2.1.

Principles of Enhanced Backscattering

For a plane wave illuminating a semi-infinite random medium, every photon scattered from the medium in the backward direction has a time-reversed photon traveling along the same path in the opposite direction, i.e., the paths formed by exactly opposite sequences of scattering centers. The phase difference between these two waves following the identical path in time-reversed order is $\Delta \phi =({\kappa }_{\text{in}}+{\kappa }_{\text{out}})\bullet (r_{\text{in}}-r_{\text{out}})$ , as shown in Fig. 1 . The phase difference must be sufficiently small for constructive interference to occur. For backscattered light, ${\kappa }_{\text{in}}+{\kappa }_{\text{out}}\to 0$ , the phase difference becomes very small, and thus, the two waves following the time-reversed paths interfere with each other constructively. This constructive interference occurs in the backscattering direction, whereas in directions sufficiently away from the backward direction, the constructive interference vanishes. In principle, the peak EBS intensity can be twice as high as the incoherent intensity scattered outside the EBS peak (in EBS literature, this intensity is frequently referred to as the “background intensity”).

Fig. 1

Illustration of the EBS phenomenon. EBS originates from the constructive interference between pairs of photons propagating along a scattering light path (solid arrows) and its time-reversed path (dashed arrows) in the case when the photons exit the medium in directions close to the backscattering $\left(\theta \to 0\mathrm{deg}\right)$ . When both waves emerge from the medium, they have an identical phase. Thus, they interfere constructively with each other, resulting in an enhanced scattered intensity in the backward direction.

The EBS phenomenon can be understood in the context of Young’s double pinhole interference experiment. Conceptually, the first and last scatterers in EBS can be considered the double pinholes in the Young’s double pinhole experiment. A plane wave passing the double pinholes produces a sinusoidal fringe pattern in the far field. Its maximum intensity is formed at $\theta =0\mathrm{deg}$ and its fringe spacing is inversely proportional to the distance between the pinholes. Similarly, in EBS a plane wave passing the first and last scatterers through the time-reversed paths would produce a sinusoidal fringe pattern with its maximum intensity in the backward direction. Its fringe spacing is inversely proportional to the transverse distance between the first and last scatterers. In EBS, all possible transverse distances between the first and last scatterers average out the fringe patterns except the backward direction, resulting in an enhanced peak. Therefore, the width of the enhanced peak is primarily dependent on the average distance between the first and last scatterers [i.e., the transport mean free path $l_s^{*}=l_s∕(1-g)$ , where $l_s$ is the scattering mean free path and $g$ is the anisotropy factor], such that the full width half maximum (FWHM) of the EBS peak in a semi-infinite disordered medium $w\approx \lambda ∕\left(3\pi l_s^{*}\right)\approx 0.7∕\left(\kappa l_s^{*}\right)$ , where $\lambda$ and $\kappa$ are the wavelength and wavevector of light, respectively. Thus, in conventional EBS measurements, the profile of the EBS peak is mainly determined by the inverse of the disorder parameter $\kappa l_s^{*}$ (or $l_s^{*}∕\lambda$ ).

2.2.

Angular Profile of Enhanced Backscattering

Quantitatively, the angular profile of an EBS peak $I_{\mathrm{EBS}}\left(\theta \right)$ can be expressed as a 2-D Fourier transform of the radial intensity distribution of EBS^{32, 33}:

2.3.

Principles of Low-Coherence Enhanced Backscattering

In EBS, the conjugated time-reversed waves can interfere with each other only when they are spatially coherent, that is, the first and last points on the scattering path must be within the coherence area.^{30, 34} Most previous EBS measurements have been conducted using coherent laser light sources with spatial coherence length $L_{\mathit{sc}}⪢l_s^{*}$ . Under such spatially coherent illumination, essentially any conjugate time-reversed waves emerging from the surface of the sample are capable of interfering with each other. However, if light incident on a sample has a finite spatial coherence length, the conjugated time-reversed waves can interfere constructively with each other only if they are spatially coherent.³⁴ Thus, the angular profile of an EBS intensity $I_{\mathrm{EBS}}\left(\theta \right)$ under low spatial coherence illumination can be expressed as:

Incoherent waves have no correlations in phase and generate only the incoherent background intensity. If $L_{\mathit{sc}}$ is sufficiently short, i.e., $L_{\mathit{sc}}⪡l_s^{*}$ , low spatial coherence illumination allows only low-order scattering to give rise to the EBS peak, which in turn results in a dramatic (e.g., several orders of magnitude) broadening of an EBS peak. As discussed later, such an anomalously broad width of EBS could not be explained by simple diffusion-theory-based models of EBS.

3.1.

Low-Coherence Enhanced Backscattering Instrument

To overcome the limitations in conventional EBS measurements in tissue, we have recently developed low-coherence enhanced backscattering (LEBS) spectroscopy by combining EBS measurements with low spatial coherence, broadband illumination, and spectrally resolved detection.^{28, 30} Figure 2 illustrates the design of an LEBS instrument. A beam of broadband cw light from a $100\text{-}\mathrm{W}$ xenon lamp (Spectra-Physics Oriel Instruments, Stratford, Connecticut) was collimated using a 4- $f$ lens system (divergence angle ranging from $\sim 0.04\mathrm{deg}$ for $L_{\mathit{sc}}=200\mu \mathrm{m}\text{to}\sim 0.30\mathrm{deg}$ for $L_{\mathit{sc}}=35\mu \mathrm{m}$ ), polarized, and delivered onto a sample at $\sim 10\mathrm{deg}$ angle of incidence to prevent the collection of specular reflection. By changing the aperture size in the 4- $f$ lens system, we varied the spatial coherence length $L_{\mathit{sc}}$ of the incident light from $200\text{to}35\mu \mathrm{m}$ . The spatial coherence length was confirmed by double-slit interference experiments.³⁵ The light backscattered by the sample was collected by a sequence of a lens, a linear analyzer (Lambda Research Optics, Incorporated, Costa Mesa, California), and an imaging spectrograph (SpectraPro-150, Acton Research, Acton, Massachusetts). The spectrograph was positioned in the focal plane of the lens and coupled with a charge-coupled device (CCD) camera (CoolSnap HQ, Princeton Instruments Incorporated, Trenton, New Jersey). The lens projected the angular distribution of the backscattered light onto the slit of the spectrograph. Then, the imaging spectrograph dispersed this light according to its wavelength in the directions perpendicular to the slit, and projected it onto the CCD camera. Thus, the CCD camera recorded a matrix of light-scattering intensities as a function of wavelength $\lambda$ and backscattering angle $\theta$ . In each CCD pixel, collected light was integrated within a certain narrow band of wavelengths around $\lambda$ . The width of this band was determined by the width of the spectrograph slit. Because the temporal coherence of light $L_{\mathit{tc}}$ was related to its spectral composition, finite-spectral band detection resulted in low temporal coherence detection. Accordingly, $L_{\mathit{tc}}$ was dependent on the spectral resolution of the spectrograph, such that $L_{\mathit{tc}}=\sqrt{\left(2\ln 2\right)∕\pi }\bullet ({\lambda }^2∕\Delta \lambda )=30\mu \mathrm{m}$ , where $\Delta \lambda =9\mathrm{nm}$ is the FWHM of the detection bandpass. We also used a He-Ne laser (Melles Griot, Carlsbad, California) to compare our measurements to the conventional EBS measurements under coherent illumination.

Fig. 2

LEBS spectroscopy instrument. C: condenser, L: lenses, A: apertures, P: polarizers, M: mirror, B: beamsplitter, SS: sample stage, and SP: imaging spectrograph. The entrance slit of the imaging spectrograph is in the focal plane of lens L3. Thus, all scattered light rays with an identical scattering angle $\theta$ are focused into the same point on the entrance slit. The spectrograph disperses this light in the direction perpendicular to the slit according to its wavelengths. Thus, the CCD records a matrix of scattered intensities as a function of wavelength and scattering angle $I_{\mathrm{EBS}}(\theta ,\lambda )$ .

