpander unit) of the beam deexpander 1610. The collimated beam 1630 may include a diameter d1 before entering the input 1615 of the beam deexpander 1610. The collimated beam 1630 may include a diameter d2 after exiting the output 1620 of the beam de-expander 1610. As shown in FIG. 16, the beam de-expander 1610 (e.g., a beam de-expander unit) may reduce the diameter d1 of the collimated beam 1630 before entering the input 1615 to the diameter d2 of the collimated beam 1630 after exiting the output 1620 while amplifying the rejection of off-axis light. In some embodiments, the diameter d1 is greater than the diameter d2. In some embodiments, the beam de-expander 1610 may comprise two positive focal length lenses. In some embodiments, the beam de-expander 1610 may comprise two negative focal length lenses. Alternatively or additionally, the beam de-expander 1610 may comprise a combination of positive and negative focal length lenses.

[0221] The beam deexpander (e.g., beam deexpander unit) 1610 can comprise two or more lenses. The beam deexpander 1610 can comprise three or more lenses, four or more lenses, five or more lenses, six or more lenses, seven or more lenses, eight or more lenses, nine or more lenses, or ten or more lenses. The beam deexpander (e.g., beam deexpander unit) 1610 can comprise two or less lenses. The beam deexpander 1610 can comprise three or less lenses, four or less lenses, five or less lenses, six or less lenses, seven or less lenses, eight or less lenses, nine or less lenses, or ten or less lenses. The beam deexpander 1610 can alternatively or additionally comprise a curved mirror (e.g., the chamber of the beam deexpander).

[0222] 17A-17D, a beam de-expander (e.g., beam de-expander unit) 1700 can be disposed between the input 220 of the optical filtering device 200, which can lead to the chamber 210 of the optical filtering device 200 described herein with reference to FIGS. 1-9, and the collected optical beam (e.g., return light) 1750. As shown in FIG. 17A, the collected optical beam 1750 can pass through a beam splitter 287, and at least a portion of the return light can pass through the beam de-expander 1700 before being directed to the input 220 of the optical filtering device 200. The collected optical beam 1750 can include a collimated beam before entering the beam de-expander 1700. The collected optical beam 1750 can include a collimated beam after exiting the beam de-expander 1700.

[0223] 17A-17D, a beam deexpander (e.g., beam deexpander unit) 1700 may comprise lenses 1711, 1712 attached to either end of a chamber of a beam deexpander 1730. A collected light beam 1750 may be collected by a probe or imaging device as described herein with reference to FIGS. 1-9. The collected light beam 1750 may pass through the lenses 1711, 1712 and the chamber of the beam deexpander 1730. The chamber of the beam deexpander 1730 may comprise at least one wall structure 1735. The chamber of the beam deexpander 1730 may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten wall structures 1735.

[0224] The wall structure 1735 may be deformable. In some embodiments, the wall structure 1735 may be outwardly deformable. In some embodiments, the wall structure 1735 may be inwardly deformable. Alternatively or additionally, in some embodiments, the wall structure 1735 may be inwardly and outwardly deformable. In some embodiments, the wall structure 1735 may be outwardly deformable to reduce the distance between the lenses 1711, 1712 (e.g., to reduce the length of the chamber of the beam deexpander 1730 between the first lens 1711 and the second lens 1712 from a first length L1 (as shown in FIG. 17C) to a second length L2 (as shown in FIG. 17D)).

[0225] 17B and 17D, the wall structure 1735 of the chamber of the beam deexpander 1730 can include threaded openings 1741, 1742. The threaded openings can include an orientation of opposing threaded openings 1741, 1742. The opposing threaded openings 1741, 1742 may or may not be perpendicular to an axis between two lenses of the chamber of the beam deexpander 1730 (e.g., the axis may correspond to an optical path of a beam passing through a first lens 1711, through the chamber of the deexpander 1730, and exiting through a second lens 1712). The opposing threaded openings 1741, 1742 can receive screws 1751, 1752 (as shown in 17B and 17C), respectively. The screws 1751, 1752 can be rotated to buckle the wall structure 1735 of the chamber of the beam deexpander 1730 outward. Alternatively or additionally, the screws 1751, 1752 can be rotated to buckle inward the wall structure 1735 of the chamber of the beam deexpander 1730. The screws 1751, 1752 can pull the first lens 1711 and the second lens 1712 towards each other, shortening the first length L1 to the second length L2 of the chamber of the beam deexpander 1730.

[0226] 17B, ​​the adjustment structure (e.g., adjustment unit, adjustment structure unit, adjustment device, adjustment structure device, adjustment mechanism) can include a T-shaped structure 1760. The T-shaped structure can include a block portion 1765 that can be inserted into an opening 1770 in the wall structure 1735. The block portion 1765 can extend into a chamber of the beam deexpander 1730 and can be disposed between the first lens 1711 and the second lens 1712. The block portion 1765 can include an opening 1775 that can be aligned with the optical path 1631 of the beam. The opening 1775 of the block portion 1765 can allow the optical beam to pass through the block portion 1765. The block portion 1765 can engage with a first end 1753 of the screw 1752 and a second end 1754 of the screw 1751 when the screws 1751, 1752 rotate. Rotation of the screws 1751 , 1752 allows for symmetrical adjustment of the deformation of the chamber of the wall structure 1735 of the beam deexpander 1730 .

[0227] The chamber of the beam deexpander 1730 can include a deformable rigid material that can maintain the shape of the chamber of the beam deexpander 1730 when deformed. The deformable rigid material can include one or more metals, such as stainless steel, aluminum, or any other metal or material that can maintain a rigid deformable shape.

[0228] 17C, the distance between the first lens 1711 and the second lens 1712 may be initially set (e.g., during manufacturing) to be slightly greater than a desired distance. For example, the desired distance may be the sum of the focal lengths F1, F2 of the lenses 1711, 1712, respectively. In other embodiments, the initial distance between the first lens 1711 and the second lens 1712 may be set to be slightly less than or equal to the distance that comprises the sum of the focal lengths of the lenses 1711, 1712.

[0229] The beam deexpander (e.g., beam deexpander unit) 1700 can be adjusted (e.g., by an adjustment structure). The adjustment structure (e.g., adjustment unit, adjustment structure unit, adjustment device, adjustment structure device, adjustment mechanism) may adjust the spacing between the first lens 1711 and the second lens 1712 to provide a collimated beam of focused light to the input of the optical filtering device 200 and subsequently to the location of the collection fiber or collector at the end of the reflecting chamber 210 of the optical filtering device 200 (as shown in FIG. 17A).

[0230] To adjust the beam deexpander 1700, the adjustment structure can be used to determine the beam diameter. In some embodiments, the adjustment structure can be used to determine a small beam diameter. According to some embodiments, a beam profiler can be used to determine a smaller beam diameter. As shown in FIG. 17C, the beam diameter can be monitored by adjusting the screws 1751, 1752. The beam diameter can indicate an amplified removal of off-axis light (e.g., a smaller beam diameter can indicate a greater amplified removal of off-axis light). When the beam deexpander (e.g., beam deexpander unit) 1700 is adjusted by the adjustment structure, elements of the adjustment structure (e.g., the screws 1751, 1752 and the T-shaped structure 1760) may or may not be removed (as shown in FIG. 17D).

[0231] 18A-18C show a beam de-expander (e.g., beam de-expander unit) 1800 configured to be deformed to change the length between a first lens 1811 and a second lens 1812, as similarly described with reference to FIGS. 17A-17D. The lenses 1811, 1812 may be mounted on either side of a chamber of the beam de-expander 1830. A collected (e.g., returned) light beam may pass through the lenses 1811, 1812 and the chamber of the beam de-expander 1830. The collected light beam may include a collimated beam before entering the beam de-expander 1800. The collected light beam may include a collimated beam after exiting the beam de-expander. The chamber of the beam de-expander 1830 may include a sidewall 1835. The chamber of the beam de-expander 1830 can include a single wall, two or more side walls, three or more side walls, four or more side walls, five or more side walls, six or more side walls, seven or more side walls, eight or more side walls, nine or more side walls, ten or more side walls, or eleven or more side walls. The side walls 1835 can include cutouts or openings 1840 that can allow for weakening or deformation of the side walls 1835. Weakening or deformation of the side walls 1835 can help reduce the distance between the lenses 1811, 1812 (e.g., shorten the length of the chamber of the beam de-expander 1830 between the first lens 1811 and the second lens 1812). The side walls 1835 can include, for example, a material that maintains its shape when deformed. The material of the side walls 1835 can include a metal such as aluminum, stainless steel, or any other metal or material that can maintain its shape when deformed.

[0232] Referring to FIG. 18A, the beam deexpander (e.g., beam deexpander unit) 1800 can include an adjustment structure. The adjustment structure may or may not be removable from the beam deexpander 1800. The adjustment structure can include a press 1850 that can include an actuator 1855. The press 1850 and the actuator 1855 can operate to adjust the beam deexpander 1800. The press 1850 can include a first jaw 1861 and a second jaw 1862. The first jaw 1861 and the second jaw 1862 can include an orientation that includes opposed parallel jaws. The first jaw 1861 and the second jaw 1862 can be movable. The first jaw 1861 and the second jaw 1862 can be fixed. Alternatively or further, the first jaw 1861 can be fixed and the second jaw 1862 can be movable. Alternatively and additionally, the first jaw 1861 may be movable and the second jaw 1862 may be fixed. The first jaw 1861 and the second jaw 1862 may face each other in an orientation perpendicular to an axis between two lenses of the chamber of the beam deexpander (e.g., the axis may correspond to the path of the beam passing through the beam deexpander 1800 (e.g., the beam deexpander unit)). The first jaw 1861 may comprise a first die element 1881 and the second jaw 1862 may comprise a second die element 1882. The die elements 1881, 1882 may be fixed to the inner surface of the jaws 1861, 1862, respectively. The jaws 1861, 1862 may be disposed on each side of the beam deexpander 1830, respectively, with the die elements 1881, 1882 in contact with the sidewall 1835 of the chamber of the beam deexpander 1800. The jaws 1861, 1862 may be movably coupled by slidable alignment screws 1863, 1864 (as shown in FIG. 18A ) that may align the jaws 1861, 1862 as they move toward and away from one another. The jaws 1861, 1862 may be moved toward and away from one another by adjustment structure, which may include an actuator 1855.Alternatively or additionally, the adjustment structure may comprise a knob, a lever, one or more screws, one or more jaws, a press, one or more block elements, a die element, or a combination thereof. In some cases, throughout, a block element may refer to an element of a die press (e.g., a die element). The die element may contact a wall of a chamber of the beam deexpander. In some embodiments, the actuator 1855 may comprise a knob 1870 (or lever) and an additional screw 1875 that may drive the press block 1890 toward the second jaw 1862, which may move the second jaw 1862 inward toward the first jaw 1861. In some embodiments, the actuator 1855 may comprise a knob 1870 (or lever) and an additional screw 1875 that may drive the press block 1890 away from the second jaw 1862, which may move the second jaw 1862 outward from the first jaw 1861. When the press block 1890 is actuated by the actuator 1855, the gap between the jaws 1861, 1862 can be decreased or increased. When the gap between the jaws 1861, 1862 is decreased, the first block element 1883 and the second block element 1884 (as shown in FIG. 18A ) can symmetrically compress and inwardly deform the side walls 1835 of the chamber of the beam deexpander 1830.

