Handheld imaging system for identify and locating tissue

Abstract

A handheld imaging system comprises a light delivery member optically coupled with a light source for delivering light emitted from the light source to tissue of a target region to illuminate the tissue; and an imaging member for detector for collecting the images of optical signals emitted from the tissue responsive to the light illumination. The light delivery member and the imaging member are incorporated into a handpiece.

Description

HANDHELD IMAGING SYSTEM FOR IDENTIFY AND LOCATING TISSUESTATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCHThis invention was made with government support under Grant No. CA212147 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED PATENT APPLICATIONSThis application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 542,550, both filed October 5, 2023.This application is a continuation-in-part application of U.S. Patent Application Serial No. 18 / 379,251, filed October 12, 2023, which is a continuation application of U.S. Patent Application Serial No. 17 / 289,323, filed April 28, 2021, which is a national stage entry of PCT Patent Application Serial No. PCT / US2019 / 059610, filed November 4, 2019, which itself claims priority to and the benefit of U.S. Provisional Patent Application Serial No.62 / 754,717, filed November 2, 2018, and is a continuation-in-part application of U.S. Patent Application Serial No. 16 / 471,049, filed June 19, 2019, now U.S. Patent No. 11,589,801, which is a national stage entry of PCT Patent Application Serial No. PCT / US2017 / 068907, filed December 29, 2017, which claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62 / 440,112, filed December 29, 2016, and U.S. Provisional Application Serial No. 62 / 540,767, filed August 3, 2017, and is a continuation-in-part application of U.S. Patent Application Serial No. 15 / 391,046, filed December 27, 2016, now U.S. Patent No. 10,258,275, which is a divisional patent application of U.S. Patent Application Serial No. 13 / 056,469, filed June 14, 2011, now U.S. Patent No. 9,687,190, which is a national stage entry of PCT Patent Application Serial No. PCT / US2009 / 052304, filed July 30, 2009, which claims priority to and the benefit of U.S. provisional patent application Serial Nos. 61 / 137,520, and 61 / 137,584, both filed July 30, 2008.This application is a continuation-in-part application of U.S. Patent Application Serial No. 18 / 577,298, filed January 8, 2024, which is a national stage entry of PCT Patent Application Serial No. PCT / US2022 / 036633, filed July 11, 2022, which itself claims priority to and the benefit of U.S. Provisional Patent Application Serial No.63 / 219,886, filed July 9, 2021, and is a continuation-in-part application of U.S. Application Serial No. 17 / 289,323, filed April 28, 2021, which is a national stage entry of PCT Application Serial No. Entry of PCT / US2019 / 059610, filed November 4, 2019, which itself claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62 / 754,717, filed November 2, 2018; and is a continuation-in-part application of U.S. Application Serial No. 16 / 471,049, filed June 19, 2019, now U.S. Patent No. 11,589,801, which is a national stage entry of PCT Application Serial No. PCT / US2017 / 068907, filed December 29, 2017, which claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62 / 440,112, filed December 29, 2016, and U.S. Provisional Application Serial No. 62 / 540,767, filed August 3, 2017.Each of the above-identified applications is incorporated herein by reference in its entirety, respectively.FIELD OF THE INVENTIONThe invention relates generally to optical assessments of bio-objects, and more particularly, to a handheld imaging system and applications of the same.BACKGROUND OF THE INVENTIONThe background description provided herein is for the purpose of generally presenting the context of the invention. The subject matter discussed in the background of the invention section should not be assumed to be prior art merely as a result of its mention in the background of the invention section. Similarly, a problem mentioned in the background of the invention section or associated with the subject matter of the background of the invention section should not be assumed to have been previously recognized in the prior art. The subject matter in the background of the invention section merely represents different approaches, which in and of themselves may also be inventions. Work of the presently named inventors, to the extent it is described in the background of the invention section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the invention.Endocrine glands identification and margin analysis: Complications in endocrine surgery arising from accidental endocrine gland removal due to inability to visually distinguishendocrine glands from surrounding tissues. Endocrine glands can be visualized through fluorescence imaging of glands’ autofluorescence as shown in FIG. 1. An alternative method for endocrine gland identification is through exogenous dye injection, such as indocyanine green (ICG). Fluorescent imaging with ICG has been recently introduced and has been suggested as a useful tool for the identification and preservation of the endocrine glands.For example, the inability to accurately locate parathyroid glands can lead to complications such as post-operative hypocalcemia. The incidence of inadvertent parathyroidectomy during thyroidectomy ranges from 8 - 19% and may result in post-operative hypocalcemia. Surgical success for patients with hyperparathyroidism is dependent on adequate resection of hypersecreting parathyroid glands. Development of preoperative parathyroid imaging modalities has improved surgical success rate; however, surgeons must still rely on visual assessment during surgery to identify parathyroid glands. The accuracy of this approach is highly dependent on the experience level of the surgeon.Parathyroid detection presents greatest difficulty in cases such as total thyroidectomy (TT), completion thyroidectomy (CT), central neck lymph node dissection, and reoperative thyroid and parathyroid procedures. The larger extent of dissection during TT and central neck lymphadenectomy poses greater risk of inducing iatrogenic surgical trauma and compromised blood supply to the parathyroid glands. Difficulty in reoperative and CT procedures is due to the formation of scar tissue and fibrosis, which distorts normal dissection planes and anatomic relationships. During parathyroidectomy, ectopic glands and multiple gland disease also present obstacles to adequate resection and surgical success. Intraoperative rapid parathyroid hormone assay is one technique for confirming complete removal of hyperfunctioning glands and in some cases to confirm the correct identification of parathyroid tissue. Intraoperative parathyroid aspiration is another technique for confirmation that requires finding the gland, aspirating it with a needle, and analyzing the aspirate for parathyroid hormone (PTH). There remains a clinical need for an intraoperative technique to detect the parathyroid gland instantly and with high accuracy.To fill this clinical need, we have previously demonstrated the capability of a novel parathyroid detection technique using near-infrared (NIR) fluorescence spectroscopy. The parathyroid glands have been shown to emit a NIR autofluorescence signal that is stronger than that of the thyroid and all surrounding tissues in the surgical bed. Unlike current preoperative parathyroid detection modalities, this method provides real-time, intraoperative feedback withhigh accuracy. Intrinsic fluorescence is the basis of this technique, which obviates the need for potentially non-specific external contrast agents. The fluorophore in the parathyroid gland is currently unknown. A more thorough understanding of the sources contributing to variability in parathyroid fluorescence intensity is necessary and may provide insight into the fluorescence etiology.Soft tissue sarcomas margin analysis: Soft tissue sarcomas (STS) are uncommon and diverse cancers arising from mesenchymal progenitors and include over 50 subtypes with varying prognoses. Often found as localized masses, STS treatment typically involves surgical resection. Complete surgical excision with a wide margin of normal tissue is crucial, as incomplete resection commonly leads to local recurrence. Existing methods to evaluate surgical margins, like preoperative MRI and intraoperative frozen section biopsy, have limitations including resolution, sensitivity, time consumption, and risk of sampling errors. The definitive margin status is determined postoperatively, which can delay further treatment if a positive margin is found.Previous findings by our group sought to address these limitations by using fluorescence spectroscopy, which measures emissions from natural biological fluorophores under specific light excitation. This technique does not require external contrast agents and has previously demonstrated the ability to distinguish between neoplastic and normal tissue based on changes in fluorophore concentrations due to disease progression. The spectral data obtained was processed and analyzed to differentiate among normal muscle, normal fat, and STS tissue types. It was found that normal muscle tissue had the highest autofluorescence intensities, while normal fat tissue had the lowest, and STS intensities lay in between. An exception was well-differentiated liposarcomas, which had autofluorescence profiles like normal fat, leading to lower detection accuracy for STS.A classifier algorithm based on multinomial logistic regression was then developed. With the exclusion of well-differentiated liposarcomas, the algorithm significantly improved in accurately classifying normal muscle, normal fat, and STS. Real-time identification of other STS tissues could provide surgeons immediate feedback on suspicious tissue margins. In summary, the study suggests that NIR autofluorescence spectroscopy has potential as a nondestructive, rapid surgical guidance tool capable of improving the accuracy of STS resections. This new approach could lead to more precise excisions, reducing local recurrence and the associated need for additional treatment, ultimately benefiting patient outcomes and reducing healthcare costs.Brain tumor margin analysis: The most common initial therapy for primary and secondary brain tumors is surgical resection. Many studies have shown that the degree of resection significantly influences the time to recurrence and the overall survival of brain tumor patients. Although primary brain tumors account for only 1.4% of all cancer, the five-year survival rate of these patients -35% is low. The goal of surgical resection, therefore, is to remove the maximum amount of tumor mass without sacrificing the patient’s neurologic function. Accurate differentiation between brain tumor tissue and normal brain tissue during surgery is vital but challenging. Traditional methods like surgical navigation and ultrasonography are limited by factors like registration error, brain shifts during surgery, and difficulty in distinguishing tumorous tissue from edema.Previous findings by our group suggest that optical spectroscopy can detect subtle changes in brain tissue related to tumor presence, by identifying a fluorescence peak differential between normal and tumor tissues. Such a peak in fluorescence spectra indicates that optical spectroscopy could be an intraoperative tool for more accurate demarcation of brain tumors compared to existing methods. The significance of this work is the possibility for improved surgical outcomes, by achieving more complete tumor resections while minimizing harm to normal brain tissue.Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies.SUMMARY OF THE INVENTIONIn one aspect, the invention relates to a handheld imaging system comprising a light delivery member optically coupled with a light source for delivering light emitted from the light source to tissue of a target region to illuminate the tissue; and an imaging member for detector for collecting the images images of optical signals emitted from the tissue responsive to the light illumination. The light delivery member and the imaging member are incorporated into a handpiece. The target region comprises an adrenal gland, a pancreas gland, a thyroid gland, a parathyroid gland, small bowel, a brain, or any other organs.In one embodiment, the handpiece is rigid or flexible.In one embodiment, the light emitted from the light source has a wavelength in an NIR range of about 600-1500 nm, or in a visible range of about 300-700 nm.In one embodiment, the light delivery member comprises free space optics and / or lightguide.In one embodiment, the light guide comprises one or more optical fibers.In one embodiment, the light guide comprises a single optical fiber formed with a beveled tip positioned in relation to the imaging member such that the angle polished optical fiber aims the illumination at the center of the field of view.T In one embodiment, the light guide comprises a plurality of optical fibers positioned around the imaging member to achieve a pseudo-uniform illumination across the field of view.In one embodiment, the imaging member comprises a detector for detector for collecting the images the images of the optical signals.In one embodiment, the detector comprises at least one camera, and / or a spectrometer.In one embodiment, the at least one camera is aligned on-axis with the axis of the handpiece.In one embodiment, the at least one camera has an image depth of 0 to 5 feet from a proximal end of the handpiece to the target tissue.In one embodiment, the at least one camera is aligned at an angle from the axis of the handpiece, allowing sharp angle imaging and 360-degree imaging when rotated around the axis.In one embodiment, the at least one camera comprises at least one charge-coupled device (CCD) camera, at least one complementary metal oxide semiconductor (CMOS) camera, at least one single-photon avalanche diode (SPAD) array, at least one photosensor array, or a combination thereof.In one embodiment, the imaging member further comprises imaging optics.In one embodiment, in a proximal camera configuration, the at least one camera is integrated into the handpiece, wherein the imaging optics relays an area of interest onto the at least one camera.In one embodiment, the at least one camera comprises a chip on tip camera.In one embodiment, in the proximal camera configuration, the filters, the imaging optics, and the at least one camera are integrated into the proximal tip of the handpiece, wherein once the image is relayed onto the at least one camera and processed, the processed information is transferred wirely or wirelessly to an external device for processing and rendering.In one embodiment, the information is transferred wirelessly to the external device through Wi-Fi, Bluetooth®, and / or near field communication (NFC).In one embodiment, in a distal camera configuration, the imaging member furthercomprises an output optical light guide including an imaging fiber bundle configured to relay the image from the imaging field to the imaging optics and the at least one camera.In one embodiment, in the distal camera configuration, the imaging optics is configured to relay the field of view onto the imaging fiber bundle and the imaging fiber bundle relays the field of view to a distal camera, enabling imaging the field of interest from a distance.In one embodiment, the imaging member further comprises one or more of long pass, notch, and band pass optical filters positioned between the tissue and the detector to block unwanted wavelengths, ambient light, illumination light, and / or reflected light from the illuminated tissues to ensure the optical signals to be detected.In one embodiment, the filter is incorporated into the handpiece or positioned with the imaging optics before the at least one camera.In one embodiment, the handpiece is provided with at least one of a protruding contact probe attached on the side of the fluorescent imaging probe; a range finder attached next to the fluorescent camera; and a stereoscopic camera set integrated into the fluorescent imaging probe.In one embodiment, the range finder is adapted for measuring spatial data including distance and angle between the fluorescent imaging system and the target tissue, accounting for intensity fall-off due to distance, which ensures that images acquired from varying distances.In one embodiment, using the camera readout along with the measured spatial data, the system can model true fluorescence, enabling universal fluorescent assessment.In one embodiment, the fluorescent is quantitatively assessed through the contact probe which is off set from the rest of the elements.In one embodiment, the handheld imaging system can function without an optical filter, directly imaging diffused reflectance light based on the illumination.In one embodiment, the output optical light guide can be coupled to a source with controllable wavelengths, including a tunable light source for different wavelengths illumination.In one embodiment, the handheld imaging system further comprises a controller configured to receive the acquired images from the imaging member and process the acquired images for intraoperatively identifying and locating the tissue.In one embodiment, the handheld imaging system further comprises a display in communication with the controller for displaying the images.In another aspect, the invention relates to a non-transitory tangible computer-readable medium storing instructions which, when executed by one or more processors, cause fluorescentimages, acquired with the above disclosed handheld imaging system, to be processed and rendered in real-time.In one embodiment, in a contact quantitative fluorescent design, a point-based readout is presented alongside the fluorescent image.In one embodiment, in a range-finding quantitative design, the readout is derived from distance measurements and camera data, then displayed accordingly.In one embodiment, an end user is presented with a point-based system as well as a form of graphic user interface that includes values to discriminate fluorescent level between highly fluorescent tissue and less fluorescent tissue.These and other aspects of the invention will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the invention.BRIEF DESCRIPTION OF THE DRAWINGSThe following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.FIG. 1 shows tissues with intrinsic fluorescence, where pancreas, adrenal glands, and thyroid emit intrinsic fluorescence with excitation and emission. (A) Fluorescence measured using a fluorescence imaging system. (B) Fluorescence measured using fluorescence spectroscopy.FIG. 2 shows raw autofluorescence spectra resulting from a 785 nm excitation source. (A) Average raw spectra for each tissue type. (B) Average raw spectra with 95% confidence intervals for normal muscle, normal fat, and all STS’s averaged into a single spectrum.FIG. 3 shows typical in vitro fluorescence spectra of different types of brain tissues.FIG. 4 shows schematically a fluorescence spectroscopy system including a spectrometer, a diode laser, a laptop / computer, and a hand-held fiber optic probe.FIG. 5 shows a prototype of the probe based parathyroid guidance device.FIG. 6 shows fluorescence images of parathyroid and thyroid acquired with an imaging system with NIR Viewer and Cooled CCD camera, respectively.FIGS. 7A-7C show schematically a handheld fluorescent imaging probe according to embodiments of the invention. FIG. 7A: Clinical interface of the imaging probe and the patient. The source, computer, and monitor could be integrated into one device. FIG. 7B: Handpiece with beveled tip (angled illumination) optical illumination. FIG. 7C: Handpiece with multioutput. In FIGS. 7B and 7C, the grey components 120 represent the casing of the handpiece 110, the blue component 130 represents the fluorescent camera, white components 140 represent the optical light guides, red cones 150 represent the illumination light, and the yellow cones 160 represent the image field of view.FIGS. 8A-8D show schematically a handheld imaging probe according to embodiments of the invention. FIG. 8A: Handpiece with a protruding contact quantitative fluorescent probe 170 attached on the side of the fluorescent imaging probe 110 from FIG. 7C. FIG. 8B: Handpiece with a protruding contact quantitative fluorescent probe 170 imbedded on the side of the fluorescent imaging probe 110 from FIG. 7C. FIG. 8C: Handpiece with a reflection-based range finder 180 attached next to the fluorescent camera 130. FIG. 8D: Handpiece with a stereoscopic camera set 190 integrated into the fluorescent imaging probe 110 from Fig. 7C.FIG. 9 shows schematic of a handheld imaging probe in a proximal camera configuration according to embodiments of the invention.FIGS. 10A-10B show schematically a handheld imaging probe according to embodiments of the invention. FIG. 10A: The source, computer, and monitor could be integrated into one device. FIG. 10B: Handpiece 210 with imaging fiber bundle 230 as the image acquisition (Distal camera). In FIG. 10B, the grey components 220 represent the casing of the handpiece, the blue component 230 represents the imaging optical guides, white components 240 represent the optical light guides, red cones 250 represent the illumination light, and the yellow cones 260 represent the image field of view. The green circles 270 are laser pointer that helps with aiming and the red circle 280 is the long wavelength illumination for wide field imaging with the same camera. However, both the green circle 270 and the red circle 280 here are optional features that assist with the operation.FIG. 11 shows schematic of a handheld imaging probe in a distal camera configuration according to embodiments of the invention.FIG. 12 shows schematic of a handheld imaging probe according to one embodiment ofthe invention. 3D render of the quantitative fluorescent imaging probe with bended fluorescent fiber illumination, fluorescent camera, and range finder.FIG. 13 shows schematic of a handheld imaging probe according to another embodiment of the invention. 3D render of the quantitative fluorescent imaging probe with bended fluorescent fiber illumination, fluorescent camera, and range finder.FIG. 14 shows, Top: Fluorescent images of two ex vivo parathyroid glands tissue and two ex vivo thyroid glands tissue. Bottom: The mean and variance of the pixels' readout from region of interest from each image acquisition. The images are taken by the handheld imaging system (Prototype #1 in EXAMPLE 4), in laboratory settings. The ex vivo tissue samples are collected under IRB from VUMC. The images and the plot show that the device is capable of distinguishing the parathyroid glands and the thyroid glands based on the fluorescence imaging and corresponding readout from the device.DETAILED DESCRIPTION OF THE INVENTIONThe invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and / or quotation marks. The use of highlighting and / or capital letters has no influence on the scope and meaning of a term; the scope and meaning of a term are the same, in the same context, whether or not it is highlighted and / or in capital letters. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. Theuse of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.It will be understood that, although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below can be termed a second element, component, region, layer or section without departing from the teachings of the invention.It will be understood that, as used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Also, it will be understood that when an element is referred to as being “on,” “attached” to, “connected” to, “coupled” with, “contacting,” etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on,” “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” to another feature may have portions that overlap or underlie the adjacent feature.It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” or “has” and / or “having” when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.Furthermore, relative terms, such as “lower” or “bottom” and “upper” or “top,” may be used herein to describe one element's relationship to another element as illustrated in the figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation shown in the figures. For example, if the device in one of the figures is turned over, elements described as being on the “lower” side of other elementswould then be oriented on the “upper” sides of the other elements. The exemplary term “lower” can, therefore, encompass both an orientation of lower and upper, depending on the particular orientation of the figure. Similarly, if the device in one of the figures is turned over, elements described as “below” or “beneath” other elements would then be oriented “above” the other elements. The exemplary terms “below” or “beneath” can, therefore, encompass both an orientation of above and below.Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.As used in this disclosure, “around”, “about”, “approximately” or “substantially” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about”, “approximately” or “substantially” can be inferred if not expressly stated.As used in this disclosure, the phrase “at least one of A, B, and C” should be construed to mean a logical (A or B or C), using a non-exclusive logical OR. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.As used in this disclosure, the term “subject” refers to a human being such as a patient, or a mammal animal such as a monkey.As used herein, the term “Raman spectroscopy” refers to an optical technique that probes the specific molecular content of a sample by collecting inelastically scattered light. As photons propagate through a medium, they undergo both absorptive and scattering events. In absorption, the energy of the photons is completely transferred to the material, allowing either heat transfer (internal conversion) or re-emission phenomena such as fluorescence and phosphorescence to occur. Scattering, however, is normally an elastic process, in which the incident photons retain their energy. In Raman scattering, the photons either donate or acquire energy from the medium, on a molecular level making it an inelastic process. In contrast to fluorescence, where the energy transfers are on the order of the electronic bandgaps, the energy transfers associated with Raman scattering are on the order of the vibrational modes of the molecule. These vibrational modes aremolecularly specific, giving every molecule a unique Raman spectral signature.Raman scattering is a very weak phenomena, and therefore practical measurement of Raman spectra of a medium requires high power excitation laser sources and extremely sensitive detection hardware. Even with these components, the Raman spectra from tissue are masked by the relatively intense tissue auto-fluorescence. After detection, post processing techniques are required to subtract the fluorescence background and enable accurate visualization of the Raman spectra. Raman spectra are plotted as a function of frequency shift in units of wavenumber (cm’J). The region of the Raman spectra where most biological molecules have Raman peaks is from 500 to 2000 cm’1. In contrast to fluorescence spectra, Raman spectra have sharp spectral features that enable easier identification of the constituent sources of spectral peaks in a complex sample. In the context of detecting the changes that cancerous tissues undergo, differences in the Raman spectral features that correlate to the increased nucleic acid content in neoplastic cells has been observed.The term “fluorescence spectroscopy” or its synonyms “fluorometry” or “spectrofluorometry” refers to a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy.Fluorescence spectroscopy utilizes that fact that molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states of energy. Generally, the sample being examined will have a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. Within each of these electronic states are various vibrational states.In fluorescence spectroscopy, the sample is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state.The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies. Therefore, by analyzing the different frequencies of light emitted influorescence spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined.In a typical measurement, the different frequencies of fluorescence light emitted by a sample are measured, holding the excitation light at a constant wavelength. This is called an emission spectrum. An excitation spectrum is measured by recording a number of emission spectra using different wavelengths of excitation light.As used in this disclosure, the term “auto-fluorescence” refers to the fluorescence produced by a molecule of interest without the use of exogenous markers. Auto-fluorescence may serve as a useful diagnostic indicator such as in the case of “biological auto-fluorescence”, which refers to the fact that cells contain molecules, which become fluorescent when excited by UV / VIS (typically 400 - 700 nm) radiation of suitable wavelength. This fluorescence emission, arising from endogenous fluorophores, is an intrinsic property of cells and is called autofluorescence to be distinguished from fluorescence signals obtained by adding exogenous markers. The majority of cell auto-fluorescence originates from mitochondria and lysosomes. Together with aromatic amino acids and lipo-pigments, the most important endogenous fluorophores are pyridinic (NADPH) and flavin coenzymes. In tissues, the extracellular matrix often contributes to the auto-fluorescence emission more than the cellular component, because collagen and elastin have, among the endogenous fluorophores, a relatively high quantum yield. Changes occurring in the cell and tissue state during physiological and / or pathological processes result in modifications of the amount and distribution of endogenous fluorophores and chemicalphysical properties of their microenvironment. Therefore, analytical techniques based on autofluorescence monitoring may be utilized in order to obtain information about morphological and physiological state of cells and tissues. Moreover, auto-fluorescence analysis can be performed in real time because it does not require any treatment of fixing or staining of the specimens.As used in this disclosure, the term, “laser speckle contrast imaging” or its abbreviation “LSCI” refers to a technique for imaging flow for assessment of parathyroid gland vascularity during endocrine surgery, which utilizes intrinsic tissue contrast from dynamic light scattering and provides a relatively simple technique for visualizing detailed spatiotemporal dynamics of blood flow changes in real-time. Laser speckle is the random interference pattern produced when coherent light scatters from a random medium and can be imaged onto a detector. Motion from scattering particles, such as red blood cells in the vasculature, leads to spatial and temporal variations in the speckle pattern. Speckle contrast analysis quantifies the local spatial variance, orblurring, of the speckle pattern that results from blood flow. Areas with greater motion have more rapid intensity fluctuations and therefore have more blurring of the speckles during the camera exposure time. LSCI can be used to quantify relative changes in blood flow.The LSCI technique analyzes the interference pattern produced when coherent light is incident on a surface. Minute differences in path length created by the light waves scattering from different regions of the surface produce bright and dark spots of constructive and destructive interference respectively, termed as a speckle pattern. This speckle pattern fluctuates depending on how fast particles are moving within a few microns of the surface. Blurring of the speckle pattern occurs when the motion is fast relative to the integration time of the detector. Analyzing this spatial blurring provides contrast between regions of faster versus slower motion and forms the basis of LSCI. This technique is sensitive to microvascular perfusion and has been employed in a variety of tissues where the vessels of interest are generally superficial, such as the retina, skin and brain. Parathyroid glands are densely packed with blood vessels, given that they secrete PTH to the entire body. Furthermore, their small size (3-8 mm) makes many of these vessels superficial, making these glands suitable targets for assessment using LSCI. Certain aspects of this invention disclose a combined auto-fluorescence and LSCI system for assessment of parathyroid gland vascularity during endocrine surgery.As used in this disclosure, “charge-coupled device” or “CCD” refers to an analog shift register that enables the transportation of analog signals (electric charges) through successive stages (capacitors), controlled by a clock signal. Charge-coupled devices can be used as a form of memory or for delaying samples of analog signals. Today, they are most widely used in arrays of photoelectric light sensors to serialize parallel analog signals. In a CCD for capturing images, there is a photoactive region (an epitaxial layer of silicon), and a transmission region made out of a shift register (the CCD, properly speaking).An image is projected through a lens onto the capacitor array (the photoactive region), causing each capacitor to accumulate an electric charge proportional to the light intensity at that location. A one-dimensional array, used in line-scan cameras, captures a single slice of the image, while a two-dimensional array, used in video and still cameras, captures a two- dimensional picture corresponding to the scene projected onto the focal plane of the sensor. Once the array has been exposed to the image, a control circuit causes each capacitor to transfer its contents to its neighbor (operating as a shift register). The last capacitor in the array dumps its charge into a charge amplifier, which converts the charge into a voltage. By repeating thisprocess, the controlling circuit converts the entire semiconductor contents of the array to a sequence of voltages, which it samples, digitizes and stores in some form of memory.The description below is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. The broad teachings of the invention can be implemented in a variety of forms. Therefore, while this invention includes particular examples, the true scope of the invention should not be so limited since other modifications will become apparent upon a study of the drawings, the specification, and the following claims. For purposes of clarity, the same reference numbers will be used in the drawings to identify similar elements. It should be understood that one or more steps within a method may be executed in different order (or concurrently) without altering the principles of the invention.In view of the foregoing, the objective of this invention is to develop an imaging technique based on fluorescence to intra-operatively detect endocrine tissue, sarcomas, and brain tumors