3.2.

Characteristics of Low-Coherence Enhanced Backscattering

3.2.1.

Spectroscopic measurements

Using LEBS spectroscopy, EBS profiles can be observed as a function of wavelength as shown in Fig. 3a . The design of the LEBS spectroscopy instrument enables simultaneous measurement of the spectral $(\lambda =400\text{to}700\mathrm{nm})$ and scattering angle ( $\theta =-5\text{to}5\mathrm{deg}$ ) distributions of backscattered light. Several spectroscopic techniques have been shown to be highly useful for tissue diagnosis and characterization including reflectance, light scattering, fluorescence, and other types of spectroscopy. Consequently, the analysis of EBS spectra may be used to provide additional information about tissue.

Fig. 3

(a) Representative LEBS intensity signal $I_{\mathrm{EBS}}(\theta ,\lambda )$ obtained from human colonic tissue using the LEBS spectroscopy instrument $(L_{\mathit{sc}}=110\mu \mathrm{m})$ . (b) Typical LEBS intensity spectrum in the backward direction $I_{\mathrm{EBS}}(\theta =0\mathrm{deg},\lambda )$ , from (a). A linear fit to $I_{\mathrm{EBS}}\left(\lambda \right)$ using linear regression accounts for the most spectral variance of the LEBS intensity signal.

3.2.2.

Speckle reduction

The experimental observation of EBS requires ensemble or configuration averaging because of speckle, which arises from random interference effects. For example, rotating the sample mechanically or averaging independent measurements are commonly used in conventional EBS measurements. Speckle becomes more severe in the absence of Brownian motion, hampering EBS studies in biological tissue.²¹ However, LEBS overcomes this problem. Figure 4a shows the angular distribution of the backscattered light obtained from ex vivo human colon tissue using coherent illumination (i.e., He-Ne laser). Evidently, the speckle pattern masks an EBS peak. To uncover an EBS peak from the background of speckle, averaging over thousands of independent measurements is required. On the other hand, Fig. 4b shows that in the case of the LEBS signal recorded from the same tissue site, speckle is negligible and a cusp of an enhanced backscattering peak can be easily identified. Both low spatial coherence illumination and low temporal coherence detection result in speckle reduction. In our LEBS measurements, a single LEBS reading averages over multiple independent coherence volumes (each coherence volume generated an independent speckle volume). For example, for $L_{\mathit{sc}}\approx 150\mu \mathrm{m}$ , the number of independent coherence volumes ${(D∕L_{\mathit{sc}})}^2\times (l∕L_{\mathit{tc}})\approx 1000$ , where $D$ is the diameter of illumination area on the sample and $l$ is the average path length. Therefore, LEBS measurements are easily achieved in random media, even in the absence of Brownian motion without the need for ensemble or configuration averaging.

Fig. 4

LEBS enables substantial reduction of speckle. (a) The angular distribution of backscattered intensity obtained from ex vivo human colonic tissue using a coherent illumination (He-Ne laser, $\lambda =632\mathrm{nm}$ ). An EBS peak is masked by severe speckle. (b) The angular profile of backscattered intensity from the identical tissue site recorded using the LEBS spectroscopy instrument (xenon lamp, $L_{\mathit{sc}}=110\mu \mathrm{m}$ , $\lambda =647\mathrm{nm}$ ). A cusp of LEBS not obstructed by speckle can be clearly identified.

3.2.4.

Depth-selective low-coherence enhanced backscattering measurements

Depth-selective LEBS spectroscopy of tissue can be achieved by three means: 1. varying coherence length $L_{\mathit{sc}}$ , 2. analysis of LEBS spectra $I_{\mathrm{EBS}}\left(\theta \right)$ at different scattering angles $\theta$ , and 3. analysis of the probability of the radial intensity distribution of LEBS photons $P\left(r\right)$ , which can be obtained via the Fourier transform of $I_{\mathrm{EBS}}\left(\theta \right)$ . In brief, $L_{\mathit{sc}}$ determines the maximum penetration depth. Then, detailed depth-resolution can be obtained by either means 2 or 3.

