Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength

Chen, Ruimin

Maslov, Konstantin

Wang, Lihong V.

Yao, Da-Kang

Zhou, Qifa

Da-Kang Yao

Ruimin Chen

Konstantin I. Maslov

Qifa Zhou

Lihong V. Wang

Crossref

In vivo imaging of cell nuclei by photoacoustic microscopy without staining

Caltech Authors - Main

PROCEEDINGS OF SPIE
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In vivo imaging of cell nuclei by
photoacoustic microscopy without
staining
Da-Kang  Yao, Ruimin  Chen, Konstantin  Maslov, Qifa
Zhou, Lihong V. Wang
Da-Kang  Yao, Ruimin  Chen, Konstantin  Maslov, Qifa  Zhou, Lihong V.
Wang, "In vivo imaging of cell nuclei by photoacoustic microscopy without
staining," Proc. SPIE 8223, Photons Plus Ultrasound: Imaging and Sensing
2012, 82231X (23 February 2012); doi: 10.1117/12.906211
Event: SPIE BiOS, 2012, San Francisco, California, United States
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In Vivo Imaging of Cell Nuclei by Photoacoustic Microscopy 
without Staining 
 
Da-Kang Yao1, Ruimin Chen2, Konstantin Maslov1, Qifa Zhou2, and Lihong V. Wang1, * 
 
1Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. 
Louis, One Brookings Drive, St. Louis, Missouri 63130, USA 
2Department of Biomedical Engineering, University of Southern California, 1042 Downey Way, Los 
Angeles, CA 90089, USA 
*Corresponding author: lhwang@biomed.wustl.edu 
 
 
ABSTRACT 
 
Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution 
noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA 
to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained 
UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic 
coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength. 
 
Keywords: DNA, RNA, label-free, histology, cancer diagnosis, UV light 
 
 
1. INTRODUCTION 
 
Imaging of cell nuclei plays an important role in cancer diagnosis. The nuclei in cancer cells are different from normal 
nuclei in morphology. For example, two morphological characteristics of cancer cell nuclei are large size and mitotic 
figure [1]. Besides eccentric nuclear morphology, irregular distribution of cell nuclei is characteristic of cancer. Right 
now, these characteristics are applied as the gold standard by pathologists to determine cancer grade and evaluate 
prognosis [2]. Classical imaging of cell nuclei in tissues goes through a complicated histological process, including 
tissue biopsying, processing, embedding, sectioning, and staining before imaging with optical microscopy [3]. Without 
staining, in vivo imaging of cell nuclei has been achieved by the ultraviolet photoacoustic microscopy (UV-PAM) [4]. 
 
UV-PAM is a new photoacoustic imaging technique capable of specially imaging cell nuclei in live tissue. In UV-PAM, 
a pulsed laser beam of ultraviolet light (UV) is focused into the tissue. DNA and RNA, two major components of cell 
nuclei, strongly absorb UV light at wavelengths around 260 nm [5, 6]. The absorbed light is converted into heat in the 
nuclei. Thermoelastic expansion generates ultrasonic waves, which are detected as photoacoustic signals by a focused 
ultrasonic transducer. The photoacoustic signals are then processed to produce a nuclear image with positive contrast. 
Using light with a wavelength of 266 nm, we have demonstrated that UV-PAM can generate high contrast images of cell 
nuclei [4]. Besides the 266 nm wavelength, various UV wavelengths are available within the absorption spectra of DNA 
and RNA. In order to find the optimal UV-PAM wavelength, we imaged cell nuclei in vivo at various wavelengths, using 
a UV-PAM system equipped with a tunable laser. 
 
 
2. MATERIALS AND METHODS 
 
2.1 Animal Preparation 
 
Six female athymic nude mice (Harlan Laboratories, Indianapolis, IN) were used for in vivo imaging. The mice were 
anesthetized with 1% isoflurane (Butler Animal Health Supply, Dublin, OH) delivered in pure oxygen (Airgas, St. Louis, 
Photons Plus Ultrasound: Imaging and Sensing 2012, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8223, 82231X · © 2012 SPIE · CCC code: 1605-7422/12/$18 · doi: 10.1117/12.906211
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MO) at a flow rate of 0.5 L/min. Before imaging, the mouse ear skin was gently washed three times using distilled water. 
After imaging, the mice recovered in ten minutes. All experimental animal procedures were carried out in conformity 
with the laboratory animal protocol of the Animal Studies Committee of Washington University in St. Louis. 
 