with minimized surgical error and improved patient outcome by supplementing the standard of visual inspection. The broader scope of the invention is to provide real-time image overlay of the tissues in surgical setting with automated differentiation and high sensitivity that can be used to guide surgeons for many applications including endocrine glands, sarcoma, brain tumor, etc. Particularly, in one aspect, the invention provides a handheld quantitative fluorescent imaging probe and bring it to clinical use, for example, for intraoperative tissue or tumor detection.The invention is based on a highly innovative discovery by Dr. Mahadevan-Jansen of Vanderbilt that the endocrine glands and sarcomas has unique and strong autofluorescence in the near-infrared (NIR) and tumorous brain tissues in the visible. These findings suggest the potential for developing diagnostic techniques leveraging the characteristic autofluorescence signatures of these tissue types.The invention presents a unique application of intrinsic optical spectroscopy and imaging for a critical surgical need in surgery today. The innovation is in the application of an optical technique for surgical guidance that is not focused on not only the disease detection but also on anatomical identification of physiological organs. Further, the technique of fluorescence at visible to near-infrared excitation and corresponding emission has never been implemented in an intrinsic manner, i.e., using natural biological fluorophores as the contrast agent. To inventors’ best knowledge, biological fluorophores emitting in the endocrine glands, sarcoma, and brain tumors have not previously been reported in other literatures. Translating this technology as apractical surgical guidance device requires innovative thinking. Numerous groups have developed fluorescent imaging devices particularly with the growth of indocyanine green as a contrast agent. It should be noted that the emission wavelength of endocrine and sarcoma fluorescence is similar to that of ICG. However, all recently reported fluorescent imaging devices are designed for high contrast images acquired with ICG, which makes the devices incapable of imaging intrinsic fluorescence. In addition, these devices rely on conventional display technology to provide a visual image for the clinician. Here we present the innovative handheld quantitative fluorescence imaging probe that can not only image intrinsic fluorescence by the tissue or tumor, but also admitted dye (e.g. ICG) for real time guidance of the procedure.In the application we develop a two-step modular process for the integration of the device in the surgeon’s hand. Such an approach, which considers and implements the surgeon’s perspective during each step of the development process is a key component to technological development and portends well to its successful acceptance. The application of auto-fluorescence imaging for endocrine tissue, sarcomas, brain tumors are highly innovative.Referring to FIGS. 7A-7C, 8A-8D, 9, 10A-10B and 11, the handheld imaging system 100 / 200 comprising a light delivery member 140 / 240 optically coupled with a light source for delivering light emitted from the light source to tissue of a target region to illuminate the tissue; and an imaging member 130 / 230 for detector for collecting the images images of optical signals emitted from the tissue responsive to the light illumination. The light delivery member 140 / 240 and the imaging member 130 / 230 are incorporated into a handpiece 110 / 210. The target region comprises an adrenal gland, a pancreas gland, a thyroid gland, a parathyroid gland, small bowel, a brain, or any other organs.In one embodiment, the handpiece is rigid or flexible.In one embodiment, the optical signals are fluorescent signals, auto-fluorescent signals, Raman signals, and / or laser speckle contrast imaging (LSCI) signals from the illuminated tissue responsive to the illumination.In one embodiment, the light emitted from the light source has a wavelength in an NIR range of about 600-1500 nm, or in a visible range of about 300-700 nm. In some embodiments, the wavelength is in a range of about 300-400 nm, about 400-500 nm, about 500-600 nm, about 600-700 nm, about 700-800 nm, about 800-900 nm, about 900-1000 nm, about 1000-1100 nm, about 1100-1200 nm, about 1200-1300 nm, about 1300-1400 nm, or about 1400-1500 nm.In one embodiment, the light delivery member comprises free space optics, and / or lightguide. The free space optics may include, but is not limited to, lenses, mirrors, splitters and / or filters.In one embodiment, the light guide comprises one or more optical fibers.In one embodiment, the light guide comprises a single optical fiber (FIG. 7B) formed with a beveled tip positioned in relation to the imaging member such that the angle polished optical fiber aims the illumination at the center of the field of view.In one embodiment, the light guide comprises a plurality of optical fibers (FIGS. 7C and 8A-8D) positioned around the imaging member to achieve an approximately uniform illumination across the field of view.In one embodiment, the imaging member comprises a detector for detector for collecting the images the images of the optical signals.In one embodiment, the detector comprises at least one camera, and / or a spectrometer.In one embodiment, the at least one camera is aligned on-axis with the axis of the handpiece.In one embodiment, the at least one camera has an image depth of 0 to 5 feet from a proximal end of the handpiece to the target tissue.In one embodiment, the at least one camera is aligned at an angle from the axis of the handpiece, allowing sharp angle imaging and 360-degree imaging when rotated around the axis.In one embodiment, the at least one camera comprises at least one charge-coupled device (CCD) camera, at least one complementary metal oxide semiconductor (CMOS) camera, at least one single-photon avalanche diode (SPAD) array, at least one photosensor array, or a combination thereof.In one embodiment, the imaging member further comprises imaging optics.In one embodiment, in a proximal camera configuration (FIGS. 7A-7C, 8A-8D and 9), the at least one camera is integrated into the handpiece, wherein the imaging optics relays an area of interest onto the at least one camera.In one embodiment, the at least one camera comprises a chip on tip camera.In one embodiment, in the proximal camera configuration, the filters, the imaging optics, and the at least one camera are integrated into the proximal tip of the handpiece, wherein once the image is relayed onto the at least one camera and processed, the processed information is transferred wirely or wirelessly to an external device for processing and rendering.In one embodiment, the information is transferred wirelessly to the external devicethrough Wi-Fi, Bluetooth®, and / or near field communication (NFC).In one embodiment, in a distal camera configuration (FIGS. 10A-10B and 11), the imaging member further comprises an output optical light guide including an imaging fiber bundle configured to relay the image from the imaging field to the imaging optics and the at least one camera.In one embodiment, in the distal camera configuration, the imaging optics is configured to relay the field of view onto the imaging fiber bundle and the imaging fiber bundle relays the field of view to a distal camera, enabling imaging the field of interest from a distance.In one embodiment, the imaging member further comprises one or more of long pass, notch, and band pass optical filters positioned between the tissue and the detector to block unwanted wavelengths, ambient light, illumination light, and / or reflected light from the illuminated tissues to ensure the optical signals to be detected.In one embodiment, the filter is incorporated into the handpiece or positioned with the imaging optics before the at least one camera.In one embodiment, the handpiece is provided with at least one of a protruding contact probe attached on the side of the fluorescent imaging probe; a range finder attached next to the fluorescent camera; and a stereoscopic camera set integrated into the fluorescent imaging probe, as shown in FIGS. 8A-8D, respectively.In one embodiment, the range finder is adapted for measuring spatial data including distance and angle between the fluorescent imaging system and the target tissue, accounting for intensity fall-off due to distance, which ensures that images acquired from varying distances.In one embodiment, using the camera readout along with the measured spatial data, the system can model true fluorescence, enabling universal fluorescent assessment.In one embodiment, the fluorescent is quantitatively assessed through the contact probe which is off set from the rest of the elements.In one embodiment, the handheld imaging system can function without an optical filter, directly imaging diffused reflectance light based on the illumination.In one embodiment, the output optical light guide can be coupled to a source with controllable wavelengths, including a tunable light source for different wavelengths illumination.In one embodiment, the handheld imaging system further comprises a controller configured to receive the acquired images from the imaging member and process the acquired images for intraoperatively identifying and locating the tissue.In one embodiment, the handheld imaging system further comprises a display in communication with the controller for displaying the images.It should be noted that all or a part of the steps / methods according to the embodiments of the invention is implemented by hardware or a program instructing relevant hardware. Yet another aspect of the invention provides a non-transitory tangible computer-readable medium storing instructions which, when executed by one or more processors, cause fluorescent images, acquired with the above disclosed handheld imaging system, to be processed and rendered in real-time.In one embodiment, in a contact quantitative fluorescent design, a point-based readout is presented alongside the fluorescent image.In one embodiment, in a range-finding quantitative design, the readout is derived from distance measurements and camera data, then displayed accordingly.In one embodiment, an end user is presented with a point-based system as well as a form of graphic user interface that includes values to discriminate fluorescent level between highly fluorescent tissue and less fluorescent tissue.The computer executable instructions or program codes enable a computer or a similar computing system to complete various operations in the above disclosed method for privilege management. The storage medium / memory may include, but is not limited to, high-speed random access medium / memory such as DRAM, SRAM, DDR RAM or other random access solid state memory devices, and non-volatile memory such as one or more magnetic disk storage devices, optical disk storage devices, flash memory devices, or other non-volatile solid state storage devices.Without intent to limit the scope of the invention, exemplary methods and their related results according to the embodiments of the invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.EXAMPLE 1:CLINICAL APPLICABILITY OF FLUORESCENT IN ENDOCRINE GLANDSDETECTIONPortable fluorescence spectroscopy system: This new intra-operative technique must be proven to be capable of handling the full spectrum of interpatient variability before its clinical adoption. The goal of our study, then, was to establish the clinical utility for this novel intraoperative parathyroid detection technique by assessing the effects of pathologic, clinical, and demographic patient variables on the parathyroid fluorescence signal. NIR fluorescence spectra was acquired