[0233] The distance between the first lens 1811 and the second lens 1812 may be initially set (e.g., during manufacturing) to be slightly larger than the desired distance. For example, the desired distance may be the sum of the focal lengths F1, F2 of the lenses 1811, 1812, respectively. In other embodiments, the initial distance between the first lens 1811 and the second lens 1812 may be set to be slightly smaller or equal to the distance including the sum of the focal lengths of the lenses 1811, 1812. An adjustment structure (e.g., an adjustment unit, an adjustment structure unit, an adjustment device, an adjustment structure device, an adjustment mechanism) may be used to adjust the beam de-expander (e.g., a beam de-expander unit) 1800. To pivot the beam de-expander 1800, the adjustment structure may be used to determine the beam diameter (e.g., a smaller beam diameter may represent a greater amplified removal of off-axis light). According to some embodiments, a beam profiler may be used to determine the smaller beam diameter. The beam diameter may be monitored when the block press 1890 is actuated by the actuator 1855. After the beam deexpander 1800 has been tuned, elements of the tuning structure (eg, press 1850) can be removed.

[0234] 6 and 7, another subset of the one or more signals may be sent to the collection arrangement 293 via the dichroic mirror 290 and detected by one or more photodetectors described herein, for example, in the detector block 1108 of FIG. 11B. The subset of the one or more signals may comprise multi-photon signals, which may include, for example, SHG and / or two-photon autofluorescence and / or two-photon fluorescence signals. The collection arrangement 293 may include optical elements (e.g., lenses or mirrors). The collection arrangement 293 may direct the collected light through a light guide to one or more photosensors. The light guide 294 may be a liquid light guide, a multimode fiber, or a bundle of fibers.

[0235] The optical device may have sensors other than the power sensor. Information from the sensors may be used or recorded along with the depth profile to provide further enhanced information about the probe and / or subject. For example, other sensors in the optical device may include a position sensor, a GPS sensor, a temperature sensor, a camera or video sensor, a dermatoscope, an accelerometer, a contact sensor, and a humidity sensor.

[0236] With reference to Figures 1A-3B and 5-7, disclosed herein is an apparatus that may be used, among other things, to image a subject. With reference to Figures 1A-3B and 5-7, disclosed herein is an apparatus that may be used, among other things, for confocal imaging.

[0237] 1A-3B and 5-7, the present specification discloses an apparatus that can be used to generate, among other things, a tissue depth profile. In one aspect, an apparatus for generating a tissue depth profile of a subject includes an optical device that transmits an excitation light beam from a light source to a surface of the tissue, the optical device providing a signal indicative of an intrinsic property of the tissue when the excitation light beam contacts the tissue, one or more focusing units in the optical device that simultaneously adjust the depth and position of the focus of the excitation light beam along a scan path, a scan pattern, or in one or more oblique directions, one or more sensors configured to detect at least a subset of the signals generated when the tissue is contacted with the excitation light beam, and one or more computer processors operably coupled to the one or more sensors, the one or more computer processors being individually or collectively programmed to process at least a subset of the signals detected by the one or more sensors to generate a tissue depth profile.

[0238] At least a subset of the signals can include polarized light. The optical device may include one or more polarization-selective optics (e.g., polarizing filters, polarizing beam splitters, etc.). The one or more polarization-selective optics can select a particular polarization of the RCM signal such that the detected RCM signal is of a particular polarization from a particular portion of the tissue. For example, the polarization-selective optics can be used to selectively image or amplify different features within the tissue.

[0239] At least a subset of the signals may include unpolarized light. The optical device may be configured to reject up to all out-of-focus light. By rejecting the out-of-focus light, a low noise image may be generated from the RCM signals.

[0240] Using multiple refractive lenses, such as relay lenses, collimating lenses, and field lenses, ultrafast pulsed light from a light source can be focused to a small spot in tissue. The small spot of focused light can generate intrinsic tissue signals, such as second harmonic generation, two-photon autofluorescence, third harmonic generation, coherent anti-Stokes Raman spectroscopy, confocal microscopy signals, or other nonlinear multiphoton generated signals, when it contacts tissue. The probe can also transfer a scanning pattern generated by optical elements, such as mirrors and translation lenses, to the movement of a focal spot in tissue to scan the focal spot through structures and generate point-by-point images of the tissue. The probe can include multiple lenses to minimize aberrations, optimize linear mapping of the focal spot scan, and maximize resolution and field of view.

[0241] The one or more focusing units in the optical device may include, but are not limited to, a movable lens, an actuator coupled to an optical element (e.g., an afocal lens), a MEMS mirror, a relay lens, a dichroic mirror, a folding mirror, a beam splitter, a reflection chamber, or any combination thereof as described in detail herein with reference to Figures 1A-9. The alignment element for directing the confocal microscope signal into the reflection chamber may include, but is not limited to, an angle adjustment element, and / or a movable mirror. The signal indicative of the unique property of the tissue may be, for example, a confocal microscope signal, a second harmonic generation signal, a multiphoton fluorescence signal, other generated signal, or any combination thereof, as described elsewhere herein.

[0242] Apparatus consistent with the methods herein can include any element of the subject methods, including, but not limited to, an optical device, one or more light sources such as ultrashort pulsed lasers, one or more movable or adjustable lenses, one or more optical filters, one or more photodetectors, one or more computer processors, one or more marking tools, and combinations thereof.

[0243] The photodetectors may include, but are not limited to, photomultiplier tubes (PMTs), photodiodes, avalanche photodiodes (APDs), charge-coupled device (CCD) detectors, charge-injection device (CID) detectors, complementary metal oxide semiconductor detectors (CMOS) detectors, multi-pixel photon counters (MPPCs), silicon photomultiplier tubes (SiPMs), light-dependent resistors (LDRs), hybrid PMT / avalanche photodiode sensors, and / or other detectors or sensors. The system may include one or more photodetectors of one or more types, and each sensor may be used to detect the same or different signals. For example, the system may use both photodiode and CCD detectors, where the photodiodes detect SHG and multiphoton fluorescence, and the CCD detects reflectance confocal microscopy signals. The photodetectors may operate to provide a frame rate, or number of images acquired per second, of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, or more. The photodetector can be operated to provide frame rates up to about 60, 50, 40, 30, 24, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 or less.

[0244] The optical device may include a photomultiplier tube (PMT) that collects the signal. The PMT may include an electrical interlock and / or a shutter. The electrical interlock and / or shutter may be activated when contact between the epithelial tissue surface and the optical probe is broken, thereby protecting the PMT when the photomultiplier tube compartment is exposed to ambient light. The use of an activatable interlock and / or shutter may allow signals to be collected in the presence of ambient light, thereby allowing a user to generate one or more real-time pre-operative depth profiles at the patient's bedside. The optical device may also include other photodetectors.

[0245] The light source providing the ultrashort pulsed light can be a wavelength-tunable ultrashort pulse Ti:sapphire laser. The Ti:sapphire laser can be a mode-locked oscillator, a chirped pulse amplifier, or a wavelength-tunable continuous wave laser. The mode-locked oscillator can generate ultrashort pulses with durations of about a few picoseconds to about 10 femtoseconds, and in some cases about 5 femtoseconds. The pulse repetition frequency can be about 70-90 megahertz (MHz). The term "chirped pulse" generally refers to a special structure that can prevent the pulse from damaging components in the laser. In a "chirped pulse" laser, the pulse can be stretched in time so that the energy is not all located at the same time and in space, preventing damage to the amplifier optics. The pulse can then be optically amplified and recompressed in time to form a short localized pulse.

[0246] The movable lens or lenses of the device can be translated to generate a number of different scan patterns or scan paths. The movable lens may be coupled to an actuator that translates the lens. The actuator may be controlled by a programmed computer processor. The actuator may be a linear actuator, such as a mechanical actuator, a hydraulic actuator, a pneumatic actuator, a piezoelectric actuator, an electromechanical actuator, a linear motor, a linear electric actuator, a voice coil, or a combination thereof. Mechanical actuators can operate by converting rotary motion to linear motion, for example, by screw mechanisms, wheel and axle mechanisms, and cam mechanisms. Hydraulic actuators can include a hollow cylinder with a piston and a non-compressible liquid. Pneumatic actuators can be similar to hydraulic actuators, but include compressed gas instead of a liquid. Piezoelectric actuators can include a material that can expand under the application of a voltage. As a result, piezoelectric actuators can achieve very fine positioning resolution, but can also have a very short operating range. In some cases, piezoelectric materials can exhibit hysteresis, which can make it difficult to repeatably control their expansion. Electromechanical actuators can be similar to mechanical actuators. However, the control knob or handle of the mechanical actuator may be replaced with an electric motor.

[0247] A tunable lens may refer to an optical element whose optical properties, such as its focal length and / or the position of its optical axis, can be adjusted during use, for example by electronic control. An electrically tunable lens may comprise a thin layer of a suitable electro-optic material (e.g., a material whose local effective index, i.e. the index of refraction, varies as a function of a voltage applied across the material). An electrode or an electrode array may be used to apply a voltage to locally tune the index of refraction to that value. The electro-optic material may comprise a liquid crystal. A voltage may be applied to modulate the birefringence axis and effective index of the electro-optic material, including the liquid crystal. In some cases, a polymer gel may be used. A tunable lens may comprise an electrode array that defines a grid of pixels in the liquid crystal, similar to the pixel grids used in liquid crystal displays. The index of refraction of the individual pixels may be electrically controlled to impart a phase modulation profile. The phase modulation profile may refer to the distribution of local phase shifts applied to light passing through the layer as a result of the locally varying effective index over a region of the electro-optic layer of the tunable lens.

[0248] In some cases, a plurality of different scan patterns or paths can be obtained using an electrically or electromechanically adjustable lens in electrical or electromechanical communication with the optical device. The curvature of the electrically or electromechanically adjustable lens can be modulated to provide a plurality of different scan patterns or paths for the epithelial tissue. The curvature of the adjustable lens can be modulated by applying an electric current. The apparatus can also include a programmed computer processor for controlling the application of the electric current.

