In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue

Aft, Rebecca L.

Hai, Pengfei

Novack, Deborah V.

Wang, Lihong V.

Wong, Terence T. W.

Zhang, Ruiying

Caltech Authors - Main

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Photoacoustic microscopy enables
multilayered histological imaging of
human breast cancer without staining
Terence T. W. Wong, Ruiying  Zhang, Pengfei  Hai,
Rebecca L. Aft, Deborah V. Novack, et al.
Terence T. W. Wong, Ruiying  Zhang, Pengfei  Hai, Rebecca L. Aft, Deborah
V. Novack, Lihong V. Wang, "Photoacoustic microscopy enables multilayered
histological imaging of human breast cancer without staining," Proc. SPIE
10494, Photons Plus Ultrasound: Imaging and Sensing 2018, 1049443 (19
February 2018); doi: 10.1117/12.2291008
Event: SPIE BiOS, 2018, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Photoacoustic microscopy enables multilayered histological imaging of 
human breast cancer without staining  
Terence T. W. Wonga,b, Ruiying Zhangb, Pengfei Haia,b, Rebecca L. Aftc,d, Deborah V. Novacke,f, 
and Lihong V. Wang,a,* 
aCaltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department of Medical 
Engineering, Department of Electrical Engineering, California Institute of Technology, Pasadena, 
CA 91125, USA;  
bOptical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. 
Louis, St. Louis, MO 63130, USA;  
cDepartment of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, 
USA;  
dJohn Cochran Veterans Hospital, St. Louis, Missouri 63106, USA;  
eMusculoskeletal Research Center, Division of Bone and Mineral Diseases, Department of 
Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA;  
fDepartment of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, 
USA 
ABSTRACT  
In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. 
About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the 
local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer 
cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with 
remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. 
Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these 
techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming 
for large specimens. Thus, 20–60% of patients undergoing BCS still require second surgeries due to positive surgical 
margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the 
need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic 
microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser 
illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional 
histology and measuring features related to the histological landscape without the need for labels. We demonstrate that 
our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast 
tissue.    
Keywords: Cancer, translational research, photoacoustic imaging, microscopy, label-free, histology 
 
1. INTRODUCTION  
Breast cancer is the most common cancer diagnosed in women. In 2016, an estimated 250,000 new cases of invasive 
breast and non-invasive breast cancer were diagnosed in US women1. To reduce the local recurrence rate, 60–75% of 
these cases were treated with breast conserving surgery (BCS)1. BCS is defined as a lumpectomy followed by radiation 
therapy. Removal of the entire cancer is best ensured by performing histopathology and observing only normal breast 
tissue at the surgical margins, which is the current standard of care (SOC). Failure to achieve appropriate margins at the 
initial operation requires a second surgical procedure2. These re-excision surgeries are a burden to patients financially, 
physically, and psychologically, result in increased complications, and can delay recommended adjuvant therapies.  
 Complete cancer removal can only be verified by post-operative pathology. The gold standard for microscopic 
margin assessment is examination of post-operative histopathology sections. To get these sections, the preparation 
Photons Plus Ultrasound: Imaging and Sensing 2018, edited by Alexander A. Oraevsky, Lihong V. Wang, Proc.
of SPIE Vol. 10494, 1049443 · © 2018 SPIE · CCC code: 1605-7422/18/$18 · doi: 10.1117/12.2291008
Proc. of SPIE Vol. 10494  1049443-1
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Ultrasonic
transducer
z
Y
3 -axis motorized
scanning stage
Water tank
Focusing
lens
Computer
DAQ
Mirror
Lens
Lens
Pinhole
 
 
 
 
procedure can take up to hours and days, hindering the possibility of providing real-time feedbacks to surgeons for better 
patient outcome. Intraoperatively, histological examination of tissue can be performed by frozen sectioning, however, 
which is limited by the difficulties of freezing adipose-rich tissue, such as breast specimens. To address this critical need, 
we have developed and optimized our UV-PAM for breast tissue imaging. UV laser illumination has an advantage of 
highlighting cell nuclei, thus providing similar contrast as hematoxylin labeling used in conventional histology. Together 
with an acoustically defined axial resolution, UV-PAM can provide multilayered histology-like images of the surface 
and subsurface of breast tissue without physical sectioning, which could provide additional information to pathologists 
for more accurate diagnosis.  
 