from 137 patients (264 parathyroid glands) undergoing cervical endocrine surgery. Patient factors such as body mass index (BMI), metabolite levels, disease type, or hormone levels, age, and sex underwent statistical analysis for their effects on the parathyroid detection rate.A portable fluorescence spectroscopy system was designed and built by the Vanderbilt group for clinical use as shown in FIG. 4. The system is comprised of a laser coupling NIR light through a 7-around-l sterilized fiber optic probe. Tissue was irradiated with 80 mW of light in a 400 pm spot size. Fluorescence was detected by a spectrometer via the collection fibers. Inline filtering at the tip of the fiber optic and at the port of the spectrometer ensured that no visible or laser light interfered with the fluorescence signal. Fluorescence measurements were controlled by a laptop computer running custom software.Parathyroid autofluorescence was consistently greater than the thyroid and all surrounding tissues in 97% (256 of 264) of glands measured. Muscle, fat, lymph nodes, thymus, and trachea showed little to no fluorescence signal. Accuracy of parathyroid detection. NIR fluorescence spectroscopy demonstrated 100% parathyroid detection accuracy in patients with toxic and nontoxic nodular goiter, Hashimoto’s thyroiditis, follicular and Hurthle cell thyroid adenoma, medullary thyroid carcinoma, Graves’ disease, and primary and tertiary hyperparathyroidism. Patients with well-differentiated thyroid cancer showed 98% detection accuracy (56 of 57 glands), and patients with concomitant parathyroid and thyroid disease exhibited a 94% detection accuracy (16 of 17 glands). Reduced performance was recorded in the specific case of patients with renal-induced secondary hyperparathyroidism (sHPT), where NIR fluorescence spectroscopy was able to detect 54% of parathyroid glands (7 of 13 glands). The fluorescence measurements of 69 parathyroid glands were confirmed with histology. Of the glands that could not be confirmed with histology, 88% were identified as parathyroid glands with high confidence and 12% with medium confidence by the visual assessment of the surgeon. Analysis revealed body mass index (P < .01), disease state (P < .01), vitamin D (P < .05), andcalcium levels (P < .05) account greatly for variability in signal intensity. Age, sex, parathyroid hormone, and ethnicity had no effect. The results suggest NIR fluorescence spectroscopy is ready for clinical implementation as a parathyroid detection tool by showing its effectiveness over a variety of clinical presentations. We have conducted extensive studies to demonstrate that NIR fluorescence spectroscopy could distinguish the parathyroid gland from the thyroid and all other tissues in the neck during surgery, regardless of disease type.Basis for NIR Fluorescence Signal: Based electron microscopy images, and results obtained thus far, we hypothesize that a component of the secretory granules act as the endogenous fluorophore responsible for the observed fluorescence in the parathyroid. Secretory granules contain specific proteins and other macromolecules that are destined for secretion into the extracellular space such as parathyroid hormone and chromogranin-A in the parathyroid gland. Secretory granules are found in high levels in endocrine tissues such as the parathyroid gland, thyroid gland, pancreas, adrenal medulla, and pituitary gland, but they are not present in the muscle, fat or lymph of the neck region. If our hypothesis is correct, then these tissues containing secretory granules should display a NIR fluorescence signal as well. Evidence that the fluorophore is located within the secretory granules of endocrine tissues was gathered by detector for collecting the images fluorescent images and spectra from parathyroid adenoma, normal thyroid gland, normal pancreas, and adrenal pheochromocytoma tissue and muscle (FIG. 1). No fluorescence was observed in the muscle sample samples (flat line along the x-axis) as compared to the other endocrine tissue samples tested. Highest levels of fluorescence were present in the parathyroid adenoma and adrenal pheochromocytoma samples. This result provides a preliminary validation of our hypothesis that secretory granules contain the endogenous NIR fluorophore.Prototype point spectroscopy device (PTEye): PTEye is currently an FDA cleared clinical device for intraoperative parathyroid detection using near-infrared autofluorescence detection. This device is originated from a collaboration between the original manufacturing company AiBiomed, and the inventor Dr. Mahadevan-Jansen. Unlike the original prototype designed and utilized in Vanderbilt University, PTEye is designed to meet Class 3R for safety (versus Class 3B with the Vanderbilt device). In addition, the other major difference with the PTEye device is that there is no need to turn the operating room (OR) light off. Most importantly, the product has been designed for user-friendliness and with specific input from our clinical team (FIG. 5).The system incorporates the same point detection technology as used in the portablefluorescence spectroscopy system with a few exceptions such as using s micro-processor board internally instead of a laptop computer and incorporating visual and auditory feedback mechanisms through a display panel and a loudspeaker. The PTEye product uses a photo detector. The laser source can range from CW to 100s of thousands Hz modulated to pick up the fluorescence signal while avoiding ambient light. The internal software and firmware have been designed to improve the signal-to-noise ratio so that the system can differentiate small changes in fluorescence signal. The PTEye has been tested and validated in tissues using excised parathyroid and thyroid tissues in the laboratory, in vitro as well as in vivo in the operating room. PTEye can be used to successfully detect parathyroid fluorescence that is as small as 20% above thyroid fluorescence. The NIR laser power meets the laser classification required for safety. The probe assembly shown is designed to meet both steam sterilization (Autoclave) cycle as well as Sterrad 100NX sterilization method. The probe assembly is detachable for sterilization methods.Proof of Concept of NIR Imaging of Parathyroid: To assess the feasibility for translating this technology to imaging, preliminary studies were performed on three different imaging systems that has the ability to provide spatial information along with the fluorescence to the surgeon. Parathyroid and thyroid tissues were imaged in vitro using 2 different NIR imaging systems designed and built at Vanderbilt as shown in FIG. 6. A NIR Viewer with a camera and a highly sensitive camera used as a gold standard were assembled. Both systems use lens and a filter. Near-infrared light (X = 600-850 nm) excited the tissues through a fiber optic probe. The detector of each imaging system collected the images after filtering the laser line through a filter at the front of the detector head. Images obtained from each of these systems are shown in FIG. 6 with the parathyroid being the smaller, brighter tissue on the left and the thyroid is the larger tissue on the right. These results validate the ability of NIR fluorescence to be translated into an imaging device to provide spatial context and signal contrast to the detectability of the parathyroid.EXAMPLE 2:HANDHELD QUANTITATIVE FLUORESCENT IMAGING PROBE FOR INTRAOPERATIVE TISSUE OR TUMOR DETECTIONWe have already demonstrated the clinical utility of fluorescent imaging during cancer surgery in the neck as well as the feasibility of real time fluorescence imaging in a clinical setting.In this exemplary embodiment, we disclose a handheld quantitative fluorescent imaging probe and demonstrate the feasibility of the imaging probe to acquire in vivo fluorescence images of the tissue or tumor, thereby bringing the imaging probe to clinical use, for example, for intraoperative tissue or tumor detection for real time guidance of surgery.As shown in FIG. 6, our preliminary data supports construction of an imaging device to provide spatial information on the location of the tissue or tumor in real-time. This imager should allow the surgeon to quickly identify the tissue or tumor within the surgical field. One aspect of the invention is to provide a novel imaging system that augments surgical guidance by overlaying fluorescence information on to the surgical field to identify the tissue or tumor. The handheld quantitative fluorescent imaging probe prototype is tested in vitro and in a pilot study in vivo to determine its ability to identify tissue or tumor from surrounding tissues such as the nerve, lymph, fat, muscle. The goal is to evaluate the feasibility of this approach.There are numerous imaging systems that have been developed for dye-based imaging such as ICG. These systems, however, are typically not sensitive enough for tissue autofluorescence imaging whose signal to noise ratio (SNR) is typically lower than that from dyes. Further, relating imaging systems are often bulky in size, making it impractical to image hard to reach cavities without expanding the surgical opening. A more practical system should have the form factor of a flexible endoscope thus allowing maneuverability. The design relies on recent improvements in sensitivity and miniaturization of the chip-on-tip cameras to develop a compact, easy-to-use handheld quantitative fluorescent imaging probe with the resolution needed for intra-operative use.Hardware: A quantitative handheld fluorescent imaging probe 110 / 210 is designed to realize intraoperative fluorescence detection in real time (FIGS. 7A-7C, 8A-8D, 9, 10A-10B and 11). The hardware can be divided into three separate components: fluorescent illumination, miniaturized fluorescent imaging, and quantitative fluorescent assessment. The handheld probe is designed to have size that is comfortably held by the operator. One design mimic that of a pistol endoscope. Another design has a casing of 140 mm long and less than 10 mm in diameter to mimic the shape of a pen or a probe. To make the design for intuitive usage, such as a pen, the camera is aligned on-axis with the axis of the handpiece cylinder. The probe camera has image depth of 0 to 3 feet from the probe tip to the target and offers sub-optimum image quality when the operator holds the probe at a distance outside of the mentioned range. In operation, the operator holds the probe like a pen and aim at the target of interest. Another design is to havethe camera aim at an angle from the axis of handpiece cylinder, allowing sharp angle imaging and 360-degree imaging when rotated around the cavity.For clarity, the on-axis camera-handpiece design is the primary focus of the disclosure, though the design criteria and concepts apply equally to all variations. The design of the probe can be straight or at an angle / arch. The casing can also be flexible or with an articulating tip.Fluorescent illumination: The purpose of the fluorescent illumination is to excite the target fluorophores. In some embodiments, fluorescent excitation ranges from 600 to 850 nm (NIR), 300 to 700 nm (visible) in wavelength with no limitation with its bandwidth. LED, laser diodes, VECSEL (laser on chip), TO-CAN, or any light source that emits the mentioned wavelength can act as the illumination. There is also no minimum required power for the light source. The illumination can be guided to the area of interest either through free space or light guide, such as optical fibers. The optical light guide can vary in form such as beveled tip (angled illumination), multi-output, etc.In a beveled tip design, as shown in FIG. 7B, an angle polished fiber 140 can aim the illumination at the center of the