characterizing the origin of autofluorescence in human esophageal
TRANSCRIPT
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
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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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
#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 21075
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.
#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 21076
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
#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 21077
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
#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 21078
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 image by the I400lp
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 image by the I400lp
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.
#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 21079
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
#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 21080
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
#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 21081
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.
#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 21082