Longitudinal, multimodal functional imaging of microvascular response to photothermal therapy.
ABSTRACT Although studies have shown that photothermal therapy can coagulate selectively abnormal vasculature, the ability of this method to achieve consistent, complete removal of the vasculature is questionable. We present the use of multimodal, wide-field functional imaging to study, in greater detail, the biological response to selective laser injury. Specifically, a single-platform instrument capable of coregistered fluorescence imaging and laser speckle imaging was utilized to monitor vascular endothelial growth factor gene expression and blood flow, respectively, in a transgenic rodent model. Collectively, the longitudinal, in vivo data collected with our instrument suggest that the biological response to selective laser injury involves early-stage redistribution of blood flow, followed by increased vascular endothelial growth factor promoter activity to stimulate pro-angiogenic events.
[show abstract] [hide abstract]
ABSTRACT: Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were able to identify genes that may participate in formation of PWS and PDL effects. Our preliminary results indicate changes in gene expression of angiogenesis-related genes, suggesting that dysregulation of angiogenic signals and/or components may contribute to PWS pathology. Lasers Surg. Med. 45: 67-75, 2013. © 2012 Wiley Periodicals, Inc.Lasers in Surgery and Medicine 02/2013; 45(2):67-75. · 2.75 Impact Factor
Longitudinal, multimodal functional
imaging of microvascular
response to photothermal therapy
Albert K. Bui,1,2Kathleen M. Teves,2,3Elmer Indrawan,2,4Wangcun Jia,2and Bernard Choi2,4,5,*
1Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
2Beckman Laser Institute and Medical Clinic, Department of Surgery, University of California, Irvine, California 92612, USA
3Department of Neurobiology, University of California, Irvine, California 92697, USA
4Department of Biomedical Engineering, University of California, Irvine, California 92697, USA
5Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, California 92697, USA
*Corresponding author: firstname.lastname@example.org
Received July 2, 2010; accepted August 11, 2010;
posted September 2, 2010 (Doc. ID 131062); published September 22, 2010
Although studies have shown that photothermal therapy can coagulate selectively abnormal vasculature, the ability
of this method to achieve consistent, complete removal of the vasculature is questionable. We present the use of
multimodal, wide-field functional imaging to study, in greater detail, the biological response to selective laser
injury. Specifically, a single-platform instrument capable of coregistered fluorescence imaging and laser speckle
imaging was utilized to monitor vascular endothelial growth factor gene expression and blood flow, respectively,
in a transgenic rodent model. Collectively, the longitudinal, in vivo data collected with our instrument suggest that
the biological response to selective laser injury involves early-stage redistribution of blood flow, followed by in-
creased vascular endothelial growth factor promoter activity to stimulate pro-angiogenic events.
Society of America
170.2655, 170.3880, 170.6480.
© 2010 Optical
Biological tissue requires a dense vascular system to sup-
ply nutrients and oxygen to tissue and remove toxic by-
products produced by normal cellular processes. Various
diseases lead to progressive alterations in microvascular
architecture. For example, with port wine stain (PWS)
birthmarks, abnormal neuronal signaling is implicated in
development of capillary malformations in the skin .
The gold-standard treatment for these birthmarks has in-
volved the use of pulsed lasers to photocoagulate selec-
tively the abnormal vasculature .
Although studies have shown that pulsed-laser therapy
can photocoagulate PWS vessels, the ability of this
method to achieve consistent, complete removal of the
birthmark is questionable [3,4]. We associate the limited
efficacy to our incomplete understanding of the vascular
repair response to selective laser-induced damage.
In this Letter, we present the use of multimodal, wide-
field functional imaging (WiFI) to study, in greater detail,
the biological response to selective laser injury. Specifi-
cally, a single-platform instrument capable of coregis-
tered fluorescence imaging and laser speckle imaging
(LSI), was utilized to monitor vascular endothelial
growth factor (VEGF) gene expression and blood flow,
To investigate the vascular repair process, a transgenic
C3H mouse model  was used. To enable direct, chronic
imaging of the microvasculature, a dorsal window cham-
ber was surgically installed on each animal [5–9]. All
animal-related procedures were approved by the Institu-
tional Animal Care and Use Committee at University of
Our animal model emits green fluorescent protein
(GFP) under control of the VEGF promoter. Because
VEGF plays a major role in angiogenesis [5,10,11], the
presence of GFP emission indicates VEGF gene expres-
sion and, thus, is indicative of an angiogenic response.
Concurrently, LSI was used to monitor changes in micro-
vascular blood flow in response to selective laser injury.
Upon completion of window chamber installation, the
animal was placed on a circulating-water heating pad po-
stray light contamination of the fluorescence and speckle
signals (Fig. 1). The window chamber was attached to a
custom aluminum mount to image directly the subdermal
of isoflurane gas (2%–5%) and oxygen was used as
promoter activity and blood flow in the dorsal window chamber
microvasculature in response to selective laser injury. A, CCD
camera; B, zoom lens; C, 532 nm emission filter; D, ring-light il-
luminator (488 nm excitation); E, window chamber; F, anesthe-
sia nose cone; G, heating bed (to water heater/circulator); H,
mirror; I, dual polarizers for attenuation; J, modular light port
(633 nm He–Ne and white halogen); K, light-tight enclosure.