2.2 Ultraviolet Photoacoustic Microscopy 
 
A detailed description of the original UV-PAM system was reported previously [4]. In order to investigate the optimal 
wavelength for in vivo photoacoustic imaging of cell nuclei, we equipped the UV-PAM system with an OPO laser 
system (NT242-SH, Altos Photonics, Bozeman, MT) that provides a wavelength tuning range from 210 to 2300 nm [7]. 
Figure 1 is a schematic of the improved UV-PAM system. A pulsed UV laser beam with a pulse width of 5 ns is emitted 
from the tunable laser system at a repetition rate of 1 kHz. After being attenuated by a neutral density filter (NDC-50C-
4M, Thorlabs, Newton, NJ), the laser beam is focused by a 100-mm-focal-length off-axis parabolic mirror (50338AL, 
Newport, Irvine, CA), and then spatially filtered by a 25 µm diameter pinhole (910PH-25, Newport). The beam is 
refocused into a water tank by a 0.1 NA objective lens (LA4280, Thorlabs), passes through a focused ring ultrasonic 
transducer (50 MHz central frequency, 7 mm focal length), and penetrates a 25 µm thick polyethylene membrane before 
focusing along the z axis to an object. The ultrasonic transducer is coaxially aligned with the objective lens to a common 
focus. The polyethylene membrane forms an optical window on the bottom of the water tank and maintains acoustic 
coupling. The laser pulse energy behind the membrane is measured by a digital power meter (PM100D, Thorlabs) with a 
silicon photodiode sensor (S120VC, Thorlabs). The water tank and the specimen are mounted on a translation stage (PT1, 
Thorlabs), which is vertically installed on a two-dimensional scanning stage (Micos USA, Irvine, CA) for manual 
focusing of the specimen. While the scanning stage performs raster scanning with a step size of 0.62 μm in the horizontal 
plane (x-y plane), photoacoustic signals are detected by the ultrasonic transducer, amplified by an amplifier (ZFL-500LN, 
Mini-Circuits, Branson, MO), and collected by a computer through a 12-bit, 200 MHz digitizer (NI PCI-5124, National 
Instruments, Austin, TX). 
  
 
Figure 1. Schematic of the ultraviolet photoacoustic microscopy system. 
Scanning stage
Parabolic mirror
Pinhole
Objective lens
Ultrasonic 
transducer
Water tank
Tissue
Tunable laser
Computer
Translational 
stage
Amplifier
x
y
z
Plastic membrane
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ss.
Is,
.
,
#
*
,
 
 
 
 
 
245 nm
248 nm
255 nm
250 nm 251 nm
252 nm
240 nm 242 nm
260 nm
290 nm
266 nm 270 nm 280 nm
282 nm 285 nm
20 µm
0 0.5 1
Normalized PA amplitude
Figure 2. In vivo en face photoacoustic images of the skin of mouse ears in the form of maximum amplitude
projection (MAP). Ultrasound gel was used for acoustic coupling. Laser pulse energy was 20 nJ.  PA, photoacoustic.
Proc. of SPIE Vol. 8223  82231X-3
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RESULTS 
 
3.1 Wavelength range 
 
We imaged cell nuclei in vivo in the ear skin of the mice using various wavelengths. An anesthetized mouse was held by 
a custom-made stereotaxic imaging stage. After the imaging stage was mounted on the translational stage, a mouse ear 
was flat placed on a plastic plate immobilized on the imaging stage. A film of ultrasound gel was used between the 
image window and the mouse ear. During the preview scanning, we adjusted the translational stage upward until a clear 
B-scan image was observed. Then, we scanned the ear skin with laser pulse energy of 20 nJ at wavelengths ranging from 
210 to 310 nm. We repeated the experiments twice at wavelengths of 245, 248, 250, 251, 252, 255, 260, 266, 270, 280, 
and 282 nm. Both experiments yielded identifiable images of cell nuclei with positive contrast. Typical images are 
shown in Fig. 2. For wavelengths of 210, 220, 230, 240, 242, 285, 290, 300, and 310 nm, we repeated imaging 
experiments two to four times, but did not obtain any identifiable image of cell nuclei. Typical images of mouse ear skin 
acquired at wavelengths of 240, 242, 285, and 290 nm are shown in Fig. 2. All of these images show that wavelengths 
ranging from 245 to 282 nm are applicable to in vivo photoacoustic imaging of unstained cell nuclei as ultrasound gel is 
used as a coupling medium. 
 
