characterizing the origin of autofluorescence in human esophageal

Characterizing the origin of autofluorescence in

human esophageal epithelium under ultraviolet

excitation

Bevin Lin1,2*

, Shiro Urayama3, Ramez M. G. Saroufeem

4, Dennis L. Matthews

1,2,

Stavros G. Demos1,5

1University of California, Davis NSF Center for Biophotonics Science & Technology, 4800 2nd Avenue,

Sacramento, CA 95817, USA 2University of California, Davis Department of Biomedical Engineering, One Shields Avenue, Davis, CA 95616, USA

3University of California, Davis Medical Center, Division of Gastroenterology and Hepatology, 4150 V Street,

Suite 3500, Sacramento, CA 95817, USA 4University of California, Davis Medical Center, Department of Pathology, 4400 V Street,

Sacramento, CA 95817, USA 5Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA

*[email protected]

Abstract: The autofluorescence under ultraviolet excitation arising from

normal squamous and columnar esophageal mucosa is investigated using

multispectral microscopy. The results suggest that the autofluorescence

signal arises from the superficial tissue layer due to the short penetration

depth of the ultraviolet excitation. As a result, visualization of esophageal

epithelial cells and their organization can be attained using wide-field

autofluorescence microscopy. Our results show tryptophan to be the

dominant source of emission under 266 nm excitation, while emission from

NADH and collagen are dominant under 355 nm excitation. The analysis of

multispectral microscopy images reveals that tryptophan offers the highest

image contrast due to its non-uniform distribution in the sub-cellular matrix.

This technique can simultaneously provide functional and structural

imaging of the microstructure using only the intrinsic tissue fluorophores.

©2010 Optical Society of America

OCIS codes: (170.1610) Clinical applications; (170.2520) Fluorescence microscopy;

(170.2680) Gastrointestinal; (170.4730) Optical pathology; (170.6510) Spectroscopy, tissue

diagnostics; (170.6935) Tissue characterization; (260.7190) Ultraviolet.

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#131007 - $15.00 USD Received 1 Jul 2010; revised 18 Aug 2010; accepted 8 Sep 2010; published 21 Sep 2010(C) 2010 OSA 27 September 2010 / Vol. 18, No. 20 / OPTICS EXPRESS 21074

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1. Introduction

A significant limitation to traditional white light endoscopy is the inability to image cellular

level epithelial morphology. Emerging techniques that address this problem include confocal

fluorescence endomicroscopy, which provides in vivo information using intravenous

fluorescein [1,2]. The combination of targeted peptide probes for complementary functional

confocal data has also been explored [3]. In addition, wide-field endomicroscopy prototype

systems include the use of contrast agents such as acriflavine hydrochloride [4], as well as

quantum dots and gold nanoparticles [5]. The use of contrast agents is an additional step that

may increase the cost and time budget of the procedure and represents an additional risk to the

patient. Consequently, the development of imaging techniques that do not require the use of

contrast agents may be desirable. Such techniques may rely on intrinsic tissue chromophores

that can be excited via linear [6] or non-linear microscopy techniques [7,8]. The latter method

offers a sectioning capability needed to image a specific layer of the tissue and reject out of

focus signal. It was shown very recently that the short penetration depth of ultraviolet (UV)

excitation gives rise to autofluorescence from only the superficial tissue layer [9]. This in turn

allows for imaging of the superficial tissue layer using wide-field microscopy approaches

without the need to stain the tissue with contrast agents, employ optical sectioning techniques

that reject most of the signal produced by the excitation light, or mandate time intensive tissue

preparation.

Several endogenous fluorophores absorb in the UV spectral region and contribute to tissue

autofluorescence (AF) emission in the visible spectrum including tryptophan, elastin,

collagen, nicotinamide adenine dinucleotide (reduced form NADH), and flavin adenine

dinucleotide (FAD) in many different organs such as the head-neck [10] and breast [11].

Investigation of AF in gastrointestinal tissues has been performed at excitation wavelengths

longer than 330 nm [12,13]. Tryptophan has been shown to dominate the emission profile

under UV excitation shorter than 310 nm [14,15].