3216 OPTICS LETTERS / Vol. 35, No. 19 / October 1, 2010
0146-9592/10/193216-03$15.00/0© 2010 Optical Society of America
inhalation anesthesia during each imaging session. A
12 bit, thermoelectrically cooled CCD camera was
mounted above the enclosure. A variable-magnification
(0:7× to 4×) lens was used to image the window chamber
onto the CCD sensor. An ∼30 mm diameter observation
port was created in one wall of the enclosure to enable
imaging of the in vivo preparation.
Images were collected with three imaging modes: (1)
architectural imaging—the window chamber was transil-
luminated with broadband light delivered from a broad-
band tungsten–halogen lamp, (2) LSI—the window
chamber was transilluminated with coherent 633 nm
He–Ne laser light, and (3) fluorescence imaging—the
window chamber was epiilluminated with 488 nm light.
For fluorescence imaging, an external bandpass filter
(center wavelength of 530 nm) was used to collect selec-
tively the GFP fluorescence emission.
Previous studies [7,12–15] have demonstrated the abil-
ity of LSI to quantify blood-flow dynamics in the dorsal
window chamber. In these studies, short exposure times
(e.g., 10 ms) were used. However, the use of short expo-
sure times hinders the ability of LSI to enable visualiza-
tion of blood flow in capillaries and small arterioles and
venules. To enable functional vascular density mapping
using LSI principles, we instead employed long expo-
sures times (e.g., >1000 ms) to enable visualization of
vessel perfusion in both large and small microvascula-
ture, albeit at a loss of quantifiable blood flow (Fig. 2).
Long-exposure LSI enables functional vascular density
mapping without the need for exogenous intravascular
contrast agents, such as fluorescein isothiocyanate
dextran or Texas Red.
We used our WiFI instrument to achieve coregistered
fluorescence and long-exposure (5000 ms) LSI images
during longitudinal experiments in which the window
chamber vasculature was irradiated with pulsed laser
light. A frequency-doubled Nd:YAG laser (DualisVP, Fo-
tona Laser, Ljublijana, Slovenia) was used to irradiate a
specific arteriole–venule pair (diameters of ∼100 μm)
using an ∼2 mm spot size. Based on previous published
data [7,15], laser parameters (3 pulses, 1 ms pulse dura-
tion, 20 Hz repetition rate, 5:0 J=cm2) that reliably
achieve acute vessel photocoagulation, were selected.
Fluorescence and LSI images were collected prior to
and immediately following laser irradiation (i.e., Day 0).
Raw speckle data were converted to maps of relative
blood flow using a simplified speckle imaging equation
. Follow-up images were collected at Days 1, 3,
and 7 postirradiation, at magnifications of 1× and 4×.
Prior to laser irradiation, LSI images showed obvious
blood flow at the site of interest (Fig. 3). After irradiation,
an immediate disruption of flow was observed, signifying
that acute photocoagulation was achieved. With subse-
quent images taken on Days 1, 3, and 7, we visualized
a progressive increase in the number of perfused vessels
surrounding the injured site. Interestingly, Day 3 showed
increased blood flow in the irradiated vessel, with flow
stoppage observed by Day 7. We hypothesize that these
events represented an initial hyperemic response in an
injured, constricted vessel, which ultimately failed to re-
main patent. As a result, recruitment of macrophages
during the ensuing wound healing response may have in-
itiated breakdown of the injured vessel, hence resulting
in an absence of blood flow at Day 7.
We concurrently observed GFP fluorescence emission
within and surrounding the irradiated site, indicative of
increased VEGF gene expression and, hence, angiogen-
esis-stimulating activity (Fig. 3). Our preliminary data
(n ¼ 3) have consistently shown peak GFP emission at
Day 7. Since this GFP signal comes from fibroblasts,
maps derived from raw speckle images, using LSI-based analy-
sis with a simplified speckle imaging equation. Blood-flow maps
are shown at 1× (left) and 2× (right) optical magnification taken
at exposure times of 10, 100, and 1000 ms. Note the increase in
discernible vasculature in the blood-flow maps taken with long-
er exposure times.
(Color online) Representative normalized blood-flow
chamber imaged on Days 0 (pre-laser and post-laser irradia-
tion), 1, 3, and 7, at 1× (top row) and 4× (middle and bottom
rows) optical magnifications, to observe the vascular repair re-
sponse to selective laser injury. Normalized blood-flow maps
were collected using a 5000 ms exposure time. Note the pre-
sence of blood flow before laser irradiation (compared to
the disruption of blood flow following laser irradiation (white
arrows in middle row). Based on subsequent image sets taken
on Days 1, 3, and 7, we observed a progressive increase in the
number of perfused vessels surrounding the injured site. Con-
currently, we observed an increase in 530 nm fluorescence
emission (bottom row) immediately surrounding the postinjury
site, indicating GFP/VEGF activity, with a peak signal on Day 7
(yellow arrow at bottom right). Scale bars: 1× images (2 mm),
4× images (1 mm).