3.2 Optimal wavelength  
 
We examined the signal-to-noise ratio (SNR) and the contrast-to-noise ratio (CNR) of the photoacoustic images of cell 
nuclei. All images were acquired with laser pulse energy of 20 nJ. The mean intensity of the image of a single cell 
nucleus was obtained by averaging the amplitude within a square window of 10×10 pixels in the image of the cell 
nucleus. In order to analyze background intensity, we chose a large square window of 30×30 pixels and a small square 
window of 20×20 pixels, both of which were concentric with the single nuclear image. The large window did not cover 
neighboring nuclei, and the small window totally covered the single nuclear image. Inside the larger window but outside 
the smaller one, we calculated the mean intensity of the background image surrounding the nuclear image. In order to 
measure image noise, we scanned the mouse ear using zero laser output in each experiment. A dummy photoacoustic 
image was formed with zero laser output and was used to compute the standard deviation of image intensity. For each 
wavelength between 245 and 282 nm, we calculated the SNR and CNR of the MAP images of 25 cell nuclei. After 
averaging, we obtained a correlation between image SNR and wavelength, as shown in Fig. 3(a), and a correlation 
between CNR and wavelength, as shown in Fig. 3(b). Both SNR and CNR significantly change with the wavelength, and 
each reaches its maximum at a wavelength of 250 nm. Particularly, the CNR at 250 nm was 2.6 times greater than that at 
266 nm. Therefore, 250 nm was the optimal wavelength for in vivo photoacoustic imaging of unstained cell nuclei. 
 
0
100
200
300
240 250 260 270 280 290
Si
gn
al
-to
-n
oi
se
 ra
tio
Wavelength (nm)
0
50
100
240 250 260 270 280 290
C
on
tra
st
-to
-n
oi
se
 ra
tio
Wavelength (nm)
(a) (b)
Figure 3. Correlations between in vivo photoacoustic image of cell nuclei and optical wavelength. At each
wavelength, laser pulse energy was 20 nJ, and 25 cell nuclei were analyzed. (a) Plot of SNR (mean ± SD) of
nuclear images versus wavelength. (b) Plot of CNR (mean ± SD) of nuclear images versus wavelength.  
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4. DISCUSSION 
 
We applied UV-PAM to in vivo imaging of unstained cell nuclei at various wavelengths and obtained nuclear images 
with positive, specific, and high contrast. In UV-PAM, DNA and RNA are excited by UV light to generate photoacoustic 
signal. In animal cells, DNA and RNA are concentrated in cell nuclei. Once DNA and RNA are excited, strong 
photoacoustic signals come from cell nuclei, which results in the positive image contrast. Outside cell nuclei, weak UV 
absorption of protein only generates weak photoacoustic signals, which form a background. Comparing the background 
signals, the strong signals only come from cell nuclei, which results in the specific contrast for cell nuclei. The high 
image contrast results from a difference between strong light absorption of nucleic acids and weak absorption of protein, 
which reaches the maximum at a wavelength of 250 nm. In addition to UV-PAM, several other modern optical 
microscopy technologies have been explored for in vivo imaging of unstained cell nuclei, such as reflectance confocal 
microscopy [8-11], multiphoton microscopy [12-14], and third-harmonic generation microscopy [15, 16], However, 
reflectance confocal microscopy is difficult to provide specific image contrast for nuclei [8], multiphoton microscopy 
produces in vivo images of cell nuclei with negative contrast [13], and third harmonic generation microscopy generates 
nuclear images with low contrast [15]. 
 
We used a film of ultrasound gel with a thickness less than 30 μm for acoustic coupling. The film was between the 
plastic membrane on the bottom of water tank and mouse skin. We found that ultrasound gel significantly absorbed UV 
light at wavelengths between 210 and 310 nm. The UV absorption plus different film thickness could cause unrepeatable 
measurements of image SNR and CNR. In order to achieve repeatable measurement, we only put a tiny drop of 
ultrasound gel on the skin of mouse ears, and then filled the tank with water and let the plastic membrane on the tank 
bottom press the gel for 10 minutes (Fig. 1). Before imaging, we checked the film thickness using time-arrival 
photoacoustic signals. After the film thickness was less than the axial resolution of our UV-PAM system (29 μm), we 
started data collection for image construction. 
 
 
5. CONCLUSIONS 
 
After equipping a UV-PAM system with a tunable laser, we imaged cell nuclei in mouse skin in vivo at wavelengths 
ranging from 210 to 310 nm. Without staining, UV-PAM produced images of cell nuclei with specific, positive, and high 
contrast. The optimal wavelength is 250 nm for in vivo photoacoustic imaging of cell nuclei. Any wavelength between 
245 and 282 nm can be used to produce in vivo images of cell nuclei. However, we did not obtain an identifiable nuclear 
image at wavelengths of 210, 220, 230, 240, 242, 285, 290, 300, and 310 nm. 
 
 
ACKNOWLEDGEMENTS 
 
This work was sponsored in part by National Institutes of Health grants R01 EB000712, R01 EB008085, R01 
CA113453901, U54 CA136398, 5P60 DK02057933, and U54 CA136398. L.W. has a financial interest in 
Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work. 
 
 
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In vivo imaging of cell nuclei by photoacoustic microscopy without staining

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