The goal of this work is to understand the origin at the microscopic level and spectral

characteristics of the AF from human esophagus tissue under 266 nm and 355 nm excitation.

The choice of these excitation wavelengths was based on recent results that demonstrated

microscopic imaging of unprocessed esophageal mucosa using wide-field microscopy to

capture the AF produced with excitation at these wavelengths [9]. As the preliminary results

suggest that this approach may enable in vivo pathological assessment with no tissue

preparation, understanding the exact mechanism giving rise to image formation is critical for

optimizing instrumentation and methodology. The experiments employ AF point spectroscopy


and microscopic narrow-band imaging (NBI) to investigate ex vivo normal squamous and

columnar mucosa of fresh, unprocessed human esophagus specimens.

2. Materials and methods

Fresh human tissue biopsy specimens were collected from consented patients with a history of

Barrett’s esophagus (BE) undergoing routine surveillance. Standard forceps were used during

endoscopy to collect one biopsy specimen from the vicinity of the squamocolumnar junction

(Z-line), and one biopsy specimen from the gastroesophageal (GE) junction for a total of two

biopsy specimens per patient. The protocol was approved by the University of California,

Davis Medical Center Institutional Review Board.

2.1 Point spectroscopy

Point spectroscopy experiments were performed with an initial population of four patients

collecting two biopsy specimens per patient, for a total of eight tissue samples. Each

unprocessed esophagus tissue biopsy specimen was individually placed between two quartz

slides to acquire the AF spectra using two excitation lasers operating at 266 nm and 355 nm

(Intelite, Inc., Minden, NV) having an average power of about 1 mW. The lasers were aligned

and coupled to a UV compatible fiber probe used to collect and transmit the emission from the

target area to a spectrometer containing a single excitation delivery fiber (400 µm diameter)

surrounded by 6 collection fibers (200 µm diameter). External shutters allowed manual

control of laser source exposure. Spectra from four different locations were taken from each

tissue specimen under each excitation wavelength and averaged to produce one mean

spectrum per specimen. The emission arising from 266 nm excitation was passed through a

280 nm long pass filter, while that under the 355 nm laser was passed through a 385 nm long

pass filter in order to reject the excitation light. A flip mirror was aligned to steer the

excitation beam along an alternate path for AF NBI image collection described in the next

section. Tungsten and deuterium lamps (Oriel Instruments, Stratford, CT) were used to

generate calibration curves to correct for the spectral response of the system used to record the

AF spectra.

2.2 Narrow-band AF

Narrow-band AF multispectral microscopic images were acquired from an initial population

of thirteen patients. Two biopsy specimens per patient were collected for a total of twenty six

tissue samples. A minimum of three AF images were recorded from each specimen using each

narrow-band filter. A description of our AF microscopy approach and experimental system

has been previously described [9]. Baseline AF images from each specimen were acquired

under 266 nm and 355 nm excitation using a 400 nm long pass filter under 5 second exposure.

Subsequently, each site was imaged using the set of narrow-band filters with a peak

transmission wavelength centered from 450 nm to 600 nm in 50 nm increments and a

bandwidth of ± 20 nm at full width half maximum (FWHM). Each filter was manually

interchanged to collect corresponding spectral images under 30 second exposure time. Images

were processed using WinView software (Princeton Instruments, Trenton, NJ). Ratio images

were obtained via pixel-by pixel division of NBI results recorded at different spectral bands to

highlight the contribution and localization of different fluorophores [16]. Tissue samples were

immediately placed in formalin after completion of the experiments and returned to the

grossing lab for histopathology diagnosis.


3. Experimental results

3.1 Point spectroscopy

The spectroscopy experiments show characteristic emission bands that can be assigned to

three main tissue fluorophores. Under 266 nm excitation, emission band 1 is observed as

shown in Fig. 1 centered between 320 nm – 350 nm. This emission band is characteristic of

tryptophan emission. The tail of the emission at longer wavelengths does not contain features

that can be identified as the contribution from additional tissue fluorophores. This may

indicate that tryptophan is the main contributor in the emission spectrum under 266 nm

excitation through the entire spectral range of our measurement. However, arrows 1 indicate a

region of visible spectral difference in the tail of the emission spectra obtained from different

specimens that may arise from other tissue fluorophores, or can be an artifact arising from

variation in blood concentration within each specimen (via re-absorption of the emission by

blood cells).