[0249] The apparatus for identifying disease in epithelial tissue of a subject may include an optical device. The optical device may transmit an excitation light beam from a light source toward a surface of the epithelial tissue. Upon contact with the epithelial tissue, the excitation light beam may generate a signal related to an intrinsic property of the epithelial tissue. The light source may comprise an ultrafast pulsed laser, such as a Ti:sapphire laser. The ultrafast pulsed laser may provide a pulse duration of 500 femtoseconds, 400 femtoseconds, 300 femtoseconds, 200 femtoseconds, 100 femtoseconds or less. The pulse repetition frequency of the ultrashort light pulses may be at least 10 MHz, 20 MHz, 30 MHz, 40 MHz, 50 MHz, 60 MHz, 70 MHz, 80 MHz, 90 MHz, 100 MHz or more.

[0250] The tissue may be an epithelial tissue. The depth profile may allow for the identification of a disease or condition of the epithelial tissue of the subject. Diseases in the tissue of the subject are disclosed elsewhere herein.

[0251] The scanning path or pattern may be in one or more oblique directions and one or more oblique planes. The oblique planes may be arranged along a direction oblique to the optical axis of the optical device. The angle between the oblique planes and the optical axis may be at most 45°. The angle between the oblique planes and the optical axis may be at least about 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 55°, 60°, 65°, 70°, 75°, 80°, 85° or more. In other cases, the angle between the oblique planes and the optical axis may be at most about 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 35°, 30°, 25°, 20°, 15°, 10°, 5° or less.

[0252] The optical device may further comprise one or more optical filters, which may be configured to collect a subset of signals. As described elsewhere herein, the optical filters may be used to collect one or more specific subsets of signals related to one or more unique properties of the epithelial tissue. The optical filters may be beam splitters, polarizing beam splitters, notch filters, dichroic filters, long-pass filters, short-pass filters, band-pass filters, or response flattening filters. The optical filters may be one or more optical filters. These optical filters may be coated glass or plastic elements that can selectively transmit light of specific wavelengths, such as autofluorescence wavelengths, and / or light with other specific attributes, such as polarization. The optical filters may collect at least one signal selected from the group consisting of second harmonic generation (SHG) signals, third harmonic generation (THG) signals, polarized signals, reflectance confocal microscopy (RCM) signals, and autofluorescence signals. The subset of signals may include at least one of a second harmonic generation (SHG) signal, a third harmonic generation (THG) signal, and an autofluorescence signal.

[0253] The light source can include an ultrafast pulsed laser with a pulse duration of less than about 200 femtoseconds. The ultrafast pulsed laser can generate pulses of light with a pulse duration of up to 500 femtoseconds, 450 femtoseconds, 400 femtoseconds, 350 femtoseconds, 300 femtoseconds, 250 femtoseconds, 200 femtoseconds, 150 femtoseconds, 100 femtoseconds, or less. In some cases, the pulse duration is about 150 femtoseconds. In some cases, the ultrafast pulsed laser can generate pulses of light with a pulse duration of at least 100 femtoseconds, 150 femtoseconds, 200 femtoseconds, 250 femtoseconds, 300 femtoseconds, 350 femtoseconds, 400 femtoseconds, 450 femtoseconds, 500 femtoseconds, or less. The pulse repetition frequency of the ultrafast pulsed laser can be at least 10 MHz, 20 MHz, 30 MHz, 40 MHz, 50 MHz, 60 MHz, 70 MHz, 80 MHz, 90 MHz, 100 MHz, or more. In some cases, the pulse repetition frequency of the ultrafast pulsed laser can be up to 100 MHz, 90 MHz, 80 MHz, 70 MHz, 60 MHz, 50 MHz, 40 MHz, 30 MHz, 20 MHz, 10 MHz, or less. In some cases, the pulse repetition frequency is about 80 MHz.

[0254] During use, the probe of the optical device may be in contact with the surface of the tissue. Alternatively or additionally, the probe may be configured to penetrate the tissue. The probe may include one or more objective lenses. The objective lenses may contact the surface of the tissue. The objective lenses may be configured to collimate the light. The contact may be direct or indirect. If the contact is direct, performing the contact may include placing the optical device next to the tissue of the subject without an intervening layer. If the contact is indirect, performing the contact may include placing the probe of the optical device next to the tissue of the subject with one or more intervening layers. The one or more intervening layers may include, but are not limited to, clothing, medical gauze, bandages, and the like. The contact may be monitored such that when contact between the surface of the epithelial tissue and the optical device is interrupted, a shutter positioned in front of the detector (e.g., relative to the path of the light) may be activated to block the incident light. In some cases, the light detector includes an electrical interlock and / or a shutter. The electrical interlock and / or shutter can be activated when contact between the epithelial tissue surface and the optical probe is broken, thereby protecting the photodetector when the photomultiplier compartment is exposed to ambient light. The use of an activatable interlock and / or shutter allows signals to be collected in the presence of ambient light, thereby enabling a user to generate one or more real-time pre-operative depth profiles at the patient's bedside.

[0255] The device may include a sensor that detects the displacement between the probe and the tissue surface and that may protect the photodetector, e.g., the photodetector, from ambient light by activating a shutter or temporarily shutting down the photodetector if the ambient light exceeds the detection capability of the photodetector to prevent the ambient light from reaching and damaging the photodetector.

[0256] The optical device may include a power meter. The power meter may be optically coupled to the light source. The power meter may be used to correct for variations in the power of the light source. The power meter may be used to control the power of the light source. For example, an integrated power meter may allow the power of the light source to be set according to the amount of power used for a particular imaging session. The power meter may ensure consistent illumination over a period of time so that images obtained over a period of time have similar illumination conditions. The power meter may provide information about the power of the illumination light to the system processing, which may record the depth profile. The power information may be included in the machine learning described elsewhere herein. The power meter may be, for example, a photodiode, a pyroelectric power meter, or a thermal power meter. The power meter may be multiple power meters.

[0257] The device may further comprise a marking tool for outlining a boundary indicating the location of the disease in the epithelial tissue of the subject. The marking tool may be a pen or other writing implement with an FDA approved skin marking ink such as Genetian Violet Ink, a preparation resistant ink that can be used with aggressive skin preparations such as CHG / isopropyl alcohol treatment, a waterproof permanent ink, or an ink that is easily removable with alcohol or the like. The pen may have a fine tip, an extra fine tip, or a wide tip. The marking tool may be a sterile pen. Alternatively, the marking tool may be a non-sterile pen.

[0258] The device may be a portable device. The portable device may be battery powered. The portable device may include wheels. The portable device may be housed within a housing. The housing may be approximately 0.1 ft 2 ,0.2ft 2 ,0.3ft 2 ,0.4ft 2 ,0.5ft 2 ,1ft 2Alternatively, the housing may have a footprint of about 1 ft 2 ,0.5ft 2 ,0.4ft 2 ,0.3ft 2 ,0.2ft 2 or 0.1 ft 2 It may have the following footprint: The handheld device may include a filtered light source that emits light within a wavelength range that cannot be detected by optical devices.

[0259] A portable device may weigh up to 50 pounds, 45 pounds, 40 pounds, 35 pounds, 30 pounds, 25 pounds, 20 pounds, 15 pounds, 10 pounds, 5 pounds or less. In some cases, a portable device may weigh at least 5 pounds, 10 pounds, 15 pounds, 20 pounds, 25 pounds, 30 pounds, 35 pounds, 40 pounds, 45 pounds, 50 pounds, 55 pounds or more.

[0260] The optical device may include a handheld housing configured to interface with a user's hand. The translatable optical device can include a handheld and portable housing, allowing a surgeon, doctor, nurse, or other medical personnel to inspect the location of disease, e.g., cancer in skin tissue, in real time at a patient's bedside. A portable device can be mounted on a surface that is approximately 0.1 ft. 2 ,0.2ft 2 ,0.3ft 2 ,0.4ft 2 ,0.5ft 2 or 1ft 2 Alternatively, the handheld device may have a footprint of about 1 ft 2 ,0.5ft 2 ,0.4ft 2 ,0.3ft 2 ,0.2ft 2 or 0.1 ft 2 It may have the following footprint:

[0261] The probe may have a tip diameter of less than about 10 millimeters (mm), 8 mm, 6 mm, 4 mm, or 2 mm. The handheld device may have a mechanism that allows for easy connection and disconnection of the disposable probe. The mechanism may have an alignment feature to allow for precise optical alignment between the probe and the handheld device. The handheld device may be shaped like an otoscope or dermatoscope with a gun-like form factor. The handheld device may have a weight of up to about 8 pounds (lbs), 4 pounds, 2 pounds, 1 pound, 0.5 pounds, or 0.25 pounds. A screen may be incorporated into the handheld device for point-of-care viewing. The screen may be detachable and rotatable. The handheld device may be attached to a portable system that may include a rolling cart or a briefcase type form. The portable device may include a screen. The portable device may include a laptop computing device, a tablet computing device, a computing device coupled to an external screen (e.g., a desktop computer with a monitor), or a combination thereof. The portable system may include a laser, electronics, a light sensor, and a power system. The laser can provide light at an optimal frequency for transmission. The handheld device can include a second harmonic frequency doubler to convert light from a frequency useful for transmission (e.g., 1,560 nm) to a frequency useful for imaging tissue (e.g., 780 nm). For example, the transmission frequency can be at least about 800 nm, 900 nm, 1,000 nm, 1,100 nm, 1,200 nm, 1,300 nm, 1,400 nm, 1,500 nm, 1,600 nm, 1,700 nm, 1,800 nm, 1,900 nm or more, and the imaging frequency can be at least about 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm or more. The laser can be low powered enough to operate the system on battery power.The system may further include a charging dock or mini-stand to hold the portable unit during operation. There may be many mini-stands in a single clinic and a single portable system that can be transported between rooms.

[0262] The housing may further comprise an image sensor. Alternatively, the image sensor may be located outside the housing. In either case, the image sensor may be configured to position the optical device housing in space. The image sensor may position the optical device housing in space by tracking one or more features around the optical device. The image sensor may be a video camera. The one or more features may be features of tissue (e.g., freckles, birthmarks, etc.) or markers on or in the tissue placed by a physician. The one or more features may be features of the space in which the optical device is used (e.g., furniture, walls, etc.). For example, the housing may incorporate several cameras therein that use computer algorithms to track the position of the housing by tracking the movement of furniture in the room in which the optical device is used, and the tracking may be used to help generate a complete 3D image of the portion of tissue. By simultaneously tracking the position of the housing or the position of the optical device while recording an image of the tissue, the computer can reconstruct the position of the image in the tissue as the housing translates. In this way, a larger mosaic area of ​​tissue may be imaged and digitally reconstructed. Such an area may be a 3D volume or a 2D mosaic or any surface in the tissue. The image sensor may be configured to detect near infrared light. The housing may be configured to project a number of points to generate a map for the image sensor to use for tracking. In addition to using an image sensor, one or more position sensors, one or more other guides, or one or more sensors may be used with or by the optical device or housing to locate the probe position relative to the location of the tissue feature or tissue characteristic. The processor may identify the position of the optical device relative to currently or previously collected data. For example, the identified characteristics of the tissue may be used to identify, mark, or record the probe position. Currently or previously placed tags or markers may also be used to identify the probe position relative to the tissue.Such tags or markers may include, but are not limited to, dyes, wires, fluorescent tracers, stickers, imprints, incisions, sutures, mechanical fiducials, mechanical anchors, or other elements that can be sensed. The guide may be used in conjunction with an optical device to indicate, mechanically reference, and / or track the optical probe position. The optical probe position data may be incorporated into the image data collected to create a depth profile.