Control of tissue depth probed with LEBS via coherence length

Only photons emerging from the tissue surface at distances $r<\sim L_{\mathit{sc}}$ from their point of entry into the tissue can contribute to LEBS, which limits the depth of penetration of LEBS photons to up to $\sim L_{\mathit{sc}}$ .

Control of tissue depth probed with LEBS via the analysis of $I_{\mathrm{EBS}}\left(\theta \right)$ at different $\theta$

As discussed before, because $I_{\mathrm{EBS}}\left(\theta \right)$ is the Fourier transform of $P\left(r\right)$ , short light paths (small $r$ ) mainly give rise to the periphery of an EBS peak (large $\theta$ ), while longer light paths $(r\approx L_{\mathit{sc}})$ give rise to the top (or center) of the EBS peak $(\theta \to 0\mathrm{deg})$ .²⁹ This property of LEBS can be used to sample various depths using a single LEBS measurement by analyzing $I_{\mathrm{EBS}}\left(\theta \right)$ at different $\theta$ . Figure 5a shows LEBS penetration depth $L_p\left(\theta \right)$ for $l_s=81\mu \mathrm{m}$ and $l_s^{*}=614\mu \mathrm{m}$ . As expected, small $\theta$ correspond to deeper penetration depths, whereas large $\theta$ correspond to shorter depths. Therefore, different depths can be selectively assessed by probing a corresponding scattering angle. For example, in the case of colonic mucosa, $I_{\mathrm{EBS}}(\theta =0.25\mathrm{deg})$ enables assessment of an epithelial cell layer $(\sim 40\mu \mathrm{m})$ , whereas $I_{\mathrm{EBS}}(\theta =0\mathrm{deg})$ allows probing the entire mucosa $(\sim 70\mu \mathrm{m})$ .

Fig. 5

(a) LEBS depth of penetration $L_p\left(\theta \right)$ as a function of $\theta$ ( $l_s=81\mu \mathrm{m}$ and $l_s^{*}=614\mu \mathrm{m}$ ). ( $\theta$ is measured from the backscattering direction, i.e., $\theta =0\mathrm{deg}$ corresponds to the direction opposite to that of the incident light.) $L_p\left(\theta \right)$ decreases with $\theta$ . (b) LEBS depth of penetration $L_p\left(r\right)$ as a function of $r$ ( $l_s=81\mu \mathrm{m}$ and $l_s^{*}=614\mu \mathrm{m}$ ). $L_p\left(r\right)$ increases with $r$ . Thus, different depths of penetration can be assessed by probing different $\theta$ or $r$ within an EBS peak (see text for details).

Control of tissue depth probed with LEBS via the analysis of $P\left(r\right)$ at different $r$

A more precise control of tissue depth probed with LEBS can be achieved by means of the analysis of $P\left(r\right)$ ,²⁹ which in turn can be obtained from the Fourier transform of $I_{\mathrm{EBS}}\left(\theta \right)$ . It is well known that the depth of penetration of photons increases with $r$ . As shown in Fig. 5b, tissue depths from $\sim 40\mu \mathrm{m}$ (a single cell layer) to $\sim 100\mu \mathrm{m}$ (thickness of colonic mucosa) can be selectively assessed by means of the analysis of $P(r,\lambda )$ by choosing appropriate parameter $r$ . We also point out that a direct measurement of $P\left(r\right)$ for small $r$ $(r<300\mu \mathrm{m})$ is extremely difficult.³⁶

The three means to achieve depth-selective measurements of tissue discussed before are complimentary. Thus, LEBS spectroscopy opens up the possibility of performing spectroscopic measurements at any given depth within the maximum penetration depth determined by $L_{\mathit{sc}}$ .