2. METHODOLOGY 
Ultraviolet photoacoustic microscopy (UV-PAM) 
Our UV-PAM system (Fig. 1), which can handle specimens of various thicknesses, produces images by detecting 
acoustic waves generated by laser-induced rapid thermoelastic expansion3. A focused ultrasonic transducer detects the 
one-dimensional (1D) depth-resolved PA signals (A-lines). Cross-sectional images (B-scans) or volumetric images (C-
scans) can be produced respectively by linear or raster scanning using a motorized scanning stage. The UV-PAM system 
records and displays the PA B-scan images in real time during data acquisition. Because different absorbers intrinsically 
possess different optical absorption spectra, when one absorber dominates at a chosen wavelength, a single-wavelength 
laser can be used to probe a specific biological component without resorting to exogenous labels. Therefore, UV light 
(266 nm), which DNA and RNA in cell nuclei absorb by an order of magnitude more strongly than other biological 
components, is used for illumination in our imaging system4,5. At this short wavelength, high spatial resolution can also 
be achieved. The lateral imaging resolution was experimentally measured to be ~330 nm4, comparable to that of a 
conventional bright-field optical microscope with a high numerical aperture (NA) and sufficient to identify individual 
cell nuclei. Hence, UV-PAM images should be able to reveal individual cell nuclear information without labeling. 
 
 
Figure 1. Schematic of the UV-PAM system for surgical margin imaging. The UV laser beam is spatially filtered and 
expanded by a pair of lenses and a pinhole. The beam is then focused by a focusing lens and illuminated onto the bottom of 
the breast tissue specimen (Sample).  The forward propagating acoustic waves are detected by a focused ultrasonic 
transducer. The received acoustic pressure is transduced into an electric signal, amplified, and recorded by a data acquisition 
(DAQ). 
Proc. of SPIE Vol. 10494  1049443-2
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018
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3. RESULTS 
Label-free UV-PAM of fixed, unprocessed human breast tumors versus traditional microscopy of specimens 
processed with standard histology 
To show the full potential of UV-PAM, we imaged formalin-fixed but unprocessed human breast tumors (Figs. 2 and 3). 
After surgical excision, the breast tumors were fixed in formalin to prevent tissue degradation. Small samples were taken 
from the tumor/normal interface and sent for PA imaging. Subsequently the specimens were processed according to 
standard tissue preparation protocol (i.e., paraffin embedding, sectioning, and H&E staining). Sections corresponding to 
the UV-PAM images were analyzed by conventional bright-field optical microscopy. Since the UV-PAM system images 
the surface and subsurface of a specimen (Fig. 4), to prepare the best corresponding H&E-stained images, superficial 
sections were utilized. These H&E-stained sections did not exactly replicate the surface imaged by UV-PAM, but the 
image features are still remarkably similar.  
 For the fixed, unprocessed breast tumor specimen from the first patient, the region of interest (ROI) was 10.0 x 
4.2 mm2 (Fig. 2a). Fig. 2b shows a UV-PAM image of the ROI. The specimen was put in a lab-made sample holder, 
which flattened the tissue for wide field-of-view (FOV) in-focus imaging. After PA imaging, the corresponding sections 
were prepared histologically and images were acquired (Fig. 2c). The close-up UV-PAM images (Fig. 2d and f) reveal 
features similar to their corresponding H&E-stained images (Fig. 2e and g). Each image centers on expanded breast 
ducts with a cribriform pattern of cellular proliferation, characteristic of ductal carcinoma in situ, that can be readily 
discerned in both the UV-PAM and H&E-stained images. Smaller nests of densely packed nuclei, representing invasive 
ductal carcinoma, can also be seen along the top edge in Fig. 2d–g).  
 