[0263] The housing can include optics configured to direct at least a subset of the signals to one or more detectors. The one or more detectors may be optically coupled to the housing via one or more optical fibers. The housing can house the one or more detectors as well as the light source, thus having a completely handheld imaging system.

[0264] 12-14 show an example of an optical device housing coupled to a support system. FIGS. 11A and 11B show the interior of an example of a support system. A portable computing device 1101 may be placed on top of the support system. The support system may include a laser 1103. The support system may include multiple support electronics, such as, for example, a battery 1104, a controller 1102 for an afocal lens actuator, a MEMS mirror driver 1105, a power supply 1106, one or more transimpedance amplifiers 1107, a photodetector block 1108, multiple operating electronics 1109, a data acquisition board 1110, other sensors or sensor blocks, or any combination thereof.

[0265] FIG. 13 shows an example of the portability of the example of FIG. 12. FIG. 14 shows an example of the system in use. The support system can send multiple light pulses to the housing via a connecting cable. The multiple light pulses can interact with the tissue generating multiple signals. The multiple signals can travel along the connecting cable back to the support system. The support system may include a portable computer. The portable computer can process the signals to generate and display an image that can be formed from the collected signals and depth profiles described herein. Preparing the subject for imaging can include using an alcohol swab to clean the subject's tissue for imaging. Additionally, a drop of an optical liquid or gel, such as glycerol or oil, can be applied to the subject's tissue. Imaging can be performed in the absence of hair removal, stains, drugs, or fixation.

[0266] The one or more computer processors may be operatively coupled to one or more sensors, which may comprise an infrared sensor, an optical sensor, a microwave sensor, an ultrasonic sensor, a radio frequency sensor, a magnetic sensor, a vibration sensor, an acceleration sensor, a gyro sensor, a tilt sensor, a piezoelectric sensor, a pressure sensor, a strain sensor, a flex sensor, an electromyographic sensor, an electrocardiographic sensor, an electroencephalographic sensor, a thermal sensor, a capacitive touch sensor, or a resistive touch sensor.

[0267] According to some embodiments of the methods and devices herein, the image can be a depth profile as described herein and can include additional data as described herein. The depth profile can be an image. The image can also be part of a depth profile as described herein and can be in the form of a tile or part of image data. The image can be obtained in vivo. The signal can be collected and the image, depth profile, tile, or data set can be created without removing tissue from the subject's body or fixing the tissue to a slide. The image can extend below the surface of the tissue. The image can have a resolution of at least about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000 micrometers or more. The image may have a resolution of up to about 1,000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 75, 50, 25, 10, 5, 1 micrometer or less. The image may include an optical image.

[0268] The images obtained using the optical device can be used to generate a trained algorithm and a generated trained algorithm dataset for detecting or identifying tissue characteristics within a subject or for classifying images of tissue from the subject. The trained algorithm can be, for example, as described with reference to PCT / US2019 / 061306, filed November 13, 2019, and PCT / US2020 / 060302, filed November 12, 2020, each of which is incorporated by reference in its entirety.

[0269] The imaging probe may be configured to measure one or more electronic signals. The electronic signals may be or may indicate a current, a voltage, a charge, a resistance, a capacitance, a conductivity, an impedance, any combination thereof, or a change therein. The imaging probe may include imaging optics. The imaging probe may be configured to measure one or more optical signals. Examples of imaging probes including handheld optical devices are provided elsewhere herein. The signals received by the imaging probe may be used to generate an image of the tissue area from which the signals were received. The imaging probe may be handheld. The imaging probe may be translated, lifted, or reoriented. For example, the imaging probe may be placed at an angle on the subject's skin and rotated to view tissue at different locations.

[0270] The imaging probe can be operably coupled to one or more computer processors. For example, the imaging probe can be plugged into a computer that includes a processor. In another example, the imaging probe can be connected to one or more computer processors via a network. The imaging probe can be handheld. The imaging probe can be configured to deliver therapy to tissue as described elsewhere herein.

[0271] The signals may be generated approximately simultaneously (e.g., signals generated within a period of about 30 seconds (s), 20 seconds, 10 seconds, 1 second, 0.5 seconds, 0.4 seconds, 0.3 seconds, 0.2 seconds, 0.1 seconds, 0.01 seconds, 0.005 seconds, 0.001 seconds or less; signals generated by the same pulse or beam of light, etc.) within a single region of tissue (e.g., signals generated within about 1, 1E-1, 1E-2, 1E-3, 1E-4, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10, 1E-11, 1E-12, 1E-13 cubic centimeters or less). The signals may be generated by the same pulse or beam of light. The signals may be generated by multiple light beams that are synchronized in time and location, as described elsewhere herein. Two or more signals may be combined to generate a composite image. A signal or a subset of signals may be generated within a single region of tissue using the same or similar scan pattern or scan plane. Each signal of the plurality of signals may be independent of other signals of the plurality of signals. A user may decide which subset (or subsets) of signals to use. For example, if both RCM and SHG signals are collected in a scan, a user may decide whether to use the RCM signals, the multiphoton signals, or any combination thereof. Additionally, video tracking of the position of the housing or optical device as described herein above may be recorded simultaneously with the generated signals.

[0272] The data can be stored in a database. The database can be stored in a computer readable format. The computer processor can be configured to access the data stored in the computer readable memory. The computer system can be used to analyze the data and obtain a result. The result can be stored remotely or internally on the storage medium and communicated to personnel such as pharmaceutical professionals. The computer system can be operatively coupled with a component for transmitting the result. The component for transmitting can include wired and wireless components. Examples of wired communication components can include a Universal Serial Bus (USB) connection, a coaxial cable connection, an Ethernet cable such as a Cat5 or Cat6 cable, an optical fiber cable, or a telephone line. Examples of wireless communication components can include a Wi-Fi receiver, a component for accessing a mobile data standard such as a 3G or 4G LTE data signal, or a Bluetooth receiver. All of these data in the storage medium can be collected and archived to build a data warehouse.

[0273] The depth profile may include a monochromatic image displaying colors derived from a single base hue. Alternatively or additionally, the depth profile may include a multi-color image displaying two or more colors. In a multi-color image, the color components may correspond to multiple depth profiles using signals or subsets of signals that are synchronized in time and position. Such depth profiles may be generated using an optical device, for example, as described elsewhere herein. Such depth profiles may include individual components, images, or depth profiles formed from multiple subsets of the collected and processed generated signals. In some embodiments, the depth profile corresponds to different positions of the optical probe on the tissue. The depth profile may include multiple layers formed from multiple subsets of images collected from the same position and time. Each of the multiple layers may include data identifying anatomical structures and / or features that are different from the anatomical structures and / or features of the other layer(s). In some embodiments, the multiple depth profiles correspond to different scanning patterns when detecting signals. In some embodiments, the different scanning patterns correspond to the same time and probe position. In some embodiments, at least one of the different scanning patterns comprises a tilted scanning pattern. In some embodiments, the tilted scan pattern forms an inclined plane.

[0274] Such a depth profile can include multiple subset depth profiles. In this way, multiple colors can be used to highlight different elements of tissue such as cells, nuclei, cytoplasm, connective tissue, vasculature, pigments, and tissue layer boundaries. Contrast can be adjusted in real time to provide and / or enhance structure-specific contrast. Contrast can be adjusted by a user (e.g., a surgeon, doctor, nurse, or other medical professional) or a programmed computer processor can automatically optimize contrast in real time. In a multicolor image, each color can be used to represent a specific subset of collected signals such as second harmonic generation signals, third harmonic generation signals, signals resulting from polarized light, and autofluorescence signals. The colors of the multicolor depth profile can be customized to reflect image patterns that a surgeon and / or pathologist can see when using standard histopathology. Pathologists can more easily interpret the results of a depth profile when the depth profile is displayed similar to how a traditional histological sample, e.g., a sample stained with hematoxylin and eosin, can be seen.

[0275] The optical device and the one or more computer processors may comprise the same device. The device may be a mobile device. The device may be multiple devices that may be operatively coupled to each other. For example, the system may be a handheld optical device optically connected to a laser and a detection box, and the box may also include a computer.

[0276] The optical device may be part of a device and the one or more computer processors may be separate from the device. The one or more computer processors may be part of a computer server. The one or more processors may be part of a distributed computer infrastructure. For example, the system may be a handheld optical device that includes all of the optical components wirelessly connected to a remote server that processes data from the optical device.

[0277] Method for optical filtering and imaging - Patents.com Disclosed herein, among other things, is a method for generating a depth profile of a tissue of a subject. In one aspect, a method for generating a depth profile of an imaging target (e.g., a tissue of a subject) includes using an optical device to transmit an excitation light beam from a light source toward a surface of the tissue, the excitation light beam generating a signal indicative of an intrinsic property of the tissue upon contacting the tissue; simultaneously adjusting the depth and position of the focal point of the excitation light beam in a scanning pattern using one or more focusing units in the optical device; detecting at least a subset of the signals generated upon contacting the tissue with the excitation light beam; and using one or more computer processors programmed to process at least a subset of the detected signals to generate a depth profile of the tissue. The scanning pattern can include multiple focal points. The method described herein for generating a depth profile can alternatively utilize a combination of two or more light beams, continuous or pulsed, juxtaposed at the focal points.

[0278] In one aspect, the present disclosure provides a method for filtering and imaging light. The method for filtering light can include providing a light filtering device. The light filtering device can include a chamber, a first reflector, and a second reflector. A distance in the chamber can separate the first reflector and the second reflector. The method can further include directing a light beam to the first reflector in the chamber, using the first reflector to direct at least a portion of the light beam from the first reflector to the second reflector in the chamber, and using the second reflector to direct at least another portion of the light beam from the second reflector in the chamber to the first reflector. The light beam can repeatedly traverse between the first reflector and the second reflector. A path length that the light beam travels between the first reflector and the second reflector can be at least three times the distance that separates the first reflector and the second reflector.

[0279] In another aspect, the present disclosure provides a method for filtering and imaging light. The method for imaging an object can include providing a probe in optical communication with an optical filtering device that includes a chamber and an object. The method can further include using the probe to provide an optical beam to the object and to collect the optical beam from the object, directing the optical beam from the object to the chamber of the optical filtering device, repeatedly directing the optical beam from a first position to a second position in the chamber of the optical filtering device directly or indirectly such that a path length that the optical beam travels between the first position and the second position is at least three times the distance that separates the first position and the second position, and processing the optical beam for generating and imaging the object.