field of view realize fluorescent illumination. However, with angled illumination, the light distribution might not be uniform enough that the system would require calibration to realize equal fluorescent read-out of the same fluorescent sample across the field of view (illumination intensity on one side of the field of view is different from than that on the other side). However, the beveled illumination method is still a valid way of imaging fluorescent.In a multi-output design, as shown in FIG. 7C, multiple output fibers 140 (white circles) are positioned around the image acquisition channel (blue circle) to achieve a pseudo-uniform illumination across the field of view. The increased number and strategic arrangement of the fibers enhance the illumination uniformity, facilitating the imaging based quantitative fluorescent assessment. However, the system response could be calibrated and modeled based on the ununiform illumination, allowing flexibility in the pattern design and number of the fiber required. Nonetheless, multi -output design offers an easy design for uniform illumination cross the field of view. In FIGS. 7B and 7C, the grey components 120 represent the casing of the handpiece 110, the blue component 130 represents the fluorescent camera, white components 140 represent the optical light guides, red cones 150 represent the illumination light, and the yellow cones 160 represent the image field of view.Miniaturized fluorescent imaging: The miniaturized fluorescent imaging system needslong pass, notch, or band pass optical filters to block the fluorescent illumination light. The optical filter is not limited to absorption and interference-based filter, but also optomechanical filters such as an acoustic optical tunable filter. The filtering wavelength depends on the illumination wavelength. For example, if the illumination wavelength is centered at 785 nm and has spectral linewidth of 785 ± 10 nm, the filter needs to block off wavelength from 775 nm to 795 nm. The wavelength and range can vary depending on the application. In practice, optical illumination spectrum profiles are not strictly a rectangular function but any spectral-intensity distribution profile. In these cases, the optical filter needs to provide blocking of the corresponding illumination wavelengths.Besides the fluorescent filter, the miniaturized camera is composed of optics and detection chip / s. In one embodiment, the detection chip is integrated into the handheld probe where imaging optics relay the area of interest onto the detector (proximal camera). In the proximal camera configuration (FIGS. 7A-7C, 8A-8D and 9), filter, imaging optics, and the camera chip are integrated into the tip of the probe. Once the image is relayed onto the chip and processed, the digital information is transferred to an external evaluation component (e.g., computer) for processing and rendering as shown in FIGS. 7A-7C.In another embodiment, as shown in FIGS. 10A-10B, an optical light guide, such as an imaging fiber bundle 230, is used to relay the image from the imaging field to the imaging optics and detector (distal camera). In the distal imaging fiber bundle camera configuration (FIGS. 10A-10B and 11), a relay imaging optics needs to relay the field of view onto the imaging fiber bundle and the imaging fiber bundle would relay the field of view to a distal camera, enabling imaging the field of interest from a distance. In this setup, the filter can be incorporated either into the handpiece or positioned with the imaging optics before the camera. In FIG. 10B, the grey components 220 represent the casing of the handpiece, the blue component 230 represents the imaging optical guides, white components 240 represent the optical light guides, red cones 250 represent the illumination light, and the yellow cones 260 represent the image field of view. The green circles 270 are laser pointer that helps with aiming and the red circle 280 is the long wavelength illumination for wide field imaging with the same camera. However, both the green circle 270 and the red circle 280 here are optional features that assist with the operation.Utilizing either the chip-on-tip design or an optical light guide minimizes the size of the fluorescent imaging component of the handheld probe. The optical light guide design removes constraints on the weight and size of the camera, allowing for greater flexibility in cameraselection. For example, a single-photon avalanche diode (SPAD) array eliminates the need for an optical filter, which alter the design.Quantitative fluorescent assessment: To enable universal assessment of tissue fluorescent, the device must acquire data that is comparable across different targets and individuals. Achieving this requires a standardized assessment procedure, which includes maintaining consistent excitation illumination intensity and wavelength, exposure time, detector filtering, and the relative positioning and angle of the illumination, detection, and target. The end user is presented with a point-based system as well as a form of graphic user interface (GUI) that includes values to discriminate fluorescent level between highly fluorescent tissue and less fluorescent tissue.In one embodiment, the system measures the distance and angle between the fluorescent imaging system and the target tissue, accounting for intensity fall-off due to distance. This ensures that images acquired from varying distances, which otherwise would yield inconsistent fluorescence readouts, are corrected for comparability. Using the camera readout along with measured spatial data, the system can model true fluorescence, enabling universal fluorescent assessment. These spatial measurements can be obtained using methods such as range finders, including stereoscopic, ultrasonic, infrared, or laser-based techniques.In another embodiment, fluorescent is quantitatively assessed through a contact probe which is off set from the rest of the elements (protrusion of the section is optional). This probe includes the fluorescent illumination and fluorescence collection (as shown in FIGS. 8A-8B). To use the system, the operator first acquires the fluorescent image with the handheld probe. Once the target tissue is identified with the fluorescent images, the operator contacts the contact probe to the target tissue and make a measurement the same way as the point-based spectroscopy device mentioned above. Note that for this component, spectroscopy is not necessary. For example, a photodetector with filtering capability could be used to detect fluorescence.Miniaturized reflectance imaging: The miniaturized imaging system can function without an optical filter, directly imaging the diffused reflectance light based on the illumination. The system without filter shares design with fluorescent imaging (either a chip on tip camera or optical light guide). Additionally, the output optical light guides can be coupled to a source with controllable wavelengths, such as a tunable light source for different wavelengths illumination.Software: A software program is designed for real-time processing of the fluorescent images. Images collected by the fluorescent camera are sent to the computer and an overlayimage of the fluorescent and image are displayed. In the contact quantitative fluorescent design, a point-based readout is presented alongside the fluorescent image. In the range-finding quantitative design, the readout is derived from distance measurements and camera data, then displayed accordingly. The end user is presented with a point-based system as well as a form of graphic user interface that includes values to discriminate fluorescent level between highly fluorescent tissue and less fluorescent tissue.EXAMPLE 3:FIBER-BUNDLE BASED SYSTEM AND MINI-CAMERA BASED SYSTEMNear infrared auto-fluorescent (NIRAF) imaging is a widefield imaging technique capable of identifying tissue, such as parathyroid glands, adrenal glands, neuroendocrine tumors, etc. The inventors have shown it is an effective method for distinguishing target tissue from the rest. However, the previous work involved a static or bulky imaging device that is not always ideal for small surgical incisions and to image hard-to-reach targets. This example describes, among other things, two approaches to a handheld NIRAF imaging system for intraoperative identifying target tissues combining with an approach that quantitatively assesses the strength of their fluorescence.In one embodiment, as shown in FIG. 11, an imaging fiber bundle is used to relay images from the surgical site to the sensor of a CCD or CMOS camera. The end of the fiber bundle at the surgical site is referred to as a distal end, while the end connected to the sensor is referred to a proximal end. In this exemplary embodiment, a rod lens or lens system is placed on the distal end of the fiber bundle to optimize the collection of light and image formation at the chosen working distance. At the proximal end, a lenses system images the end of the fiber bundle through a long pass filter onto the camera sensor. The lenses are chosen to magnify the image from the distal end of the fiber bundle to fill the camera sensor. For example, in the fiber bundle system schematic, a 20x objective is paired with an 80 mm focal length converging lens to magnify the image from a 1 mm diameter fiber bundle onto an 8.7 mm diameter CMOS sensor, while having a long pass filter between the two lenses. To provide illumination for fluorescent imaging, an optical fiber is connected to a laser module of a specific wavelength and is run alongside the fiber bundle. In addition, a diffuser can be attached at the distal end of the illumination optical fiber to produce a more uniform illumination pattern on the target tissue to be imaged. By bundling the illumination fiber and imaging fiber into one rigid or flexible probe,parathyroid glands can be more easily illuminated and detected in cases with small surgical incisions. An alternative to the optical fiber for illumination could be a small laser diode at the distal end of the probe.Besides an imaging fiber bundle, a long pass filtering micro-camera could also be employed for this purpose. In one embodiment as shown in FIG. 9, the camera sensor would be at the distal end of the probe, adjacent to the illumination fiber, while the wiring connecting the camera will run to the distal end outside the surgical field where it will be connected to the appropriate electronics for recording images. For example, in the mini-camera system schematic, a mini-camera with 249x250 pixels is deployed at the distal end. To provide illumination for fluorescent imaging, an optical fiber is connected to a laser module of a specific wavelength and runs alongside the imaging channel. A diffuser can be attached at the distal end of the illumination optical fiber to produce a more uniform illumination on the target tissue to be imaged. An optional white light fiber can be attached to both modalities for white light illumination and better focusing. Bundling the imaging channel, fiber optics, and the optional white light fiber into one rigid or flexible probe, parathyroid glands can be more easily evaluated in cases with small surgical incisions.To quantitatively assess the strength of fluorescence, a fiber optic probe delivers laser light to the tissue site and simultaneously collects signal from the site. The signal is subsequently separated to the corresponding NIRAF by optics such as a spectrometer, relayed to the laptop, and displayed quantitatively. Such quantitative assessment method combining with the imaging modality will provide surgeons with a single tool to quantitatively identify parathyroid glands during operations. This has the potential to improve patient