Chapter 1 Introduction
1.1 Skin Aging
Aging is a process of structural integrity loss and physiological changes caused by
both intrinsic and extrinsic factors. Intrinsic aging of skin is a natural process genetically
determined and inalterable. However, extrinsic aging of skin is affected by relatively
controllable factors, and the effects of sunlight exposure are estimated to account for up to
90% of the visible skin aging [1]. Morphologic changes related to intrinsic aging in older
skin are relatively subtle and consist of primary laxity, fine wrinkling, and a variety of
benign neoplasms [2]. The aging-related changes include fewer basal keratinocytes,
decreased vascularity, fewer sweat glands, and flattened dermal-epidermal junction [2].
These changes increase the susceptibility of skin and reduced the skin barrier function,
which increases incidence of inflammatory or infectious skin disorders in the elderly
people [2]. In fact, most people in America over 65 have at least one skin disorder, and
many have two or more [3]. As the proportion of people who are elderly is increasing, the
issue of skin aging is becoming more and more important.
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1.2 Harmonic Generation Microscopy
Although skin biopsy is the most generally used technique to evaluate morphologic
changes in dermatological study, its invasive nature makes it not a suitable method to
investigate skin aging. The harmonic generation microscopy could be a more adaptive
technique for revealing the changes of intrinsic skin aging due to its capability of
non-invasive in vivo imaging with a high spatial resolution. The harmonic generations are
nonlinear optical processes, in which virtual-level transitions are involved. Through the
induction of electric polarization, the second-harmonic generation (SHG) process is the
generation of light with the frequency that is twice the frequency of the excitation
(fundamental) light, and the third-harmonic generation (THG) is the generation of light
with the frequency that is triple the frequency of the excitation light [4]. Due to only the
virtual-level transition involved, the SHG and THG processes leave no energy and cause
no photodamage [5, 6]. Moreover, the intensities of the SHG and THG generated were
proportional to square and cubic of the excitation light intensity respectively and both
signals are generated only in close proximity to the focal point. Therefore, the confined
excitation volume can provide high resolution of three-dimensional image [7, 8]. In
theory, SHG only occurs from optically non-centrosymmetric media, such as collagen
fibers and muscle fibers [9-11]. Therefore, SHG microscopy is ideal for investigate
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collagenous structures in the dermis. THG is a process that is dipole allowed and occurs
in all materials. Moreover, THG occurs at interfaces of any media and is free from the
constraint of a phase-matching condition and wavelength restriction [12]. Therefore,
THG can provide the information of bio-tissues and cellular morphology. In the previous
studies, THG was reported to arise from the cell membrane [13] and the cytoplasmic
organelles [8], and to be enhanced by melanin [14], oxy-hemoglobin [15], and elastin [16]
through resonance enhancement [17]. In our previous studies, a femtosecond Cr:forsterite
laser at 1230 nm was used as the excitation source of the harmonic generation
microscopy combining both SHG and THG, which was used for the non-invasive in vivo
optical virtual biopsy on human skin without damage. This harmonic generation biopsy
could clearly reveal in vivo skin structure from the outermost stratum corneum to the
upper dermis [16, 18]. In this study, the harmonic generation biopsy system was applied
to investigate structural changes of epidermis and dermis related to intrinsic aging.
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1.3 Comparison between SHGM and SHOCT
Second harmonic optical coherence tomography (SHOCT) is an extension of the
OCT that can provide additional information by collecting SHG signals for imaging
[19-22]. Compared with second harmonic generation microscopy (SHGM), SHOCT also
collects SHG signals from non-centrosymmetric media and possesses the advantages of
harmonic generation, such as no photodamage and confined excitation volume. For the
system setup, because SHOCT needs to split the source light first to generate a reference
second-harmonic light by a nonlinear crystal and then recombine it with the
second-harmonic signal generated from sample, SHOCT requires a more complicated
system setup. For the spatial resolutions, SHOCT could achieve axial resolution of
several micrometers determined by the coherence length and lateral resolution of few
micrometers determined by the spot size, such as 4.2 ȝm and 1.9 ȝm reported by Jiang et
al. [22]. However, using high NA objective, SHGM could achieve axial and lateral
resolution of ~1 ȝm and sub-micrometer [14, 18]. To improve the lateral resolution of
SHOCT, adopting a higher NA objective could narrow the spot size but results in a
smaller depth of focus [23]. To improve the axial resolution of SHOCT, the coherence
length should be reduced, and the coherence length lc was proportional to square of the
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center wavelength Ȝ0 and the inverse of bandwidth ǻȜ (ୡ ൌ ଶ ୪୬ ଶఒగοఒబమ) [23]. In order to
prevent large scattering and absorption of the human skin, the center wavelength is
preferred in some ranges and could not be too small [23]. Therefore, wide bandwidth is
needed to acquire higher resolution. For example, to achieve axial resolution of ~1 ȝm,
bandwidth should be over 70 nm at center wavelength of 400 nm [24]. For the
penetration depth, OCT was generally reported to achieve penetration of few millimeters
[23]. For SHOCT, the penetration depth was reported to be 280-350 ȝm in hydrated
type I collagen [19] and ~700 ȝm in fish scales. For SHGM, the penetration depth was
reported to be ~1.5 mm through zebra fish embryo [25], ~700 ȝm inside the mouse eye
[26], and above 300 ȝm in the human skin [14, 18]. Therefore, SHOCT and SHGM seem
to provide similar depth of penetration in bio-tissues.
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