[0280] The methods and systems disclosed herein may be used to form a depth profile of a tissue sample by utilizing a scanning pattern that moves the imaging beam focal point through the sample in a tilted or oblique direction relative to the optical axis to improve the resolution of the optical system imaging the sample (e.g., in vivo biological tissue). The scanner may move the focal point in one or more lines and / or one or more planes tilted relative to the optical axis to form a depth profile of the tissue. The depth profile may provide a projected vertical cross-sectional image that is generally or approximately representative of a cross-section of the tissue that may be used to identify possible disease states of the tissue. The methods and systems may provide a projected vertical cross-sectional image of an in vivo sample of intact biological tissue formed from the depth profile image components (e.g., a scanning pattern of the focal point). The methods and systems disclosed herein may also generate images of tissue cross-sections that may be viewed as tissue sections, but that may represent different XY positions.

[0281] According to some embodiments, the methods and systems disclosed herein can utilize one or more tilted planes (or one or more tilted focal planes) formed by a scanning pattern of the focal spot in one or more tilted planes. A system capable of simultaneously controlling the X, Y, and Z positions of the focused spot can move the focal spot through a trajectory in the tissue. The trajectory can be predetermined, modifiable, or arbitrary. Scanning at an angle to the vertical Z axis (e.g., optical axis) can significantly improve resolution. This effect can occur, for example, because the intersection of the tilted plane with the point spread function (PSF) is much smaller than the PSF projection in the XZ or YZ planes. Thus, the effective PSF of a focused beam moved along or in a tilted line becomes smaller as the tilt angle increases, and can approach the lateral PSF resolution at an angle of 90° (at which point the scan direction line or plane can be in the XY (lateral) plane). Tilt scanning or imaging as described herein can be used with any type of return signal. Non-limiting examples of return signals may include generated signals described elsewhere herein.

[0282] To ensure that part of the scan trajectory moves the focal point in the Z direction, the depth profile through the tissue can be scanned at an angle relative to the optical axis (e.g., greater than 0° and less than 90°). In some instances, moderate tilt angles can result in substantial improvements in resolution. The effective PSF size is: lateral / sin(θ), where θ is the angle between the z-axis and the imaging axis. Further details can be found in FIG. 3. Thus, at a scan angle of 45°, the resolution along the depth axis of the tilt plane is 1.414 times greater than the lateral resolution. With sub-micron lateral resolution, tilt resolutions of near microns or sub-microns can be achieved, depending on the scan angle. This process can result in cross-sectional resolution achievable with optical systems of much higher numerical aperture (NA). By operating at a more modest NA, the optical elements can be more robust to off-axis aberrations and can scan a larger field of view and / or a greater depth. Furthermore, by operating at a more modest NA, the footprint of the imaging device can be reduced while maintaining high resolution.

[0283] Once a projected cross-sectional image is constructed, the depth profile information derived from the generated signals resulting from the tilted scan can be projected onto the XZ or YZ plane to form an image plane. In this situation, there may be no distortion of the depth of the structure relative to the surface of the tissue. This projected cross-sectional image, in some representative embodiments, may include data corresponding to planes optically sliced ​​at one or more angles relative to the normal. The projected cross-sectional image may have significantly improved resolution while still representing the depth of the imaged structure or tissue.

[0284] The depth profile can be generated by scanning the focal point in a scan pattern that includes one or more tilted directions. The scan may or may not be in a single plane. The scan may be in one or more tilted planes. The scan may be a complex shape, such as a spiral, or may be a predetermined, variable, or random array of points. The scan pattern, scan plane, tilt plane, and / or focal plane may be a different plane than the visual or image cross-section that can be formed from the processed generated signals. The image cross-section can be formed from the processed generated signals that result from moving the imaging focal point through a vertical plane, a tilt plane, a non-planar pattern, a shape (e.g., a spiral, a wave, etc.), or a random or pseudo-random combination of focal points.

[0285] The depth profile can be generated in real time. For example, the depth profile can be generated while an optical device transmits one or more excitation light beams from a light source toward a surface of the tissue. The depth profile can be generated at a frame rate of at least 1 frame per second (FPS), 2 FPS, 3 FPS, 4 FPS, 5 FPS, 10 FPS, or more. In some cases, the depth profile can be generated at a frame rate of up to 10 FPS, 5 FPS, 4 FPS, 3 FPS, 2 FPS, or less. The frame rate can refer to the rate at which an imaging device displays successive images, called frames. The image frames of the depth profile can provide cross-sectional images of the tissue.

[0286] The image frame or area of ​​the image may be a quadrilateral having any suitable dimensions. The image frame may be a rectangle, possibly with equal sides (e.g., a square), depicting, for example, a 200 μm×200 μm cross section of tissue. The image frame may depict a cross section of tissue having dimensions of at least about 50 μm×50 μm, 100 μm×100 μm, 150 μm×150 μm, 200 μm×200 μm, 250 μm×250 μm, 300 μm×300 μm, or more. In some cases, the image frame may depict a cross section of tissue having dimensions of about 300 μm×300 μm, 250 μm×250 μm, 200 μm×200 μm, 150 μm×150 μm, 100 μm×100 μm, 50 μm×50 μm, or less. The image frame may not have equal sides.

[0287] The image frame may be at any angle relative to the optical axis. For example, the image frame may be at an angle of about 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 60°, 70°, 80°, 90°, or more relative to the optical axis. The image frame may be at an angle of about 90°, 85°, 80°, 75°, 70°, 65°, 60°, 50°, 40°, 30°, 20°, 10°, 5° or less relative to the optical axis. In some cases, the angle is between any two of the values ​​described above or elsewhere herein, for example, between 0° and 50°.

[0288] The image frame may be of any design, shape, or size. Examples of shapes or forms include, but are not limited to, mathematical shapes (e.g., circles, triangles, squares, rectangles, pentagons, or hexagons), two-dimensional geometric shapes, multi-dimensional geometric shapes, curves, polygons, polyhedra, polyhedrons, minimal surfaces, ruled surfaces, non-orientable surfaces, quadrilaterals, pseudospheres, algebraic surfaces, miscellaneous surfaces, Riemann surfaces, box-drawing characters, Cuisenaire rods, geometric shapes, shapes with common names, symbols, Unicode geometric shapes, other geometric shapes, or combinations of partial shapes or shapes thereof. The image frame may be a projection image cross-sectional image, as described elsewhere herein.

[0289] The excitation light beam may be an ultrashort pulse of light. The ultrashort pulse of light may be emitted from an ultrashort pulse laser (also referred to herein as an "ultrafast pulse laser"). The ultrashort pulse of light may have a high peak intensity and may result in nonlinear interactions in various materials. The ultrashort pulse of light may refer to light having a full width at half maximum (FWHM) on the order of femtoseconds or picoseconds. In some examples, the ultrashort pulse of light has an FWHM of at least about 1 femtosecond, 10 femtoseconds, 100 femtoseconds, 1 picoseconds, 100 picoseconds, or 1000 picoseconds or more. In some examples, the ultrashort light pulse may have an FWHM of up to about 1000 picoseconds, 100 picoseconds, 1 picoseconds, 100 femtoseconds, 10 femtoseconds, 1 femtoseconds, or less. The ultrashort pulse of light may be characterized by several parameters, including pulse duration, pulse repetition rate, and average power. Pulse duration can refer to the FWHM of optical power versus time. Pulse repetition rate can refer to the frequency of the pulses or the number of pulses per second.

[0290] Non-limiting examples of ultrashort pulse laser technologies include titanium (Ti):sapphire lasers, mode-locked diode-pumped lasers, mode-locked fiber lasers, and mode-locked dye lasers. Ti:sapphire lasers may be tunable lasers with a crystal of sapphire (Al2O3) doped with titanium ions as the lasing medium (e.g., the active laser medium, which is the source of optical gain in the laser). Lasers, such as diode-pumped lasers, fiber lasers, and dye lasers, can be mode-locked by active or passive mode-locking to obtain ultrashort pulses. Diode-pumped lasers may be solid-state lasers whose gain medium comprises a laser crystal or a piece of bulk glass (e.g., ytterbium crystal, ytterbium glass, chromium-doped laser crystal). Although the pulse duration cannot be as short as possible with Ti:sapphire lasers, diode-pumped ultrafast lasers can cover a wide parameter range in terms of pulse duration, pulse repetition rate, and average power. Fiber lasers based on glass fibers doped with rare earth elements such as erbium, ytterbium, neodymium, dysprosium, praseodymium, thulium, or combinations thereof may also be used. In some cases, dye lasers may be used that include organic dyes such as rhodamine, fluorescein, coumarin, stilbene, umbelliferone, tetracene, malachite green, etc. as the lasing medium, in some cases as a liquid solution.

[0291] The light source providing the ultrashort pulsed light can be a wavelength-tunable ultrashort pulse Ti:sapphire laser. The Ti:sapphire laser can be a mode-locked oscillator, a chirped pulse amplifier, or a wavelength-tunable continuous wave laser. The mode-locked oscillator can generate ultrashort pulses with durations of about a few picoseconds to about 10 femtoseconds, and in some cases about 5 femtoseconds. The pulse repetition frequency can be about 70-90 megahertz (MHz). The term "chirped pulse" generally refers to a special structure that can prevent the pulse from damaging components in the laser. In a "chirped pulse" laser, the pulse can be stretched in time so that the energy is not all located at the same time and in space, preventing damage to the amplifier optics. The pulse can then be optically amplified and recompressed in time to form a short localized pulse.

[0292] Ultrashort pulses of light can be produced by gain switching. In gain switching, the laser gain medium is pumped, for example, with another laser. Gain switching can be applied to various types of lasers, including gas lasers (e.g., transversely pumped atmospheric (TEA) carbon dioxide lasers). In some cases, adjusting the pulse repetition rate can be more easily achieved with gain-switched lasers than with mode-locked lasers, because gain switching can be controlled with an electronic driver without changing the laser cavity settings. In some cases, a pulsed laser can be used to optically pump the gain-switched laser. For example, a nitrogen ultraviolet laser or an excimer laser can be used for pulsed pumping of a dye laser. In some cases, Q-switching can be used to produce ultrafast pulses of light.

[0293] A type of signal that can be generated or collected to determine disease in tissue can be a reflectance confocal microscopy (RCM) signal. RCM can use light reflected from an imaging target, such as tissue, when a light beam from an optical device is directed at the imaging target. The RCM signal can be a small portion of the light that is directed at the sample. The RCM signal can be collected by filtering out out-of-focus light or by collecting focused light from a collimated beam, for example, as described herein with reference to Figures 1A-7. The interaction of the imaging target with the light beam may or may not change the polarization of the RCM signal. Different components of the sample can change the polarization of the RCM signal to different degrees. The use of polarization selective optical elements in the optical path of the RCM signal can allow a user to select the RCM signal from a given component of the sample. The system can select, split, or amplify RCM signals that correspond to different anatomical features or characteristics to provide further tissue data. For example, based on the change in polarization detected by the system, the system can select or amplify RCM signal components corresponding to melanin deposits by using polarization selective optics to select or amplify RCM signals associated with melanin. Other tissue components, including but not limited to collagen, keratin, and elastin, can be identified using polarization selective optics. Non-limiting examples of generated signals that may be detected are described elsewhere herein.