The significance of depth-selective spectroscopic measurements for tissue characterization and diagnosis is underscored by the following reasons. 1. The most superficial tissue layer (i.e., the epithelium) is the origin of nearly 90% of human cancers and the epithelial cells are the first affected in carcinogenesis.³⁷ Thus, obtaining diagnostic information from most superficial tissue is crucial for the early diagnosis of epithelial precancerous lesions. 2. Hemoglobin absorption in the blood vessels located underneath the epithelium is a particularly notorious problem, as it obscures the endogenous spectral signatures of epithelial cells. 3. The depth-dependent biological heterogeneity of the epithelium underscores the need to selectively assess the epithelial cells at different depths. For example, in the colon (the major unit of organization of the mucosa is the crypt), the epithelial cells at the base of the crypt ( $\sim 80\mu \mathrm{m}$ below the tissue surface) are capable of proliferation, while the epithelial cells at the top of the crypt $(\sim 40\mu \mathrm{m})$ undergo apoptosis, as illustrated in Fig. 6 . In adenomatous colonic mucosa, the apoptotic activity is reduced in the base of the crypt while the proliferative activity is increased in the lumenal surface of the colon.³⁸ The cells that are initially involved in neoplastic transformations are located in a specific area of the crypt: the base of the crypt is known as the location for initiation of colon carcinogenesis.³⁹ Similar considerations apply to most other types of epithelia, including stratified squamous epithelium (such as the epithelium of uterine cervix, oral cavity, etc.).

Fig. 6

The crypt: the major unit of the colonic mucosa. The epithelial cells have distinct cell activities at various depths. Typical depth of a colonic crypt is $70\text{to}90\mu \mathrm{m}$ .

4.1.

Colorectal Cancer

Colorectal cancer (CRC) will be diagnosed in approximately 140,000 Americans this year, leading to 50,000 deaths, which makes CRC the second leading cause of cancer deaths in the United States.⁴⁰ Given that CRC is curable if diagnosed early, widespread screening has been proposed to reduce CRC fatalities. For example, colonoscopy has shown to dramatically reduce CRC fatalities through both early detection of CRC and cancer prevention. Although colonoscopy is remarkably effective in reducing CRC, screening the entire at-risk population ( $>60\text{million}$ Americans over age 50) through colonoscopy is impossible for a variety of reasons, including expense (the cost of detection of a single colon cancer case would be $>\$\mathrm{1,000,000}$ ), patient reluctance, and complication rate. Therefore, development of novel initial-screening (risk-stratification) techniques is imperative. Many screening techniques take advantage of the “field effect” (or field cancerization) in CRC. The field effect refers to the proposition that the genetic/environmental milieu that results in a neoplastic lesion in one area of the colon also leads to alterations to tissue composition, structure, and function in the entire colon. Accordingly, detection of such changes in an easily accessible part of the colon (e.g., the rectum or sigmoid colon) can be used to infer the risk of carcinogenesis in the rest of the colon.⁴¹ Unfortunately, the most widely used initial screening techniques such as fecal occult blood tests and flexible sigmoidoscopy (i.e., visual evaluation of the rectal and sigmoid colonic mucosa, which is the easiest to access) are suffered by low sensitivity. Thus, development of an alternative minimally invasive screening/risk-stratification test that is accurate, sensitive, cost effective, easy to perform, and does not require the need for colonoscopy and colon preparation is indeed crucial.

4.2.