outcomes after surgery. An alternative way of quantitative assessment is to incorporate a device that measures the distance between the NIRAF imaging probe and the target tissue. The system can calibrate the fluorescent strength of the imaging target, based on the distance.EXAMPLE 4: EXEMPLARY PROTOTYPES OF QUANTITATIVE HANDHELD FLUORESCENT IMAGING PROBEIn a first exemplary prototype, as shown in FIG. 12, a chip-on-tip camera is used to acquire fluorescent image (Blue front, bigger metal tube). The chip-on-tip camera (ams OSRAM, Premstaetten, Austria) is a micro-camera that have cross-section of 1 x 1 mm, locating behind along pass absorption filter at 785 nm (MidOpt, Palatine, IL). A lens system relays the field of 1x1 cm to the chip in the camera, at a working distance range of 1-3 cm. The camera acquires image at 25 ms exposure time and offers real time imaging with 250 x 249 pixels.The fluorescent illumination has an optical fiber that connects to a laser module (Innovative Photonics Solutions, Plainsboro, NJ) with center wavelength of 730 nm (white arch on top of the bigger metal tube). The optical fiber is bended at a proper degree and directs the light to the imaging field. A diffuser attached at the fiber tip to produce a more uniform illumination pattern to the imaging field.A laser-based range finder (Bridger Photonics, Bozeman, MT or Avantier, North York, ON) is attached to the side of the probe (smaller metal tube on the side), as shown in FIG. 12. The range finder would identify distance between the tip of the probe to imaging field to the computer for quantitative fluorescent modeling and calculation.By bundling the illumination fiber and imaging channel into one rigid or flexible probe, target tissue / cancer can be more easily illuminated and detection.FIG. 14 shows fluorescent images of two ex vivo parathyroid glands tissue and two ex vivo thyroid glands tissue (top panel), taken by the first prototype of the handheld imaging system in laboratory settings, and the mean and variance of the pixels' readout from region of interest from each image acquisition (bottom panel). The ex vivo tissue samples are collected under IRB from VUMC. The images and the plot show that the device is capable of distinguishing the parathyroid glands and the thyroid glands based on the fluorescence imaging and corresponding readout from the device.In a second exemplary prototype, as shown in FIG. 13, a chip-on-tip camera is used to acquire fluorescent image. The chip-on-tip camera is a micro-camera (OmniVision, Santa Clara, CA) that have cross-section of 3x3 mm, incorporating long pass interference filters at 730 nm (Semrock, Rochester, NY) as part of the camera optics. A lens system relays the field of 3x3 cm to the chip in the camera, at a working distance range of 3-6 cm. The camera acquires image at 125 ms exposure time and offers real time imaging with 1250x1249 pixels.The fluorescent illumination has a ring of optical fiber that connected to a laser module (Innovative Photonics Solutions, Plainsboro, NJ) with center wavelength of 680 nm, as shown in FIG. 13. A diffuser, optional attached at the fiber tip to produce a more uniform illumination pattern to the imaging field.A contact-based quantitative fluorescent probe is attached to the side of the probe(smaller metal tube on the side), as shown in FIG. 13 The signal is subsequently separated to the corresponding NIRAF by optics such as a spectrometer, relayed to the laptop, and displayed quantitatively. Such quantitative assessment method combining with the imaging modality will provide surgeons with a single tool to quantitatively identify parathyroid glands during operations. This has the potential to improve patient outcomes after surgery. The fluorescent probe will offer quantitative fluorescent readout upon contact.By bundling the illumination fiber and imaging channel into one rigid or flexible probe, target tissue / cancer can be more easily illuminated and detection.In a third exemplary prototype, a chip-on-tip camera is used to acquire fluorescent image. The chip-on-tip camera is a micro-camera (Sony, New York, NY) that have cross-section of 6x6 mm, incorporating band pass interference filters at 630 nm as part of the camera optics (Chroma, Bellows Falls, VT). A lens system relays the field of 1x1 cm to the chip in the camera, at contact with the imaging site. The camera acquires image at 25 ms exposure time and offers real time imaging with 800x250 pixels.The fluorescent illumination has an angle polished fiber that connected to a laser module (Laser Components, Bedford, NH) with center wavelength of 580 nm. A negative lens, optional, is attached at the fiber tip to produce a more uniform illumination pattern to the imaging field.An ultrasonic-based range finder (Adafruit, New York, NY) is attached to the side of the probe. The range finder would identify distance between the tip of the probe to imaging field to the computer for quantitative fluorescent modeling and calculation.By angling the bundle of the illumination fiber and imaging channel into a arching probe, target tissue / cancer can be more easily illuminated and detection on the wall of the cavity.In a fourth exemplary prototype, an imaging fiber bundle is used to relay images from the field of view to the sensor CCD. The end of the fiber bundle at the field of view is referred to as the distal end, and the end connected to the sensor is the proximal end. A lens system is place on the distal end of the fiber bundle to image the field of 3x3 cm at a working distance range of 1-3 cm. At the proximal end, a lenses system will image the end of the fiber bundle through a long pass filter onto the camera sensor. The lenses are chosen to magnify the image from the distal end of the fiber bundle to fill the camera sensor. For example, in the fiber bundle system schematic, a 20x objective is paired with an 80 mm focal length converging lens to magnify the image from a 1 mm diameter fiber bundle onto an 8.7 mm diameter CMOS sensor, while having a long pass filter (LP02-808RU-25) between the two lenses. To provide illumination forfluorescent imaging, an optical fiber is connected to a laser module of a specific wavelength and is run alongside the fiber bundle. A diffuser can be attached at the distal end of the illumination optical fiber to produce a more uniform illumination pattern on the target tissue to be imaged. By bundling the illumination fiber and imaging fiber into one rigid or flexible probe, parathyroid glands can be more easily illuminated and detected in cases with small surgical incisions.EXAMPLE 5:TEST THE CAPABILITY OF THE QUANTITATIVE HANDHELD FLUORESCENT PROBE TO IDENTIFY TISSUE OR TUMOR IN PATIENTS IN VIVO.In this example, the system is tested in vivo on patients undergoing related surgeries at Vanderbilt University Medical Center (VUMC).Patient Recruitment and Measurement: The protocol has been approved by the Vanderbilt Institutional Review Board (IRB). All patients referred to the Vanderbilt Endocrine Surgery Center, regardless of race or gender are screened for inclusion in the study. An initial evaluation is conducted by the treating surgeon while seeing the patients at the Vanderbilt Clinic. Eligibility for participating in the study is determined during the preoperative evaluation requiring that the individual be a safe and acceptable candidate. Only adult patients between the ages of 18-99 years with related diseases undergoing corresponding operations are considered. Informed written consent is obtained from each patient enrolled.Statistical Sample Size Estimation: To test the capability of the quantitative handheld fluorescent probe, 30 patients are recruited. Assuming a recruitment rate of 50% (based on the predicted disease category and patient consent rate), we have access to about 75 with corresponding diseases in the 6-month period. Therefore, we anticipate that recruitment of the proposed disease to take approximately 1.5-2 months.Analysis of Prototype Testing: After 30 patients have been tested with the quantitative handheld fluorescent probe, the data collected are analyzed to determine the robustness of the device. An Analysis of Variance (ANOVA) test is performed on the patient data to determine whether the observed autofluorescence signal was affected by systematic inter-patient variations. Mixed effects ANOVA are also performed separately for patient data to assess variability across probe based and imaging systems. Results determine whether the quantitative handheld fluorescent probe detects differences in the tissue or tumor in same pathology. Any issues related to the surgical field such as the effect of room lights, and reflections off surgical instruments areidentified and modifications are made in hardware and software as needed to address these issues. In this version of the device, we propose to turn the OR lights off during image acquisition. The device addresses this issue of detector for collecting the images images with the light on.The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.The embodiments were chosen and described in order to explain the principles of the invention and their practical application so as to activate others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the invention pertains without departing from its spirit and scope. Accordingly, the scope of the invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.Some references, which may include patents, patent applications, and various publications, are cited and discussed in the description of this invention. The citation and / or discussion of such references is provided merely to clarify the description of the invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.LIST OF REFERENCES[1] E. SPARTALIS et al., “Intraoperative Indocyanine Green (ICG) Angiography for the Identification of the Parathyroid Glands: Current Evidence and Future Perspectives,” In Vivo (Brooklyn), vol. 34, no. 1, pp. 23-32, Dec. 2020, doi: 10.21873 / invivo.11741.[2] S. Gourgiotis, P. Moustafellos, N. Dimopoulos, G. Papaxoinis, S. Baratsis, and E.Hadjiyannakis, “Inadvertent parathyroidectomy during thyroid surgery: the incidence of a complication of thyroidectomy.,” Langenbecks Arch Surg, vol. 391, no. 6, pp. 557-60, Nov. 2006, doi: 10.1007 / s00423-006-0079-8.[3] A. Mohebati and A. R. Shaha, “Imaging techniques in parathyroid surgery for primaryhyperparathyroidism.,” Am J Otolaryngol, vol. 33, no. 4, pp. 457-68, 2012, doi: 10.1016 / j.amjoto.2011,10.010.[4] H. Chen et al., “Operative Failures After Parathyroidectomy for Hyperparathyroidism,” Ann Surg, vol. 252, no. 4, pp. 691-695, Oct. 2010, doi: 10.1097 / SLA.0b013e3181f698df.[5] R. Bergamaschi, G. Becouarn, J. Ronceray, and J. P. Arnaud, “Morbidity of thyroid surgery.,” Am J Surg, vol. 176, no. 1, pp. 71-5, Jul. 1998, doi: 10.1016 / s0002- 9610(98)00099-3.[6] D. T. Lin, S. G. Patel, A. R. Shaha, B. Singh, and J. P. Shah, “Incidence of Inadvertent Parathyroid Removal During Thyroidectomy,” Laryngoscope, vol. 112, no. 4, pp. 608- 611, Apr. 2002, doi: 10.1097 / 00005537-200204000-00003.[7] N. D. Perrier et al. , “Intraoperative parathyroid aspiration and parathyroid hormone assay as an alternative to frozen section for tissue identification.,” World J Surg, vol. 24, no. 11, pp. 1319-22, Nov. 2000, doi: 10.1007 / s002680010218.[8] J. Q. Nguyen et al., “Near-infrared autofluorescence spectroscopy of in vivo soft tissue sarcomas,” Opt Lett, vol. 40, no. 23, p. 5498, Dec. 2015, doi: 10.1364 / OL.40.005498.[9] E. Chamma et al., “Optically-tracked handheld fluorescence imaging platform for monitoring skin response in the management of soft tissue sarcoma,” J Biomed Opt, vol. 20, no. 7, p. 076011, Jul. 2015, doi: 10.1117 / 1 JB0.20.7.076011.