Under 355 nm excitation, emission bands 2 and 3 are observed between 400 nm – 430 nm

and 440 nm – 470 nm that can be assigned to emission from collagen and NADH,

respectively. There are no additional features in the measured spectra under 355 nm excitation

that can be identified as contribution from other tissue fluorophores, such as FAD or

lipofuscin. However, variability in the relative strength of emission bands 2 and 3 was

observed. This may be due to blood re-absorption of the emission (as discussed above) and/or

on the tissue pathology. Since this is not the focus of this investigation, we will not expand the

discussion on this point. Arrow 2 indicates the spectrum of the biopsy specimen further

examined with NBI and shown in Fig. 2.

Fig. 1. Normalized mean autofluorescence spectra of esophagus mucosal biopsy specimens

under 266 nm excitation and 355 nm excitation.

3.2 Narrow-band AF imaging

Figure 2 shows a series of spectral and spectral ratio images obtained from the same location

of a biopsy specimen collected from an overlapping Z-line and GE junction at 40 cm. These

images represent a typical pattern consistently observed when imaging normal (columnar or

squamous) esophageal mucosa of different patients. The gold standard pathology for this

specimen was columnar mucosa with mild chronic inflammation.

Figures 2a and 2b are the baseline AF images (referred to as I400lp

355 and I400lp

266) acquired

under 355 nm and 266 nm excitation respectively, using a 400 nm long pass (lp) filter. These

two images are remarkably different, with the image under 355 nm excitation (2a) providing


very little contrast while the image under 266 nm excitation (2b) providing a clear

visualization of the columnar mucosa seen as the characteristic honeycomb pattern. Based on

the spectroscopy results, the main difference between these images is that under 266 nm

excitation (2b), the AF image is dominated by the tryptophan emission which is absent under

355 nm excitation (2a). Based on the broad absorption spectrum of the fluorophores excited

under 355 nm illumination, we can assume that these fluorophores also contribute to the

emission under 266 nm excitation. The ratio image I400lp

266 / I400lp

355 can remove or at least

reduce the contribution of the other fluorophores and thus provide a mostly tryptophan based

image. This ratio image is shown in Fig. 2c exhibiting a moderately enhanced contrast in the

visualization of the honeycomb pattern.

Fig. 2. 142 µm x 136 µm raw images of a single ex vivo human esophagus columnar mucosa

biopsy specimen under (a) 355 nm excitation and (b) 266 nm excitation with a 400 nm long

pass filter and (c) the ratio image of (b) divided by (a). NBI under 266 nm excitation using the

(d) 450 nm, (e) 550 nm and, (f) 600 nm filters. Ratio of spectral images (g) 450 nm/400 lp, (h)

550 nm/400 lp and, (i) 600 nm/400 lp.

Figures 2d-2f are raw AF images under 266 nm excitation using narrow-band filters

centered at 450 ± 20nm (2d), 550 ± 20nm (2e), and 600 ± 20nm (2f) referred to as I450nb

266,

I550nb

266, I600nb

266, respectively. The spectral range used to acquire image I450nb

266 (2d) is centered

at the emission peak of NADH and therefore, the relative contribution of NADH emission in

this image should be higher compared to any other narrow-band images. Similarly,

flavoproteins and/or lipo-pigments AF should provide the highest relative contribution in the

I550nb

266image (2e). Finally, the I600nb

266image shown in Fig. 2f was recorded at a spectral range


that is out of resonance with the emission peak of all major fluorophores contributing to tissue

AF, but may still be a significant contribution from lipo-pigments as their emission spectrum

is broader than that of the flavoproteins. Despite the careful selection of these narrow band

images aimed at highlighting the contribution of additional tissue fluorophores in the

formation of the AF image under 266 nm excitation, these images present very minimal

difference in image contrast suggesting that tryptophan emission dominates the

autofluorescence signal used for image formation within the entire spectral range. This also

indicates that tryptophan is the key contributor giving rise to the observed contrast between

the cytoplasm and membrane regions, allowing for visualization of the cellular morphology.