Animal and Human Studies

We have investigated the potential of depth-selective LEBS spectroscopy for accurate CRC risk stratification in two animal models of experimental CRC and pilot human studies.^{28, 29, 30, 31} First, we have performed animal studies with the azoxymethane (AOM)-treated rat model of colon carcinogenesis. This is one of the most robust and well-tested animal models of colon carcinogenesis.^{28, 29, 30} In the AOM-treated rats, colon carcinogenesis progresses through the same steps as in humans: the earliest detectable marker of colon carcinogenesis, aberrant crypt foci (ACF) develop $8\text{to}12\text{weeks}$ after the AOM injection, adenomas are observed in $20\text{to}30\text{weeks}$ , and carcinomas develop in $40\text{weeks}$ . 48 male Fisher 344 rats $\left(150\text{to}200\mathrm{g}\right)$ (Sigma, Saint Louis, Missouri) were randomized to two weekly intraperitioneal (i.p.) injections with either AOM $(15\mathrm{mg}∕\mathrm{kg})$ or saline (in the case of negative control animals). Animals were euthanized at specified time points (2, 4, and $6\text{weeks}$ after the second AOM injection). Colons were removed, opened longitudinally, and washed. LEBS measurements were performed on fresh, unfixed colons within one hour of sacrifice. We recorded LEBS signals from at least 20 tissue sites per animal equally distributed throughout colonic surfaces. Moreover, to ensure that our findings are not model specific, we used male multiple intestinal neoplasia (MIN) (Jackson Laboratory, Bar Harbor, Maine) and control C57BL6 mice (wildtype at the adenomatous polyposis coli locus) at $6\text{to}7\text{weeks}$ of age $(n=18)$ .³¹ In the MIN model, adenomatous polyposis coli (APC) truncation leads to spontaneous intestinal tumorigenesis, thus replicating the human familial adenomatous polyposis. The MIN mice develop intestinal adenomas at $9\text{to}10\text{weeks}$ of age, and most intestinal adenomas occur in the small intestine. We point out that the time points for which we have recorded LEBS signals (i.e., $2\text{to}6\text{weeks}$ after AOM injection in the case of the AOM animal model and $6\text{to}7\text{week}$ old mice in the case of the MIN mice model) correspond to very early stages of carcinogensis. At these early time points, no currently known histologic, molecular, or genetic markers have been able to detect preneoplastic transformation. Finally, to investigate whether our findings in these animal models can be translated into humans, we performed human studies of 63 subjects undergoing colonoscopy.³¹ We used six biopsies of endoscopically normal mucosa at least $3\mathrm{cm}$ away from any neoplastic lesion to validate whether LEBS spectroscopy can be used to detect the field effect of CRC. In the human studies, we defined a low-risk group as subjects without personal history of neoplasia and family history of adenomas or carcinomas, and a high-risk group $(n=12)$ as subject with adenomas on current colonoscopy (all adenomas were histologically confirmed), respectively.

4.3.

Results

We identified the optimal depth of penetration for which LEBS markers are the most diagnostic. We assessed LEBS spectra corresponding to 30-, 50-, and $70\text{-}\mu \mathrm{m}$ depths ( $\theta =0.4\mathrm{deg}$ , $\theta =0.2\mathrm{deg}$ , and $\theta =0\mathrm{deg}$ , respectively) and found that $70\text{-}\mu \mathrm{m}$ penetration depth provides the most diagnostically significant results and enables detection of CRC at the earliest time point ( $2\text{weeks}$ after AOM injection).³¹ It is of notice that this depth approximately corresponds to the depth of colonic mucosa, and several lines of evidence suggest that the base of the crypt is the location for initiation of colon carcinogenesis.

Typically, $I_{\mathrm{EBS}}\left(\lambda \right)$ is a declining function of wavelength and its steepness depends on the size distribution of submicron intraepithelial structures.⁴² Although LEBS spectra are quite complex with the scale of spectral features as small as only a few nanometers, a linear fit to $I_{\mathrm{EBS}}\left(\lambda \right)$ using linear regression from $530\text{to}640\mathrm{nm}$ accounts for the most spectral variance. For example, Fig. 3b shows a typical $I_{\mathrm{EBS}}(\lambda ,\theta =0\mathrm{deg})$ recorded from a human colon tissue and its linear fit with $R^2\approx 0.6$ . Thus, to characterize the spectrum of $I_{\mathrm{EBS}}\left(\lambda \right)$ as a biomarker, we obtained the value of the slope of the fit over wavelength, which is referred to as the LEBS spectral slope. We found that the LEBS spectral slope showed excellent temporal and spatial correlations with the progression of carcinogenesis in the animal models and the pilot human studies. Figures 7a and 7b show alterations of LEBS spectral slope obtained from the AOM-treated rats compared with control values in the two different compartments. As shown in Fig. 7a, spectral slope of the basal compartment is dramatically decreased as early as $2\text{weeks}$ after the AOM administration, and decreases over the process of carcinogenesis. On the other hand, LEBS spectral slope of the lumenal compartment remains unchanged at the $2\text{-}\text{week}$ time point but instead changes dramatically later in the time course of the experiment. These results show that the LEBS spectral slope from $\sim 70\text{-}\mu \mathrm{m}$ depth provides the earliest sensitivity between the saline-treated rats and the AOM-treated rats. We also confirmed this result in the $6\text{-}\text{week}$ -old MIN mice. As shown in Fig. 8 , the LEBS spectral slope is diagnostic for the early preadenoma stage of intestinal neoplasia in histologically normal mucosa of MIN mice ( $P$ $\text{value}=0.009$ ).