[0010] S. A. Toms, “Brain tumor demarcation using optical spectroscopy; an in vitro study,” J Biomed Opt, vol. 5, no. 2, p. 214, Apr. 2000, doi: 10.1117 / 1.429989.

[0011] M. A. McWade, M. E. Sanders, J. T. Broome, C. C. Solorzano, and A. Mahadevan- Jansen, “Establishing the clinical utility of autofluorescence spectroscopy for parathyroid detection,” Surgery, vol. 159, no. 1, pp. 193-203, Jan. 2016, doi: 10.1016 / j.surg.2015.06.047.

Claims

CLAIMSWhat is claimed is:

1. A handheld imaging system, comprising: a light delivery member optically coupled with a light source for delivering light emitted from the light source to tissue of a target region to illuminate the tissue; and an imaging member for detector for collecting the images images of optical signals emitted from the tissue responsive to the light illumination, wherein the light delivery member and the imaging member are incorporated into a handpiece.

2. The handheld imaging system of claim 1, wherein the handpiece is rigid or flexible.

3. The handheld imaging system of claim 1, wherein the light emitted from the light source has a wavelength in an NIR range of about 600-1500 nm, or in a visible range of about 300-700 nm.

4. The handheld imaging system of claim 1, wherein the light delivery member comprises free space optics and / or light guide.

5. The handheld imaging system of claim 4, wherein the light guide comprises one or more optical fibers.

6. The handheld imaging system of claim 5, wherein the light guide comprises a single optical fiber formed with a beveled tip positioned in relation to the imaging member such that the angle polished optical fiber aims the illumination at the center of the field of view.

7. The handheld imaging system of claim 5, wherein the light guide comprises a plurality of optical fibers positioned around the imaging member to achieve a pseudo-uniform illumination across the field of view.

8. The handheld imaging system of claim 1, wherein the imaging member comprises a detector for collecting the images of the optical signals.

9. The handheld imaging system of claim 8, wherein the detector comprises at least one camera, and / or a spectrometer.

10. The handheld imaging system of claim 9, wherein the at least one camera is aligned on- axis with the axis of the handpiece.

11. The handheld imaging system of claim 10, wherein the at least one camera has an image depth of 0 to 5 feet from a proximal end of the handpiece to the target tissue.

12. The handheld imaging system of claim 9, wherein the at least one camera is aligned at an angle from the axis of the handpiece, allowing sharp angle imaging and 360-degree imaging when rotated around the axis.

13. The handheld imaging system of claim 9, wherein the at least one camera comprises at least one charge-coupled device (CCD) camera, at least one complementary metal oxide semiconductor (CMOS) camera, at least one single-photon avalanche diode (SPAD) array, at least one photosensor array, or a combination thereof.

14. The handheld imaging system of claim 9, wherein the imaging member further comprises imaging optics.

15. The handheld imaging system of claim 14, wherein in a proximal camera configuration, the at least one camera is integrated into the handpiece, wherein the imaging optics relays an area of interest onto the at least one camera.

16. The handheld imaging system of claim 15, wherein the at least one camera comprises a chip on tip camera.

17. The handheld imaging system of claim 16, wherein in the proximal camera configuration,the filters, the imaging optics, and the at least one camera are integrated into the proximal tip of the handpiece, wherein once the image is relayed onto the at least one camera and processed, the processed information is transferred wirely or wirelessly to an external device for processing and rendering.

18. The handheld imaging system of claim 17, wherein the information is transferred wirelessly to the external device through Wi-Fi, Bluetooth®, and / or near field communication (NFC).

19. The handheld imaging system of claim 14, wherein in a distal camera configuration, the imaging member further comprises an output optical light guide including an imaging fiber bundle configured to relay the image from the imaging field to the imaging optics and the at least one camera.

20. The handheld imaging system of claim 19, wherein in the distal camera configuration, the imaging optics is configured to relay the field of view onto the imaging fiber bundle and the imaging fiber bundle relays the field of view to a distal camera, enabling imaging the field of interest from a distance.

21. The handheld imaging system of claim 14, wherein the imaging member further comprises: one or more of long pass, notch, and band pass optical filters positioned between the tissue and the detector to block unwanted wavelengths, ambient light, illumination light, and / or reflected light from the illuminated tissues to ensure the optical signals to be detected.

22. The handheld imaging system of claim 21, wherein the filter is incorporated into the handpiece or positioned with the imaging optics before the at least one camera.

23. The handheld imaging system of claim 14, wherein the handpiece is provided with at least one of a protruding contact probe attached on the side of the fluorescent imaging probe;a range finder attached next to the fluorescent camera; and a stereoscopic camera set integrated into the fluorescent imaging probe.

24. The handheld imaging system of claim 23, wherein the range finder is adapted for measuring spatial data including distance and angle between the fluorescent imaging system and the target tissue, accounting for intensity fall-off due to distance, which ensures that images acquired from varying distances.

25. The handheld imaging system of claim 24, wherein using the camera readout along with the measured spatial data, the system can model true fluorescence, enabling universal fluorescent assessment.

26. The handheld imaging system of claim 23, wherein the fluorescent is quantitatively assessed through the contact probe which is off set from the rest of the elements.

27. The handheld imaging system of claim 14, wherein the handheld imaging system can function without an optical filter, directly imaging diffused reflectance light based on the illumination.

28. The handheld imaging system of claim 14, wherein the output optical light guide can be coupled to a source with controllable wavelengths, including a tunable light source for different wavelengths illumination.

29. The handheld imaging system of claim 1, further comprising: a controller configured to receive the acquired images from the imaging member and process the acquired images for intraoperatively identifying and locating the tissue.

30. The handheld imaging system of claim 29, further comprising a display in communication with the controller for displaying the images.

31. The handheld imaging system of claim 1, wherein the target region comprises an adrenal gland, a pancreas gland, a thyroid gland, a parathyroid gland, small bowel, a brain,skeletal muscle, soft tissue sarcoma, or any other organs.

32. A non-transitory tangible computer-readable medium storing instructions which, when executed by one or more processors, cause fluorescent images, acquired with the handheld imaging system of any one of claims 1-31, to be processed and rendered in realtime.

33. The non-transitory tangible computer-readable medium of claim 32, wherein in a contact quantitative fluorescent design, a point-based readout is presented alongside the fluorescent image.

34. The non-transitory tangible computer-readable medium of claim 32, wherein in a rangefinding quantitative design, the readout is derived from distance measurements and camera data, then displayed accordingly.

35. The non-transitory tangible computer-readable medium of claim 32, wherein an end user is presented with a point-based system as well as a form of graphic user interface that includes values to discriminate fluorescent level between highly fluorescent tissue and less fluorescent tissue.

Images