Fig. 3. Digitized intensity profile along the same 1.2 µm zone spanning through 4 cells from a

section of the specimen shown in Fig. 2 corresponding to the images obtained (a) under 266 nm

excitation with a 400 nm long pass filter, and the spectral ratio images (b) I450nb

266 / I400lp

266 , (c)

I550nb

266 / I400lp

266 , and d) I600nb

266 / I400lp

266 .

As mentioned above, the I450nb

266 image should contain the highest relative contribution of

NADH emission compared to any other spectral image. We hypothesized that a ratio image

obtained by dividing the I450nb

266 image by the I400lp

266 (which contains the emission of all

fluorophores contributing to the detected AF under 266 nm excitation) would provide an

image that highlights the localization of NADH at the microscopic level. Similarly, the ratio

image obtained by dividing the I550nb


266 may provide visualization of the

localization of flavoproteins and/or lipo-pigments. These images are shown in Figs. 2g and

2h, respectively. The ratio image obtained from the division of the I600nb


266

is also shown in Fig. 2i. These three ratio images show the cytoplasm region with higher

intensity indicating the localization of the corresponding fluorophores within this region.


A careful examination of these ratio images indicates that the regions of higher intensity

are not generally overlapping. To better quantify this effect, the digitized intensity profile

along a small section of the images of the specimen shown in Fig. 2 is shown in Fig. 3. These

profiles represent the normalized average intensity over a zone of the imaged area that is 1.2

µm thick spanning through 4 cells. The results are represented as percent change from the

average intensity to enable a quantitative assessment of the differences in intensity within

each cell as well as facilitate assessment of the image contrast. The profile shown in Fig. 3a

shows the intensity of the I400lp

266image in this section of the specimen corresponding to the

image shown in Fig. 2b. Similarly, The profiles shown in Figs. 3b, 3c and, 3d show the

intensity along the same section of the specimen of the I450nb

266/ I400lp

266, I550nb

266/ I400lp

266 and,

I600nb

266/ I400lp

266ratio images, respectively, corresponding to the image shown in Figs. 2g, 2h and,

2i. The peaks in the profile of Fig. 3a correspond to the location of membranes. There is more

than 30% difference in intensity between the emission of the cytoplasm and that of the

membrane in the recorded images allowing for a clear visualization of the microstructure of

human esophagus columnar mucosa. Figures 3b-3d demonstrate the variation in the intensity

in the cytoplasm region in the ratio images obtained from different narrow band spectral

windows, which supports the hypothesis that these images arise from different fluorophores.

The nuclei of the esophagus columnar epithelium are not visible using this technique

because they are located deep below the surface that is outside the imaging depth of this

technique. Columnar epithelial cells appear as tall columns with elongated nuclei located

towards the basal surface. This is not the case for squamous epithelium. Stratified squamous

epithelial cells of the esophagus appear as scale-like tiles that have flattened surface layers.

Nuclei are located very close to the surface (lumen) and progressively condense and flatten

during maturation. An AF image under 266 nm excitation of a 340 µm x 180 µm region of

squamous mucosa biopsy specimen is shown in Fig. 4a. In accordance with the observations

established in the study of columnar mucosa (see Fig. 2), the enhanced emission of the

membrane and/or intercellular junctions leads to visualization of a tile-like appearance of

polygonal cells with well demarcated edges at the periphery, characteristic of this type of

tissue. In addition, the circular structures observed within the cytoplasm region of each cell

are believed to be the nuclei of the cells. It is therefore possible to obtain information about

the nucleus to cytoplasm volume ratio, which is a critical characteristic change directly related

to progression of disease such as cancer.

