Combining the concept of structured illumination with two-photon microscopy, two-photon grid scanning pattern
microscopy (TP-GPSM) was demonstrated to provide optical section power due to two-photon excitation and super-resolution
in lateral through structured illumination. Based on a laser scanning geometry, the two-photon illumination
patterns were effectively produced by temporally and spatially modulating the excitation light. Several possible ways to
produce structured patterns were proposed in this paper. For image reconstruction, sufficient phase-stepped images were
needed. With the 2-dimensional grid scanning pattern, ten images, including two orientations and five images with
different phases in each orientation are required, and a 2-fold improvement in lateral resolution can be obtained. TP-GPSM
was shown to have the potential for super-resolution imaging in thick tissues.
In this study, we performed theoretical analysis and experimental measurement of third harmonic generation (THG) in
melanin solution with different concentrations. As predicted by theory, only THG at glass/solution interface was
observed due to Guoy phase shift effect. We have shown that this interfacial THG intensity is strongly affected by index
matching condition between the two media, leading to minimal THG at a certain melanin concentration. By fitting the
dependence of THG intensity versus melanin concentration, linear and nonlinear electric susceptibilities of melanin are
obtained, providing a valuable tool to characterize optical properties of biological molecules.
Multiharmonic optical microscopy has been widely applied in biomedical research due to its unique capability to perform noninvasive studies of biomaterials. In this study, virtual biopsy based on back-propagating multiple optical harmonics, combining second and third harmonics, is applied in unfixed rat tympanic membrane. We show that third harmonic generation can provide morphologic information on the epithelial layers of rat tympanic membrane as well as radial collagen fibers in middle fibrous layers, and that second harmonic generation can provide information on both radial and circular collagen fibers in middle fibrous layers. Through third harmonic generation, the capillary and red blood cells in the middle fibrous layer are also noted. Additionally, the 3-D relationship to adjacent bony structures and spatial variations in thickness and curvature are obtained. Our study demonstrates the feasibility of using a noninvasive optical imaging system for comprehensive evaluation of the tympanic membrane.
Oral cancer ranked number four in both cancer incident and mortality in Taiwanese male population. Early disease
diagnosis and staging is essential for its clinical success. However, most patients were diagnosed in their late disease
stage as ideal prescreening procedures are yet to be developed especially when dealing with a large surface of
precancerous lesions. Therefore, how to detect and confirm the diagnosis of these early stage lesions are of significant
clinical value. Harmonic generation process naturally occurred in biological molecules and requires no energy deposition
to the target molecule. Thus harmonic generation microscopy (HGM) could potentially serve as a noninvasive tool for
screening of human oral mucosal diseases. The in vivo optical biopsy of human oral cavity with HGM could be achieved
with high spatial resolution to resolve dynamic physiological process in the oral mucosal tissue with equal or superior
quality but devoid of complicated physical biopsy procedures. The second harmonic generation (SHG) provide
significant image contrast for biomolecules with repetitive structures such as the collagen fibers in the lamina propria
and the mitotic spindles in dividing cells. The cell morphology in the epithelial layer, blood vessels and blood cells flow
through the capillaries can be revealed by third harmonic generation (THG) signals. Tissue transparent technology was
used to increase the optical penetration of the tissue. In conclusion, this report demonstrates the first in vivo optical
virtual biopsy of human oral mucosa using HGM and revealed a promising future for its clinical application for
noninvasive in vivo diseases diagnosis.
The ability to in vivo image deep tissues noninvasively with a high resolution is strongly required for optical virtual biopsy. Higher harmonic generation microscopy, combined with second- and third-harmonic generation microscopies, is applied to 17 Asian volunteers' forearm skin. After continuous observation for 30 min, no visible damage was found. Our study proves that harmonic generation biopsy (HGB) is able to satisfy the safety requirement and to provide high penetrability (~300 µm) and submicron resolution all at the same time and is a promising tool for future virtual biopsy of skin diseases. In contrast to a previous study on fixed human skin specimens, a much improved penetrability and much reduced resolution-degradation versus depth are found in this in vivo examination.