 
Figure 2. Imaging of a breast tumor from the first patient. (a) Photograph of the first breast tissue specimen. (b and c) UV-
PAM and H&E-stained images of the fixed, unprocessed breast tumor of the green dashed region in (a), respectively. Note 
that the H&E-stained histologic image was acquired after processing, sectioning, and staining the excised breast tissue. (d 
and e) Zoomed-in UV-PAM and H&E-stained images of the red dashed regions in (b) and (c), respectively. (f and g) 
Zoomed-in UV-PAM and H&E-stained images of the blue dashed regions in (b) and (c), respectively.  
                
               For the second patient, a ROI of 7.5 x 4.5 mm2 (Fig. 3a) was imaged by our UV-PAM system (Fig. 3b). The 
corresponding H&E-stained image is shown in Fig. 3c. The close-up UV-PAM image (Fig. 3d) shows a number of small 
bright cell nuclei that correspond to lymphocytes on the histology image (Fig. 3e). Lymphocytes are often associated 
with cancer. Comparing the two close-up images (Fig. 3d and e), we observe denser lymphocytes in our UV-PAM 
image. This difference can be explained by the greater imaging penetration depth of UV-PAM (~100 µm) in comparison 
to the sectioned slice thickness (~7 µm) of the H&E-stained tissue. When the cell nuclei are densely packed along the 
lateral and axial directions, individual cell nuclei cannot be resolved, resulting in a bright and dense feature, as seen in 
another close-up UV-PAM image (Fig. 3f). In this case, the corresponding H&E-stained image (Fig. 3g) demonstrates 
densely packed breast tumor cells.  
  
Proc. of SPIE Vol. 10494  1049443-3
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Figure 3. Imaging of a breast tumor from the second patient. (a) Photograph of the second breast tissue specimen. (b and c) 
UV-PAM and H&E-stained images of the fixed, unprocessed breast tumor of the green dashed region in (a), respectively. (d 
and e) Zoomed-in UV-PAM and H&E-stained images of the red dashed regions in (b) and (c), respectively. (f and g) 
Zoomed-in UV-PAM and H&E-stained images of the blue dashed regions in (b) and (c), respectively. 
 
               For a third patient sample, the ROI was 7.8 x 3.0 mm2 (Fig. 4a). As with the second patient, in our UV-PAM 
image we can see a bright signal in the top region (Fig. 4b). This region also represents a high-grade tumor. With the 
high resolution provided by UV-PAM and without any sample processing, UV-PAM can also image fat cells, which are 
clearly shown with the zoomed-in UV-PAM images (Fig. 4c and d). In Fig. 4d, we can easily identify the cell nuclei of 
the fat cells, which are normally dissolved during the standard tissue preparation protocol. 
 
  
Figure 4. Imaging of a breast tumor from the third patient. (a) Photograph of the third breast tissue specimen. (b) UV-PAM 
image of the fixed, unprocessed breast tumor of the orange dashed region in (a). (c) Zoomed-in UV-PAM image of the red 
dashed regions in (b). (d) Zoomed-in UV-PAM image of the green dashed regions in (c). 
 
 To further demonstrate the unique advantage of UV-PAM, which is to generate multilayered histology-like 
images from a single raster scan, we time-gated our measured PA signals to generate depth-resolved images of the first 
patient’s breast tumor specimen (Fig. 5). Fig. 5a shows the entire field-of-view at 8 different depths with an axial step 
size of 22 µm. Two close-up UV-PAM images of the red and blue dashed boxes (marked in Fig. 5a) are also shown with 
the 8 corresponding depths (Fig. 5b and c). This multilayered imaging capability does not come at the expense of 
scanning time as the time-of-flight information of the PA waves automatically provide the depth information of the 
specimens. This multilayered imaging feature maximizes the chance of identifying tumor cells of an excised tissue, 
which cannot be provided by bright-field optical microscopy. 
Proc. of SPIE Vol. 10494  1049443-4
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Figure 5. Series of depth-resolved images of the first patient’s breast tumor specimen imaged by UV-PAM. The depth-
resolved images are shown at 8 depths with an axial step size of 22 µm. 
 