[0294] Tissues and cellular structures within tissues can interact with the excitation light beam in a wavelength-dependent manner to generate signals related to the intrinsic properties of the tissue. The generated signals can be used to assess the normal, abnormal, cancerous, or other characteristics of the tissue, such as skin tissue, or the subject's tissue, related to the health, function, treatment, or appearance of the subject (e.g., the subject's health). The subset of signals generated and collected can include at least one of a second harmonic generation (SHG) signal, a third harmonic generation (THG) signal, a polarization signal, and an autofluorescence signal. The oblique plane imaging technique can be used with any of the generated signals, as described elsewhere herein.

[0295] High-order harmonic generation microscopy (HHGM) based on nonlinear multi-photon excitation (e.g., second harmonic generation and third harmonic generation) can be used to examine cellular structures in live and fixed tissues. SHG can generally refer to a nonlinear optical process in which photons having approximately the same frequency interact with a nonlinear material and effectively "combine" to generate new photons with approximately twice the energy of the initial photon, and therefore approximately twice the frequency and approximately half (1 / 2) the wavelength. Similarly, THG can generally refer to a nonlinear optical process in which photons having approximately the same frequency interact with a nonlinear material and effectively "combine" to generate new photons with approximately three times the energy of the initial photon, and therefore approximately three times the frequency and approximately one-third (1 / 3) the wavelength. Second harmonic generation (SHG) and third harmonic generation (THG) of ordered endogenous molecules, such as but not limited to collagen, microtubules, and muscle myosin, can be obtained without the use of exogenous labels, resulting in detailed, real-time optical reconstruction of molecules including fibrillar collagen, myosin, microtubules, and other cellular information such as membrane potential and cell depolarization. The ordering and organization of proteins and molecules in tissues, such as collagen type I and type II, myosin, and microtubules, when interacting with light, can generate signals that can be used to assess the cancerous state of tissues. SHG signals can be used to detect changes such as changes in collagen fibril / fibril structure that can occur in diseases including cancer, fibrosis, and connective tissue disorders. A variety of biological structures can generate SHG signals. In some cases, labeling of molecules with exogenous probes and contrast agents that can change the way biological systems function can be avoided. In some cases, the methods herein for identifying diseases in epithelial tissues of a subject can be performed without administering contrast agents to the subject.

[0296] Another type of signal that can be generated and collected to determine disease in tissues can be autofluorescence. Autofluorescence can generally refer to light naturally emitted by certain biological molecules, such as proteins, small molecules, and / or biological structures. Tissues and cells can contain a variety of autofluorescent proteins and compounds. Distinct wavelengths can be absorbed by chromophores such as endogenous molecules, proteins, water, and fats that are naturally present in cells and tissues. Non-limiting examples of autofluorescent fluorophores that can be found in tissues include aromatic amino acids such as tryptophan, tyrosine, and phenylalanine that can emit in the UV range, as well as polypeptides and proteins that contain vitamin derivatives that can emit at wavelengths ranging from about 400 nm to 650 nm, including the nicotinamide ring of NAD(P)H from retinol, riboflavin, and niacin, and pyridramine crosslinks found in elastin and some collagens that are based on pyridoxine (vitamin B6).

[0297] The autofluorescence signal may include multiple autofluorescence signals. One or more filters may be used to separate the multiple autofluorescence signals into one or more autofluorescence channels. For example, different parts of tissue may fluoresce at different wavelengths, and wavelength-selective filters may be used to direct each fluorescence wavelength to a different detector. One or more monochromators or diffraction gratings may be used to separate the multiple autofluorescence signals into one or more channels.

[0298] Ultrafast pulsed lasers can generate pulses of light having pulse durations of up to 500 femtoseconds, 450 femtoseconds, 400 femtoseconds, 350 femtoseconds, 300 femtoseconds, 250 femtoseconds, 200 femtoseconds, 150 femtoseconds, 100 femtoseconds, or less. In some cases, the pulse duration is about 150 femtoseconds. In some cases, ultrafast pulsed lasers can generate pulses of light having pulse durations of at least 100 femtoseconds, 150 femtoseconds, 200 femtoseconds, 250 femtoseconds, 300 femtoseconds, 350 femtoseconds, 400 femtoseconds, 450 femtoseconds, 500 femtoseconds, or less. The pulse repetition frequency of the ultrafast pulsed laser can be at least 10 MHz, 20 MHz, 30 MHz, 40 MHz, 50 MHz, 60 MHz, 70 MHz, 80 MHz, 90 MHz, 100 MHz, or more. In some cases, the pulse repetition frequency of the ultrafast pulsed laser can be up to 100 MHz, 90 MHz, 80 MHz, 70 MHz, 60 MHz, 50 MHz, 40 MHz, 30 MHz, 20 MHz, 10 MHz, or less. In some cases, the pulse repetition frequency is about 80 MHz.

[0299] The collected signals can be processed by a programmed computer processor to generate a depth profile. The signals can be wirelessly transmitted to the programmed computer processor. Alternatively, the signals can be transmitted to the programmed computer processor via a wired connection. The signals or a subset of the signals related to the unique properties of the tissue can be used to generate the depth profile with the aid of the programmed computer processor. The collected signals and / or the generated depth profile can be stored electronically. In some cases, the signals and / or the depth profile are stored until deleted by a user, such as a surgeon, doctor, nurse, or other medical professional. When used for diagnosis and / or treatment, the depth profile can be provided to the user in real time. The depth profile provided in real time can be used as a pre-operative image to identify the borders of a disease, for example skin cancer. The depth profile can provide visualization of various layers of tissue, such as skin tissue, including the epidermis, dermis, and / or subcutaneous tissue. The depth profile can extend below at least the stratum corneum, the zona pellucida, the granular layer, the spinous or squamous cell layer, and / or the basal cell layer. In some cases, the depth profile may extend at least 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm or more below the surface of the tissue. In some cases, the depth profile may extend up to 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm or more below the surface of the tissue. In some cases, the depth profile extends from about 100 μm to 1 mm, about 200 μm to 900 μm, about 300 μm to 800 μm, about 400 μm to 700 μm, or about 500 μm to 600 μm below the surface of the tissue.

[0300] The method may further include processing the depth profile using one or more computer processors to identify disease in the tissue. The identification of disease in the tissue may include one or more features. The one or more features may provide one or more quantitative values ​​indicative of one or more of the following: likelihood of diagnostic accuracy, likelihood of the presence of the disease in the subject, likelihood of the subject developing the disease, likelihood of success of a particular treatment, or any combination thereof. The one or more computer processors may also be configured to predict the risk or likelihood of developing the disease, confirm the diagnosis or presence of the disease, monitor the progression of the disease, and monitor the effectiveness of a treatment of the disease in the subject.

[0301] The method may further include contacting the subject's tissue with the optical device. The contact may be direct or indirect contact. If the contact is direct contact, performing the contact may include placing the optical device next to the subject's tissue without an intervening layer. If the contact is indirect contact, performing the contact may include placing the optical device next to the subject's tissue with one or more intervening layers. The one or more intervening layers may include, but are not limited to, clothing, medical gauze, and bandages. The contact may be monitored such that when contact between the epithelial tissue surface and the optical device is interrupted, a shutter positioned in front of the detector (e.g., relative to the light path) may be activated to block the incident light.

[0302] According to some representative embodiments, the scanning pattern can be followed by an inclined surface. The inclined surface may be disposed along a direction inclined with respect to the optical axis of the optical device. The angle between the inclined surface and the optical axis may be up to 45°. The angle between the inclined surface and the optical axis may be about 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 55°, 60°, 65°, 70°, 75°, 80°, 85° or more. In other cases, the angle between the inclined surface and the optical axis may be about 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 35°, 30°, 25°, 20°, 15°, 10°, 5° or less. In some cases, the angle between the inclined surface and the optical axis may be between any of the two aforementioned values, for example, about 5° to 50°.

[0303] According to various representative embodiments, the scan path or pattern may follow one or more patterns designed to obtain enhanced, improved, or optimized image resolution. The scan path or pattern may include, for example, one or more vertical planes, one or more inclined planes, one or more spiral focal paths, one or more zigzag or sinusoidal focal paths, or any combination thereof. The scan path or pattern may be configured to maintain the scan focus near the center of the optical element while moving in an inclined direction. The scan path or pattern may be configured to maintain the scan focus near the center of the optical axis (e.g., the focal axis).

[0304] The scanning pattern of the multiple foci may be selected by an algorithm. For example, a series of images may be acquired using a moving focal point at one or more scan angles (relative to the optical axis). The scanning pattern may include vertical scanning and / or tilt scanning. Depending on the quality of the acquired image, one or more additional images may be acquired using a different scan angle or combinations thereof selected by the algorithm. As an example, if the images acquired using vertical scanning or tilt scanning at a smaller angle are of poor quality, the computer algorithm may instruct the system to acquire images using a combination of scan directions or using a larger scan angle. If a combination of scan patterns results in improved image quality, the imaging session may continue to use that combination of scan patterns. This method may be performed without removing tissue from the subject. The method may be performed without administering a contrast agent to the subject.

[0305] The excitation light beam may include unpolarized light. In other embodiments, the excitation light beam may include polarized light. The wavelength of the excitation light beam may be at least about 400 nanometers (nm), 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or longer. In some cases, the wavelength of the excitation light beam may be up to about 950 nanometers (nm), 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm, 500 nm, 450 nm, 400 nm, or less. The wavelength of the pulses of light may be about 700 nm to 900 nm, about 725 nm to 875 nm, about 750 nm to 850 nm, or about 775 nm to 825 nm.

[0306] Multiple wavelengths can also be used. When multiple wavelengths of light are used, the wavelengths can be centered at least about 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm or more, with a bandwidth of at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm or more. For example, the wavelengths can be centered at about 780 nm with a bandwidth of about 50 nm (e.g., about ((780-(50 / 2))=755 nm) to about ((780+(50 / 2))=805 nm)). In some cases, the wavelength can be centered at a maximum of about 950 nanometers (nm), 900 nm, 850 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm, 500 nm, 450 nm, 400 nm or less, with a bandwidth of at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm or more.

[0307] The subset of signals may include at least one of the signals selected from the group consisting of second harmonic generation (SHG) signals, third harmonic generation (THG) signals, reflectance confocal microscopy (RCM) signals, and autofluorescence signals. SHG, THG, RCM, and autofluorescence are disclosed elsewhere herein. The subset of signals may include one or more of the generated signals defined herein.