Fig. 7

LEBS spectral slopes obtained from rat colonic tissue 2, 4, and $6\text{weeks}$ after the initiation of colon carcinogenesis (i.e., AOM injection) compared to those obtained from the negative control (i.e., saline-treated rats). (a) The lower compartment $(L_p\approx 70\mu \mathrm{m})$ of the crypt. (b) The upper compartment $(L_p\approx 40\mu \mathrm{m})$ of the crypts. These changes in LEBS spectra are observed in the earliest stages of colon carcinogenesis, preceding expression of currently known histological and molecular biomarkers.

Fig. 8

LEBS spectral changes confirmed in the MIN mouse model of intestinal carcinogenesis. LEBS spectral slope obtained from the microscopically normal appearing (i.e., uninvolved) intestinal mucosa in $6\text{-}\text{week}$ -old MIN mice is significantly different from one obtained from age-matched control mice (wild type) $(L_p\approx 70\mu \mathrm{m})$ .

Furthermore, we found that the change in the LEBS spectral slope is also diagnostic in humans. This is illustrated in Fig. 9 , which shows a significant decrease in the spectral slope obtained from the colonoscopically normal rectal mucosa in patients who harbored adenomas elsewhere in the colon when compared to those who were neoplasia free ( $P$ $\text{value}=0.006$ ). Adenomas were approximately equally distributed between the sigmoid colon/rectum (28% of all adenomas), transverse colon (35%), and the cecum (38%). The magnitude of decrease in LEBS spectral slope appeared to be greater if the lesion was located in the proximity to the site of LEBS analysis. However, significant differences were noted from LEBS measurements taken at distant sites as well (e.g., rectal LEBS spectral slope correlates with the presence of adenomas in the transverse colon). These results demonstrate that LEBS spectroscopy has the potential to accurately risk-stratify patients for CRC and may be translated into a practical clinical method for colon cancer screening. Translation of LEBS into clinical practice will require development of fiber optics LEBS probes that can be used to record LEBS spectra from rectal mucosa in vivo and assess LEBS markers without the need for colonoscopy or biopsy. The development of such LEBS fiber optics probes is currently underway in our research group. Rectal LEBS tests can potentially be performed without the need for colonoscopy or patients’ colonic preparation. We envision that in the future the LEBS technique may enable a primary care physician to assess LEBS biomarkers during a patient’s annual physical exam and thus determine the risk of CRC and the need for colonoscopy.

Fig. 9

Pilot human studies. LEBS spectral changes have the potential to diagnose colonic neoplasia in humans without the need for colonoscopy. LEBS spectral slope assessed in the rectum of subjects undergoing colonoscopy $(L_p\approx 70\mu \mathrm{m})$ correlates with presence of neoplasia elsewhere in the colon ( $P$ $\text{value}=0.006$ ). High-risk groups include subjects with advanced adenomas anywhere in the colon (cecal, colonic, or rectal adenoma $>10\mathrm{mm}$ ), while low-risk groups (i.e., no dysplasia) include patients without personal or family history of colonic neoplasia.