4. CONCLUSIONS 
To conclude, we have demonstrated and optimized UV-PAM for breast tissue imaging, providing images similar to that 
can be seen on H&E-stained slide without labels. With the high resolution images provided by UV-PAM, diagnostic 
features, including cell nuclear sizes and internuclear distance can also be measured. These features are important for 
cytologic and histologic grade. In the future, these useful parameters could be provided to automatic algorithms and 
recognize malignancy in UV-PAM images alone. While we have concentrated on breast tissue in this study, UV-PAM 
can be optimized for other cancer specimens where margin status is critical for patient outcome. 
 
REFERENCES 
[1] American Cancer Society, Breast Cancer Facts & Figures 2016-2017., Am. Cancer Soc. Inc, Abstract (2016). 
[2] Clarke, M. et al. “Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local 
recurrence and 15-year survival: an overview of the randomised trials,” Lancet 366, 2087-2106, (2005).  
[3] Wang, L. V., and Yao J., “A practical guide to photoacoustic tomography in the life sciences,” Nat. Methods 13, 
627–638 (2016). 
[4] Wong, T. T. W. et al. “Fast Label-free multilayered histology-like imaging of human breast cancer by photoacoustic 
microscopy,” Sci. Adv. 3, e1602168 (2017).  
[5] Wong, T. T. W. et al. “Label-free automated three-dimensional imaging of whole organs by microtomy-assisted 
 photoacoustic microscopy,” Nat. Commun. 8, 1386 (2017).  
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English

Photoacoustic microscopy enables multilayered histological imaging of human breast cancer without staining

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Photoacoustic microscopy enables
multilayered histological imaging of
human breast cancer without staining
Terence T. W. Wong, Ruiying  Zhang, Pengfei  Hai,
Rebecca L. Aft, Deborah V. Novack, et al.
Terence T. W. Wong, Ruiying  Zhang, Pengfei  Hai, Rebecca L. Aft, Deborah
V. Novack, Lihong V. Wang, "Photoacoustic microscopy enables multilayered
histological imaging of human breast cancer without staining," Proc. SPIE
10494, Photons Plus Ultrasound: Imaging and Sensing 2018, 1049443 (19
February 2018); doi: 10.1117/12.2291008
Event: SPIE BiOS, 2018, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Photoacoustic microscopy enables multilayered histological imaging of 
human breast cancer without staining  
Terence T. W. Wonga,b, Ruiying Zhangb, Pengfei Haia,b, Rebecca L. Aftc,d, Deborah V. Novacke,f, 
and Lihong V. Wang,a,* 
aCaltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department of Medical 
Engineering, Department of Electrical Engineering, California Institute of Technology, Pasadena, 
CA 91125, USA;  
bOptical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. 
Louis, St. Louis, MO 63130, USA;  
cDepartment of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, 
USA;  
dJohn Cochran Veterans Hospital, St. Louis, Missouri 63106, USA;  
eMusculoskeletal Research Center, Division of Bone and Mineral Diseases, Department of 
Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA;  
fDepartment of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, 
USA 
ABSTRACT  
In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. 
About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the 
local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer 
cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with 
remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. 
Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these 
techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming 
for large specimens. Thus, 20–60% of patients undergoing BCS still require second surgeries due to positive surgical 
margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the 
need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic 
microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser 
illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional 
histology and measuring features related to the histological landscape without the need for labels. We demonstrate that 
our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast 
tissue.    
Keywords: Cancer, translational research, photoacoustic imaging, microscopy, label-free, histology 
 