[0308] The collection may be performed in the presence of ambient light, which may refer to normal room lighting such as that provided by various types of electric lighting sources, including incandescent bulbs or lamps, halogen lamps, gas discharge lamps, fluorescent lamps, light emitting diode (LED) lamps, and carbon arc lamps, in an examination room or operating area where a surgical procedure is performed.

[0309] By simultaneously adjusting the depth and focal position of the excitation light beam along the tilt scan, scan path, or scan pattern, the maximum resolution depth of the depth profile can be increased. The increased maximum resolution depth can be at least about 1.1 times, 1.2 times, 1.5 times, 1.6 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, or more, of the original maximum resolution depth. In other embodiments, the increased maximum resolution depth can be up to about 3 times, 2.9 times, 2.8 times, 2.7 times, 2.6 times, 2.5 times, 2.4 times, 2.3 times, 2.2 times, 2.1 times, 2.0 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1 times, 4 times, or less, of the original maximum resolution depth. This increase may be for cases where the depth and position of the focus cannot be adjusted simultaneously.

[0310] A signal indicative of the intrinsic properties of the tissue can be detected by a photodetector. The power and gain of the photodetector sensor may be modulated to improve image quality. The excitation light beam can be synchronized with the sensing by the photodetector.

[0311] The RCM signal can be detected by a series of optical components in optical communication with the beam splitter. The beam splitter can be a polarizing beam splitter, a fixed ratio beam splitter, a reflective beam splitter, or a dichroic beam splitter. The beam splitter can transmit about 1%, 3%, 5%, 10%, 15%, 20%, 25%, 33%, 50%, 66%, 75%, 80%, 90%, 99% or more of the incident light. The beam splitter can transmit about 99%, 90%, 80%, 75%, 66%, 50%, 33%, 25%, 20%, 15%, 10%, 5%, 3%, 1% or less of the incident light. The series of optical components can include one or more mirrors. The series of optical components can include one or more lenses. The optical fiber can be single mode, multimode, or a bundle of optical fibers.

[0312] The method may be performed without penetrating the tissue of the subject. The methods disclosed herein for identifying disease in tissue of a subject may be used during and / or for the treatment of disease, e.g., during Mohs surgery to treat skin cancer. In some cases, disease, e.g., skin cancer, may be identified in the epithelial tissue of a subject without removing epithelial tissue from the epithelial tissue of the subject. This may advantageously prevent pain and discomfort to the subject and may facilitate detection and / or identification of disease. The location of disease may be detected in a non-invasive manner, allowing a user, such as a health care professional (e.g., a surgeon, doctor, nurse, or other practitioner), to determine the location and / or boundaries of the diseased area prior to surgery. Identification of disease in epithelial tissue of a subject may be performed in some cases without penetrating the epithelial tissue of the subject, e.g., by a needle.

[0313] The disease or condition may include cancer. In some cases, the cancer may be thyroid cancer, adrenal cortical carcinoma, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastases, central nervous system (CNS) cancer, peripheral nervous system (PNS) cancer, breast cancer, Castleman's disease, cervical cancer, childhood non-Hodgkin's lymphoma, lymphoma, colon and rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal cancer, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, childhood leukemia, acute myeloid ... The disease may include pulmonary fibrosis, chronic lymphocytic leukemia, chronic myelogenous leukemia, liver lung cancer, non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer, non-melanoma skin cancer, gastric cancer, testicular cancer, thymic cancer, uterine cancer (e.g., uterine sarcoma), vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia. The disease may be an epithelial cancer. The epithelial cancer may be skin cancer.

[0314] The method may further include processing the depth profile using one or more computer processors to classify disease in the tissue. The classification may identify the tissue as diseased with an accuracy, selectivity, and / or specificity of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or more. The classification may identify the tissue as diseased with an accuracy, selectivity, and / or specificity of up to about 99.9%, 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less. The one or more computer processors may classify the disease using one or more computer programs. The one or more computer programs may include one or more machine learning techniques. The one or more machine learning techniques may be trained on a system other than the one or more processors.

[0315] The depth profile may have a resolution of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200 microns or more. The depth profile may have a resolution of up to about 200, 150, 100, 75, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5 microns or less. For example, the depth profile may be capable of resolving 1 micron of intercellular space.

[0316] In some embodiments, the data is normalized with respect to illuminance.

[0317] The method may further include measuring the power of the excitation light beam. A power meter may be used to measure the power of the excitation light beam. The power meter may measure the power of the excitation light beam in real time. The one or more computer processors may normalize the signal of the measured power of the excitation light beam. The normalized signal may be normalized with respect to average power, instantaneous power (e.g., power read simultaneously with the signal), or a combination thereof. The one or more computer processors may generate a normalized depth profile. The normalized depth profile may be compared across depth profiles generated at different times. The depth profile may also include information regarding the illumination power when the image was acquired. The power meter may also be referred to herein as a power sensor or power monitor.

[0318] The method can allow for synchronous collection of multiple signals. The method can allow for collection of multiple signals generated by a single excitation event. A depth profile can be generated using signals generated from the same excitation event, as described elsewhere herein. A user can determine which signals to use to generate the depth profile.

[0319] The method can generate two or more layers of information. The two or more layers of information can be information generated from data generated from the same light pulse of a single probe system. The two or more layers can be from the same depth profile. Also, each of the two or more layers can form a separate depth profile from which a projected cross-sectional image can be formed or displayed. For example, each separate layer, or each separate depth profile, can correspond to a particular processed signal or signals corresponding to a particular imaging method. For example, a depth profile can be generated by acquiring confocal microscopy signals from skin tissue, another depth profile can be generated using two-photon fluorescence signals from melanin and SHG signals from collagen, and three or more depth profiles can be overlaid as multiple layers of information. Each group of signals can be filtered, processed, and used separately to form individual depth profiles and projected cross-sectional images, and can be combined into a single depth profile with data that can be used to generate a projected cross-sectional image, and data from each group of signals can be combined and the combination can be used to generate a single depth profile or any combination thereof. Each group of signals corresponding to one or more particular features of the tissue can be assigned a color that is used to display an individual cross-sectional image of the one or more features or a composite cross-sectional image including data from each signal group. The cross-sectional images or individual depth profiles can be overlaid to generate a composite image or depth profile. Thus, multi-color, multi-layer, depth profiles or images can be generated.

[0320] Image example 10A-10D show examples of images formed from a depth profile of skin. FIG. 10A shows an image displayed from a depth profile derived from a generated signal due to two-photon autofluorescence. The autofluorescence signal was generated from an excitation signal of about 780 nm and collected from a probe at the tip of an optical device into a light guide. The autofluorescence signal was detected in the range of about 415-650 nm using appropriately selected optical filters. The epidermis 1003 can be seen at the surface of the skin along with the stratum corneum 1001. Among other features, elastin 1002 at the boundary between the epidermis 1003 and the dermis 1005 layer as well as epithelial cells 1008 (keratinocytes) of the epidermis 1003 can be seen. FIG. 10B shows an image displayed from a depth profile or layer synchronized in time and position with the depth profile or layer of FIG. 10A. The image displayed from the depth profile of FIG. 10B is derived from a second harmonic generation signal at about 390 nm detected with appropriately selected optical filters. A second harmonic generation signal was generated from the excitation signal at about 780 nm and collected from the probe at the tip of the optical device into a light guide. Collagen 1004 of the dermis layer 1005 as well as other features can be seen. FIG. 10C shows an image displayed from a depth profile or layer synchronized in time and position with the depth profiles or layers of FIGS. 10A and 10B. The image displayed from the depth profile of FIG. 10C is derived from the reflected confocal signal reflected back to the RCM detector. The reflected signal at about 780 nm returns through its origin path and was split to an alignment array that focuses and aligns the reflected signal into an optical fiber for detection and processing. Melanocytes 1007 and collagen 1006 as well as other features can be seen. The images of FIGS. 10A, 10B and 10C are obtained from excitation light pulses and can be derived from a single composite depth profile with multiple layers or can be derived as a single layer from separate depth profiles. Figure 10D shows an overlay image of Figures 10A-10C. Boundaries that can be identified from features in Figures 10A and 10B can help identify the location of the melanocytes identified in Figure 10D. Diagnostic information can be included in the individual images of Figure 10D and / or the composite or overlay image.For example, some suspicious lesions could be identified based on the location and shape of melanocytes or keratinocytes in various tissue layers. The depth profiles in Figures 10A-10D can be example data for use in a machine learning algorithm as described. For example, all three layers can be input into a machine learning classifier as individual layers, and a composite image can also be used as a separate input.

[0321] Optical techniques for detecting epithelial cancer The present disclosure provides optical techniques that can be used to diagnose epithelial diseases and skin lesions. Optical imaging techniques can show nuclear and cellular morphology and can provide the capability of real-time detection of tumors in large areas of freshly excised or biopsy tissue without sample processing such as that of histology. Optical imaging methods can also facilitate non-invasive, real-time visualization of suspicious tissue without excising, sectioning, and / or staining tissue samples. Optical imaging can improve the yield of diagnosable tissue (e.g., by avoiding areas with fibrosis or necrosis), minimize unnecessary biopsies or endoscopic resections (e.g., by distinguishing neoplasms from inflammatory lesions), and can assess surgical margins in real time to confirm negative margins (e.g., to perform limited resections). The ability to evaluate tissue samples in real time, without having to wait for tissue processing, sectioning, and staining, can improve diagnostic turnaround times, especially in time-sensitive situations such as during Mohs surgery. Non-limiting examples of optical imaging techniques for diagnosing epithelial diseases and cancer include multiphoton microscopy, autofluorescence microscopy, polarized light microscopy, confocal microscopy, Raman spectroscopy, optical coherence tomography, and ultrasonography. Non-limiting examples of detectable tissue components include keratin, NADPH, melanin, elastin, flavin, protoporphyrin ix, and collagen. Other detectable components can include tissue boundaries. Exemplary images from depth profiles shown in Figures 10A-10D show several detectable components, including, but not limited to, tissue boundaries of the stratum corneum, epidermis, and dermis, melanocytes, collagen, and elastin.

[0322] Confocal microscopy can be used to examine epithelial tissues. Exogenous contrast agents may be administered to enhance visibility. Confocal microscopy can provide non-invasive images of nuclear and cellular morphology in approximately 2-5 μm thin sections of living human skin with a lateral resolution of approximately 0.5-1.0 μm. Confocal microscopy can be used to visualize in vivo microanatomical structures such as the epidermis and individual cells, including melanocytes.

[0323] Multiphoton microscopy (MPM) can be used to image unique molecular signals in biological imaging subjects, such as skin tissue of a patient. In MPM, a sample can be illuminated with light of a wavelength longer than the normal excitation wavelength, for example, twice as long or three times as long. MPM can include second harmonic generation microscopy (SHG) and third harmonic generation microscopy (THG). Third harmonic generation can be used to image neural tissue.