Figure 4b shows the digitized intensity profile along a small section of the image of the

specimen shown in Fig. 4a over a 1.5 µm wide zone of the of the imaged area of the sample

spanning through 3 cells. This profile was obtained using the same method described above to

obtain the profiles shown in Fig. 3. The peaks in the profile denoted as “N” and “M” represent

the location of nucleus and membrane, respectively. Comparison of the profiles shown in

Figs. 3a and 4b indicate that the contrast of the membrane is increased in the case of columnar

mucosa (Fig. 3a) compared to that of squamous epithelium (Fig. 4b). This can be attributed to

the increased depth of the columnar cells.

4. Discussion

The results suggest that visualization of the epithelial morphology based on its native

fluorescence under UV excitation using wide-field microscopy is based on two main

mechanisms. The first mechanism is associated with the property that UV light only

superficially penetrates epithelial tissue, on the order of 100 µm or less. As a result, the

fluorescence signal produced in this superficial tissue layer can be contained within the


Fig. 4. (a) 340 µm x 180 µm raw image of a single ex vivo human esophagus squamous mucosa

biopsy specimen under 266 nm excitation with a 400 nm long pass filter. (b) Digitized intensity

profile along a 1.5 µm zone spanning through 3 cells from a section of the specimen shown in

Fig. 2.

comparable thickness of the image plane of the microscope providing high contrast images

without using an optical sectioning technique (such as confocal microscopy) that generally

causes a large portion of the generated signal to be rejected. That is because the out of focus

(background) signal is sufficiently reduced to allow the formation of high contrast images of

tissue microstructures using the AF of the tissue cells and intracellular components. It must be

noted that this mechanism allows image acquisition based on the emission of all native tissue

fluorophores (as they can all be excited with UV light) and it is independent of the emission

wavelength. It is therefore possible to acquire images that probe the various optically active

analytes to obtain not only structural information but also functional information. In addition,

images based on the emission of contrast agents can be attained and combined with those of

native fluorophores (if their emission is outside the spectral range of native fluorophores, such

as at wavelengths longer than about 750 nm) to provide molecular (or other types) of targeting

information.

The second mechanism leading to the acquisition of images that delineates the different

compartments of cells is that there is sufficient variability in the concentration of

chromophores contained within these compartments. The experimental results presented in

this work indicate that tryptophan is the native tissue fluorophore providing the best image

contrast using this imaging approach enabling visualization of the microstructure and

organization of the superficial layer in a similar way to that provided by H&E staining.

The ratio images shown in Fig. 2 do not provide any additional information to improve the

visualization of normal human esophagus columnar mucosa. However, the target-like feature

located in the lower right corner of all images of this specimen appears with increased contrast

in the ratio images. We believe this feature may represent a villi crypt, which was observed in


multiple locations of this specimen as well as in other specimens. It is therefore possible that

spectral ratio imaging may be useful in providing additional diagnostic information that can

enhance the ability to evaluate tissue pathology.

Understanding the exact mechanism for image formation was essential for establishing the

optical criteria necessary for differentiation between Barrett’s esophagus (BE) and grades of

dysplasia, from low grade through esophageal adenocarcinoma, as well as to develop the

designing criteria for implementation in vivo using endomicroscopy. Our work in these two

areas is in progress and results will be reported in the near future.

Incorporation of NBI may be more difficult to implement in a clinical in vivo setting, but

has the potential to visualize epithelial morphology for early disease detection where

biochemical changes precede morphological changes. In addition, it can provide functional

information using intrinsic tissue chromophores while molecular targeting information can be

added with the use of contrast agents. Image multiplexing is possible using this technique and,

depending on instrumentation design, it can be implemented using parallel (simultaneous)

multi-image acquisition via image splitting to different spectral bands. This can be followed

by reconstruction of the image to its different principal components to delineate the structural,

functional, and molecular/targeting information.

Acknowledgements

This research is supported by funding from the Center for Biophotonics, an NSF Science and

Technology Center, is managed by the University of California, Davis, under Cooperative

Agreement No. PHY 0120999. This work was performed in part at Lawrence Livermore

National Laboratory under the auspices of the U.S. Department of Energy under Contract

W-7405-Eng-48. We would like to thank Professor Brian Wilson for stimulating discussions.


characterizing the origin of autofluorescence in human esophageal

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