1. INTRODUCTION  
Breast cancer is the most common cancer diagnosed in women. In 2016, an estimated 250,000 new cases of invasive 
breast and non-invasive breast cancer were diagnosed in US women1. To reduce the local recurrence rate, 60–75% of 
these cases were treated with breast conserving surgery (BCS)1. BCS is defined as a lumpectomy followed by radiation 
therapy. Removal of the entire cancer is best ensured by performing histopathology and observing only normal breast 
tissue at the surgical margins, which is the current standard of care (SOC). Failure to achieve appropriate margins at the 
initial operation requires a second surgical procedure2. These re-excision surgeries are a burden to patients financially, 
physically, and psychologically, result in increased complications, and can delay recommended adjuvant therapies.  
 Complete cancer removal can only be verified by post-operative pathology. The gold standard for microscopic 
margin assessment is examination of post-operative histopathology sections. To get these sections, the preparation 
Photons Plus Ultrasound: Imaging and Sensing 2018, edited by Alexander A. Oraevsky, Lihong V. Wang, Proc.
of SPIE Vol. 10494, 1049443 · © 2018 SPIE · CCC code: 1605-7422/18/$18 · doi: 10.1117/12.2291008
Proc. of SPIE Vol. 10494  1049443-1
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Ultrasonic
transducer
z
Y
3 -axis motorized
scanning stage
Water tank
Focusing
lens
Computer
DAQ
Mirror
Lens
Lens
Pinhole
 
 
 
 
procedure can take up to hours and days, hindering the possibility of providing real-time feedbacks to surgeons for better 
patient outcome. Intraoperatively, histological examination of tissue can be performed by frozen sectioning, however, 
which is limited by the difficulties of freezing adipose-rich tissue, such as breast specimens. To address this critical need, 
we have developed and optimized our UV-PAM for breast tissue imaging. UV laser illumination has an advantage of 
highlighting cell nuclei, thus providing similar contrast as hematoxylin labeling used in conventional histology. Together 
with an acoustically defined axial resolution, UV-PAM can provide multilayered histology-like images of the surface 
and subsurface of breast tissue without physical sectioning, which could provide additional information to pathologists 
for more accurate diagnosis.  
 
2. METHODOLOGY 
Ultraviolet photoacoustic microscopy (UV-PAM) 
Our UV-PAM system (Fig. 1), which can handle specimens of various thicknesses, produces images by detecting 
acoustic waves generated by laser-induced rapid thermoelastic expansion3. A focused ultrasonic transducer detects the 
one-dimensional (1D) depth-resolved PA signals (A-lines). Cross-sectional images (B-scans) or volumetric images (C-
scans) can be produced respectively by linear or raster scanning using a motorized scanning stage. The UV-PAM system 
records and displays the PA B-scan images in real time during data acquisition. Because different absorbers intrinsically 
possess different optical absorption spectra, when one absorber dominates at a chosen wavelength, a single-wavelength 
laser can be used to probe a specific biological component without resorting to exogenous labels. Therefore, UV light 
(266 nm), which DNA and RNA in cell nuclei absorb by an order of magnitude more strongly than other biological 
components, is used for illumination in our imaging system4,5. At this short wavelength, high spatial resolution can also 
be achieved. The lateral imaging resolution was experimentally measured to be ~330 nm4, comparable to that of a 
conventional bright-field optical microscope with a high numerical aperture (NA) and sufficient to identify individual 
cell nuclei. Hence, UV-PAM images should be able to reveal individual cell nuclear information without labeling. 
 