[0324] Autofluorescence microscopy can be used to image biomolecules that are intrinsically fluorescent (e.g., fluorophores). Non-limiting examples of endogenous biomolecules that are autofluorescent include nicotinamide adenine dinucleotide (NADH), NAD(P)H, flavin adenine dinucleotide (FAD), collagen, retinol, and tryptophan, as well as indoleamine derivatives of tryptophan. Changes in the fluorescence levels of these fluorophores, such as those associated with tumor progression, can be optically detected. The changes can be associated with altered cellular metabolic pathways (NADH, FAD) or altered structural tissue matrices (collagen).

[0325] Polarized light can be used to assess biological structures and examine parameters such as cell size and refractive index, which can provide information about the composition of cells, for example, cells in a tissue sample, and histology. Cancer can significantly alter the histology of tissues, and these changes can be optically detected using polarized light.

[0326] Raman spectroscopy can also be used to examine epithelial tissues. Raman spectroscopy can rely on the phenomenon of inelastic scattering (so-called "Raman" scattering) to detect the spectral signatures of disease progression biomarkers such as lipids, proteins and amino acids.

[0327] Optical coherence tomography may also be used to examine epithelial tissues. Optical coherence tomography can be based on interferometry, where a laser light beam is split with a beam splitter, sending part of the light to a sample and part of the light to a reference. The combination of the reflected light from the sample with the reference can result in an interference pattern that can be used to determine a reflection profile that provides information about the spatial dimensions and location of structures within the sample. Current commercially available optical coherence tomography systems have a lateral resolution of about 10-15 μm, with an imaging depth of about 1 mm or more. This technique can rapidly generate three-dimensional (3D) image volumes that reflect different layers of tissue components (e.g., cells, connective tissue, etc.), but the image resolution (e.g., similar to a ×4 objective lens on a tissue microscope) may not be sufficient for routine histopathology diagnosis.

[0328] Ultrasound can also be used to examine epithelial tissue. Ultrasound can be used to assess relevant features of epithelial cancers, such as depth and vasculature. Although ultrasound may be limited in detecting pigments such as melanin, it can complement histological analysis and provide additional details to aid in treatment decisions. Ultrasound may be used for non-invasive assessment of features such as thickness and blood flow of primary tumors and may contribute to modifying important management decisions.

[0329] The methods for diagnosing epithelial diseases and skin pathologies disclosed herein may include one or more of multiphoton microscopy, autofluorescence microscopy, polarized light microscopy, confocal microscopy, Raman spectroscopy, optical coherence tomography, and ultrasonography. In some cases, the methods for diagnosing epithelial diseases and / or skin pathologies include autofluorescence microscopy and multiphoton microscopy. Alternatively, the methods for diagnosing epithelial diseases and / or skin pathologies include autofluorescence microscopy, multiphoton microscopy, and polarized light microscopy. Both second harmonic generation microscopy and third harmonic generation microscopy can be used. In some cases, one of second harmonic generation microscopy and third harmonic generation microscopy is used.

[0330] Methods for diagnosing epithelial diseases and skin lesions disclosed herein may include using one or more depth profiles to identify anatomical features and / or other tissue properties or features, and overlaying an image from the one or more depth profiles onto an image that can identify the skin lesion.

[0331] Computer Systems The present disclosure provides a computer system that is programmed to implement the methods of the present disclosure. Figure 15 shows a computer system 1501 that is programmed or configured to direct light to the systems and devices described elsewhere herein to guide light to an imaging target and collect light from the imaging target. The computer system 1501 can be further configured or programmed to image the imaging target using light from the imaging target. The computer system 1501 can coordinate various aspects of the systems and methods of the present disclosure, such as, for example, directing light to the imaging target, focusing light at various positions or depths of the imaging target, and generating images of the imaging target. The computer system 1501 can be part of a mobile or portable imaging system. Alternatively or additionally, the computer system 1501 can be a remote device wirelessly connected to the imaging system.

[0332] The computer system 1501 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 1505, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1501 also includes memory or storage locations 1510 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1515 (e.g., hard disk), a communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1510, the storage unit 1515, the interface 1520, and the peripheral devices 1525 are in communication with the CPU 1505 via a communication bus (solid lines), such as a motherboard. The storage unit 1515 may be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network ("network") 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 1530 can in some cases be a remote communication and / or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530 can in some cases, with the aid of the computer system 1501, implement a peer-to-peer network, which allows devices coupled to the computer system 1501 to operate as clients or servers.

[0333] The CPU 1505 can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions may be directed to the CPU 1505, which may then program or otherwise configure the CPU 1505 to perform the methods of the present disclosure. Examples of operations performed by the CPU 1505 may include fetch, decode, execute, and writeback.

[0334] The CPU 1505 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0335] The storage unit 1515 can store files such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, such as user preferences and user programs. The computer system 1501 can optionally include one or more additional data storage units external to the computer system 1501, such as located on a remote server in communication with the computer system 1501 via an intranet or the Internet.

[0336] Computer system 1501 can communicate with one or more remote computer systems via network 1130. For example, computer system 1501 can communicate with a user's remote computer system (e.g., laptop, mobile phone, etc.). Examples of remote computer systems include a personal computer (such as a portable PC), a slate or tablet PC (e.g., Apple® iPad, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or a personal digital assistant. A user can access computer system 1501 via network 1530.

[0337] The methods herein may be implemented by machine (e.g., computer processor) executable code stored in electronic storage locations of the computer system 1501, such as, for example, memory 1510 or electronic storage unit 1515. The machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by the processor 1505. In some cases, the code may be retrieved from the storage unit 1515 and stored in the memory 1510 for immediate access by the processor 1505. In some circumstances, the electronic storage unit 1515 may be eliminated and machine executable instructions are stored in the memory 1510.

[0338] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or it may be compiled at run-time. The code may be provided in a programming language that may be selected to allow the code to be executed in a pre-compiled or compiled manner.

[0339] Aspects of the systems and methods provided herein, such as the computer system 1501, may be embodied in programming. Various aspects of the technology may be considered as "products" or "articles" in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine executable code may be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory of a computer, processor, etc., or associated modules, such as various semiconductor memories, tape drives, disk drives, etc., that may provide persistent storage at any time for software programming. All or part of the software may be communicated over the Internet or various other communication networks. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media that may bear software elements include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, via wired and optical land line networks, and across various air links. The physical elements that convey such waves, such as wired or wireless links, optical links, etc., may also be considered media bearing software. As used herein, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution, unless limited to persistent, tangible "storage" media.

[0340] Thus, a machine-readable medium such as a computer-executable code may take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any storage device, such as any computer(s), that may be used to implement, for example, the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire and optical fibers, including the wiring that comprises a bus within a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards paper tape, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves transmitting data or instructions, cables or links transmitting such waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0341] The computer system 1501 may include or communicate with an electronic display 1535 that may have a user interface (UI) 1540 for, for example, configuring settings for imaging a subject and controlling images of the subject. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0342] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented via software when executed by the central processing unit 1505. The algorithms may be configured, for example, to image an imaging target.

[0343] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided in the specification. Although the present invention has been described in conjunction with the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art at this point without departing from the present invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is therefore contemplated that the present invention shall cover such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. a probe configured to (i) direct a light beam from a light source to an imaging target; and (ii) collect light from the imaging target when the light beam contacts the imaging target; an optical filtering device in optical communication with the probe, the optical filtering device comprising: (a) a chamber having (i) an input configured to receive the light collected by the probe and (ii) an output configured to transmit the light away from the chamber; and (b) a plurality of reflectors disposed within the chamber, the plurality of reflectors configured to direct the received light in an optical path from the input to the output by reflection of the light between reflectors of the plurality of reflectors, the chamber configured to reject out-of-focus light along the optical path; 1. A device comprising:

2. 10. The device of claim 1, wherein the cross-sectional diameter of the output is greater than or equal to about 0.1 times the cross-sectional diameter of the input.

3. 10. The device of claim 1, wherein the chamber has a longest interior linear dimension and the optical path has a path length that is at least three times the length of the longest linear dimension.

4. The device of claim 1 , wherein the optical path has a path length of 0.25 meters, 0.5 meters, or 1 meter or more.

5. The device of claim 1 , wherein at least one reflector of the plurality of reflectors is a retroreflector.

6. The device of claim 1 , wherein the output is configured to direct a focused portion of the light to a collector.

7. The device of claim 6 , wherein the device comprises the collector.

8. 10. The device of claim 1, wherein the plurality of reflectors includes a first reflector arranged to direct focused portions of the light in an optical path between the reflectors, the optical path returning to the first reflector multiple times before reaching the output.

9. 10. The device of claim 1, wherein the plurality of reflectors comprises a first reflector at a first position within the chamber and a second reflector at a second position within the chamber, the first reflector configured to direct the light to the second reflector.

10. 10. The device of claim 9, wherein the optical path has a path length, the first reflector and the second reflector are separated by a distance, and the path length is at least five times the distance.

11. The device of claim 10, wherein the path length traversed by the light is between 5 and 30 times the distance.

12. The device of claim 1 , further comprising a scanning unit disposed between the light source and the probe, the scanning unit configured to scan the light beam across the imaging object.

13. The device of claim 1 , wherein the probe comprises an objective lens configured to collimate the light.

14. 10. The device of claim 1, further comprising an alignment unit disposed between the probe and the optical filtering device, the alignment unit configured to (i) guide the light to the optical filtering device and (ii) adjust the angle of incidence of the light into the chamber.

15. 10. The device of claim 1, further comprising a beam splitter disposed between the probe and the optical filtering device, the beam splitter configured to (i) split the light to generate split light, and (ii) direct at least a portion of the split light to the optical filtering device.

16. The device of claim 1 , wherein the device is configured for confocal imaging.

17. The device of claim 1, further comprising a beam de-expander unit positioned between the light beam and the optical filtering device.

18. 1. A method for imaging an imaging target, comprising: (a) providing (i) an optical filtering device comprising a chamber and (ii) a probe in optical communication with the imaging subject; (b) directing a light beam from a light source through the probe to an imaging object and collecting light from the imaging object as the light beam contacts the imaging object; (c) using the probe to deliver a light beam to the imaging object and collect a resulting return light beam from the imaging object; (d) directing the return light beam from the imaging target into the chamber of the optical filtering device; (e) repeatedly directing the light beam from a first reflector to a second reflector within the chamber of the optical filtering device such that a path length traversed by the light beam between the first reflector and the second reflector is at least three times the distance separating the first reflector and the second reflector; (f) processing the light beam to generate an image of the imaging target; A method comprising:

19. 20. The method of claim 18, further comprising, before (d), directing the returning optical beam to a beam deexpander unit.

20. 20. The method of claim 18, further comprising, subsequent to (d), processing the light to generate an image of the imaging target.

21. 20. The method of claim 18, further comprising using the light for confocal imaging.

22. 20. The method of claim 18, wherein the path length traversed by the light beam is at least five times the distance separating a first location and a second location.

Images