(Color online) Representative mouse dorsal window
October 1, 2010 / Vol. 35, No. 19 / OPTICS LETTERS3217
which are cells involved in extracellular matrix secretion
and wound healing, the strong fluorescent signal seen on
Day 7 (i.e., after blood flow stopped in the laser irradiated
vessel), suggests that the resulting hypoxic environment
promoted secretion of growth factors, like VEGF, to
remodel the microvasculature.
Since angiogenesis is a process that develops over
days to weeks following injury, the immediate (i.e.,
Day 0, postlaser irradiation) increase in the number of
perfused vessels surrounding the irradiated site is most
likely due to redistribution of blood flow to collateral ves-
sels, as observed previously . Therefore, in addition to
the proangiogenic activity suggested by the progressive
increase in GFP emission, increased perfusion and dila-
tion of existing vessels appear to play a key part in the
microvascular repair response after selective laser
Collectively, the longitudinal in vivo data collected
with our WiFI instrument suggest that the biological re-
sponse to selective laser injury involves early-stage redis-
tribution of blood flow, followed by increased VEGF
promoter activity to stimulate proangiogenic events. Ad-
ditional experiments are planned to study the (1) effects
of severity of microvascular injury (i.e., high irradiance
versus low irradiance), (2) type of microvascular injury
(i.e., photothermal versus photochemical), and (3) mod-
ulation of the angiogenic response using antiangiogenic
agents [15,17]. Furthermore, with integration of other
camera-based optical modalities, such as hemoglobin
oxygen saturation imaging [9,18], we plan to obtain a de-
tailed picture of the overall biological response to selec-
tive optical injury modalities.
We thank Rakesh Jain and Dai Fukumura, Steele La-
boratories, Massachussetts General Hospital, for their
generous donation of the transgenic animal model used
in the research. Funding for this research was provided
in part by the Arnold and Mabel Beckman Foundation,
the National Institutes of Health (NIH) (EB009571),
and the National Institutes of Health Laser Microbeam
and Medical Program (LAMMP, a P41 Technology
1. J. B. Mulliken and A. E. Young, Vascular Birthmarks:
Hemangiomas and Malformations (W. B. Saunders, 1988).
2. R. R. Anderson and J. A. Parrish, Lasers Surg. Med. 1, 263
3. K. V. T. Le, H. Shahidullah, and I. J. Frieden, Dermatol. Surg.
25, 127 (1999).
4. Z. F. Jasim and J. M. Handley, J. Am. Acad. Dermatol. 57,
5. D. Fukumura, R. Xavier, T. Sugiura, Y. Chen, E. C. Park,
N. F. Lu, M. Selig, G. Nielsen, T. Taksir, R. K. Jain, and
B. Seed, Cell 94, 715 (1998).
6. H. D. Papenfuss, J. F. Gross, M. Intaglietta, and F. A. Treese,
Microvasc. Res. 18, 311 (1979).
7. B. Choi, W. C. Jia, J. Channual, K. M. Kelly, and J. Lotfi, J.
Invest. Dermatol. 128, 485 (2008).
8. J. K. Barton, G. Vargas, T. J. Pfefer, and A. J. Welch,
Photochem. Photobiol. 70, 916 (1999).
9. B. S. Sorg, B. J. Moeller, O. Donovan, Y. T. Cao, and M. W.
Dewhirst, J. Biomed. Opt. 10, 044004 (2005).
10. N. Ferrara, J. Mol. Med. 77, 527 (1999).
11. T. Veikkola and K. Alitalo, Sem. Can. Biol. 9, 211 (1999).
12. B. Choi, N. M. Kang, and J. S. Nelson, Microvasc. Res. 68,
13. J. Channual, B. Choi, D. Pattanachinda, J. Lotfi, and K. M.
Kelly, Lasers Surg. Med. 40, 644 (2008).
and J. S. Nelson, Lasers Surg. Med. 39, 494 (2007).
15. W. C. Jia, V. Sun, N. Tran, B. Choi, S. W. Liu, M. C. J. Mihm,
T. L. Phung, and J. S. Nelson, Lasers Surg. Med. 42, 105
16. J. C. Ramirez-San-Juan, R. Ramos-Garcia, I. Guizar-Iturbide,
G. Martinez-Niconoff, and B. Choi, Opt. Express 16, 3197
17. T. L. Phung, D. A. Oble, W. Jia, L. E. Benjamin, M. C. Mihm,
and J. S. Nelson, Lasers Surg. Med. 40, 1 (2008).
18. A. K. Dunn, A. Devor, H. Bolay, M. L. Andermann, M. A.
Moskowitz, A. M. Dale, and D. A. Boas, Opt. Lett. 28, 28
3218 OPTICS LETTERS / Vol. 35, No. 19 / October 1, 2010