 
Figure 1. Schematic of the UV-PAM system for surgical margin imaging. The UV laser beam is spatially filtered and 
expanded by a pair of lenses and a pinhole. The beam is then focused by a focusing lens and illuminated onto the bottom of 
the breast tissue specimen (Sample).  The forward propagating acoustic waves are detected by a focused ultrasonic 
transducer. The received acoustic pressure is transduced into an electric signal, amplified, and recorded by a data acquisition 
(DAQ). 
Proc. of SPIE Vol. 10494  1049443-2
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
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3. RESULTS 
Label-free UV-PAM of fixed, unprocessed human breast tumors versus traditional microscopy of specimens 
processed with standard histology 
To show the full potential of UV-PAM, we imaged formalin-fixed but unprocessed human breast tumors (Figs. 2 and 3). 
After surgical excision, the breast tumors were fixed in formalin to prevent tissue degradation. Small samples were taken 
from the tumor/normal interface and sent for PA imaging. Subsequently the specimens were processed according to 
standard tissue preparation protocol (i.e., paraffin embedding, sectioning, and H&E staining). Sections corresponding to 
the UV-PAM images were analyzed by conventional bright-field optical microscopy. Since the UV-PAM system images 
the surface and subsurface of a specimen (Fig. 4), to prepare the best corresponding H&E-stained images, superficial 
sections were utilized. These H&E-stained sections did not exactly replicate the surface imaged by UV-PAM, but the 
image features are still remarkably similar.  
 For the fixed, unprocessed breast tumor specimen from the first patient, the region of interest (ROI) was 10.0 x 
4.2 mm2 (Fig. 2a). Fig. 2b shows a UV-PAM image of the ROI. The specimen was put in a lab-made sample holder, 
which flattened the tissue for wide field-of-view (FOV) in-focus imaging. After PA imaging, the corresponding sections 
were prepared histologically and images were acquired (Fig. 2c). The close-up UV-PAM images (Fig. 2d and f) reveal 
features similar to their corresponding H&E-stained images (Fig. 2e and g). Each image centers on expanded breast 
ducts with a cribriform pattern of cellular proliferation, characteristic of ductal carcinoma in situ, that can be readily 
discerned in both the UV-PAM and H&E-stained images. Smaller nests of densely packed nuclei, representing invasive 
ductal carcinoma, can also be seen along the top edge in Fig. 2d–g).  
 
 
Figure 2. Imaging of a breast tumor from the first patient. (a) Photograph of the first breast tissue specimen. (b and c) UV-
PAM and H&E-stained images of the fixed, unprocessed breast tumor of the green dashed region in (a), respectively. Note 
that the H&E-stained histologic image was acquired after processing, sectioning, and staining the excised breast tissue. (d 
and e) Zoomed-in UV-PAM and H&E-stained images of the red dashed regions in (b) and (c), respectively. (f and g) 
Zoomed-in UV-PAM and H&E-stained images of the blue dashed regions in (b) and (c), respectively.  
                
               For the second patient, a ROI of 7.5 x 4.5 mm2 (Fig. 3a) was imaged by our UV-PAM system (Fig. 3b). The 
corresponding H&E-stained image is shown in Fig. 3c. The close-up UV-PAM image (Fig. 3d) shows a number of small 
bright cell nuclei that correspond to lymphocytes on the histology image (Fig. 3e). Lymphocytes are often associated 
with cancer. Comparing the two close-up images (Fig. 3d and e), we observe denser lymphocytes in our UV-PAM 
image. This difference can be explained by the greater imaging penetration depth of UV-PAM (~100 µm) in comparison 
to the sectioned slice thickness (~7 µm) of the H&E-stained tissue. When the cell nuclei are densely packed along the 
lateral and axial directions, individual cell nuclei cannot be resolved, resulting in a bright and dense feature, as seen in 
another close-up UV-PAM image (Fig. 3f). In this case, the corresponding H&E-stained image (Fig. 3g) demonstrates 
densely packed breast tumor cells.  
  
Proc. of SPIE Vol. 10494  1049443-3
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/4/2018
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Normalized PA amplitude
b C d
 
 
 
 
 
Figure 3. Imaging of a breast tumor from the second patient. (a) Photograph of the second breast tissue specimen. (b and c) 
UV-PAM and H&E-stained images of the fixed, unprocessed breast tumor of the green dashed region in (a), respectively. (d 
and e) Zoomed-in UV-PAM and H&E-stained images of the red dashed regions in (b) and (c), respectively. (f and g) 
Zoomed-in UV-PAM and H&E-stained images of the blue dashed regions in (b) and (c), respectively. 
 
               For a third patient sample, the ROI was 7.8 x 3.0 mm2 (Fig. 4a). As with the second patient, in our UV-PAM 
image we can see a bright signal in the top region (Fig. 4b). This region also represents a high-grade tumor. With the 
high resolution provided by UV-PAM and without any sample processing, UV-PAM can also image fat cells, which are 
clearly shown with the zoomed-in UV-PAM images (Fig. 4c and d). In Fig. 4d, we can easily identify the cell nuclei of 
the fat cells, which are normally dissolved during the standard tissue preparation protocol. 
 
  
Figure 4. Imaging of a breast tumor from the third patient. (a) Photograph of the third breast tissue specimen. (b) UV-PAM 
image of the fixed, unprocessed breast tumor of the orange dashed region in (a). (c) Zoomed-in UV-PAM image of the red 
dashed regions in (b). (d) Zoomed-in UV-PAM image of the green dashed regions in (c). 
 
 To further demonstrate the unique advantage of UV-PAM, which is to generate multilayered histology-like 
images from a single raster scan, we time-gated our measured PA signals to generate depth-resolved images of the first 
patient’s breast tumor specimen (Fig. 5). Fig. 5a shows the entire field-of-view at 8 different depths with an axial step 
size of 22 µm. Two close-up UV-PAM images of the red and blue dashed boxes (marked in Fig. 5a) are also shown with 
the 8 corresponding depths (Fig. 5b and c). This multilayered imaging capability does not come at the expense of 
scanning time as the time-of-flight information of the PA waves automatically provide the depth information of the 
specimens. This multilayered imaging feature maximizes the chance of identifying tumor cells of an excised tissue, 
which cannot be provided by bright-field optical microscopy. 
Proc. of SPIE Vol. 10494  1049443-4
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Figure 5. Series of depth-resolved images of the first patient’s breast tumor specimen imaged by UV-PAM. The depth-
resolved images are shown at 8 depths with an axial step size of 22 µm. 
 
4. CONCLUSIONS 
To conclude, we have demonstrated and optimized UV-PAM for breast tissue imaging, providing images similar to that 
can be seen on H&E-stained slide without labels. With the high resolution images provided by UV-PAM, diagnostic 
features, including cell nuclear sizes and internuclear distance can also be measured. These features are important for 
cytologic and histologic grade. In the future, these useful parameters could be provided to automatic algorithms and 
recognize malignancy in UV-PAM images alone. While we have concentrated on breast tissue in this study, UV-PAM 
can be optimized for other cancer specimens where margin status is critical for patient outcome. 
 
REFERENCES 
[1] American Cancer Society, Breast Cancer Facts & Figures 2016-2017., Am. Cancer Soc. Inc, Abstract (2016). 
[2] Clarke, M. et al. “Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local 
recurrence and 15-year survival: an overview of the randomised trials,” Lancet 366, 2087-2106, (2005).  
[3] Wang, L. V., and Yao J., “A practical guide to photoacoustic tomography in the life sciences,” Nat. Methods 13, 
627–638 (2016). 
[4] Wong, T. T. W. et al. “Fast Label-free multilayered histology-like imaging of human breast cancer by photoacoustic 
microscopy,” Sci. Adv. 3, e1602168 (2017).  
[5] Wong, T. T. W. et al. “Label-free automated three-dimensional imaging of whole organs by microtomy-assisted 
 photoacoustic microscopy,” Nat. Commun. 8, 1386 (2017).  
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Photoacoustic microscopy enables multilayered histological imaging of human breast cancer without staining

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