The cornea functions as an optical lens and plays an important role in vision. For corneal diagnosis and treatment such as refractive surgery, a microscopic imaging system with a 3-D cellular resolution and retinal safety is strongly desired. Recently, confocal and multiphoton microscopies have been applied to corneal imaging with visible to near-infrared light sources. To increase retinal safety, an infrared light source is be needed. In this work, an infrared-based third and second harmonic generation microscopic study of mouse eyes is reported with ~700-µm penetrability and high cellular resolution. This study provides a critical reference for future development of infrared-based corneal imaging.
Cornea functions as an outermost lens and plays an important role in vision. For cornea diagnosis and treatment, a
microscopic imaging system with cellular resolution and high eye safety is strongly desired. Recently, the cell
morphology of corneal epithelium and endothelium can be revealed by confocal or two-photon fluorescence microscopy,
while the collagen fibers in the corneal stroma can be shown by second harmonic generation (SHG) microscopy.
However, in most of the developed imaging tools, visible to near-infrared light sources were used. To increase the eye
safety, a light source with longer wavelength would be needed. In this presentation, a study using an infrared laser based
nonlinear microscopy to investigate the ocular tissues of a mouse eye will be demonstrated. Since most of autofluorescence
was suppressed under infrared excitation, third harmonic generation (THG) microscopy was used to reveal
the cellular morphology and ~700μm penetrability could be achieved. Combining SHG with THG, in an intact mouse
eye, not only the cornea but also the upper half of the lens could be observed with cellular resolution. Our study
indicated that infrared-based SHG and THG microscopy could provide a useful in vivo investigating tool for
ophthalmology.
Atopic dermatitis (AD) is characterized by hyperkeratosis of epidermis and fibrosis within dermis in chronic skin lesions. Thus far, the histology of skin lesions has been evaluated only by examination of excised specimens. A noninvasive in vivo tool is essential to evaluate the histopathological changes during the clinical course of AD. We used Cr:forsterite laser-based multimodality nonlinear microscopy to analyze the endogenous molecular signals, including third-harmonic generation (THG), second-harmonic generation (SHG), and two-photon fluorescence (TPF) from skin lesions in AD. Significant differences in thickness of epidermis and stratum corneum (SC), and modified degrees of fibrosis in dermis (measured by THG signals and SHG signals, respectively), are clearly demonstrated in in vitro studies. Increased TPF levels are positively associated with the levels of the THG signals from the SC. Our in vitro observations of histological changes are replicated in the in vivo studies. These findings were reproducible in skin lesions from human AD. For the first time, we demonstrate the feasibility of preclinical applications of Cr:forsterite laser-based nonlinear microscopy. Our findings suggest that the optical signatures of THG, TPF, and SHG can be used as molecular markers to assess the pathophysiological process of AD and the effects of local treatment.
Gene expression plays an important role in embryo development and organ function. Previous studies have shown that harmonic generation microscopy (HGM) can be used as a fluorescence signal-independent, minimally invasive method with a subcellular 3-D resolution and a penetration depth in the order of millimeters for long-term continuous imaging of vertebrate embryos. We show that it is ideal to combine in vivo HGM with the morphant technology for minimally invasive, long-term continuous observation of gene expression in the nervous system of vertebrate embryos. Since second- and third-harmonic generations (SHG, THG) are virtual-state-transition-based systems that depend only on the structure of the organisms, they are not temporally limited by the expression of the fluorescence proteins. We successfully identified the expression of the zarnt2a and the hif-1, 2, and 3 genes in the nervous system of zebrafish embryos with specific knockdown genes by microscopically observing the embryos from the early stages of embryogenesis. The results from a combination of the two different modalities, i.e., SHG microscopy and THG microscopy, successfully revealed the weak cell adhesion, cell apoptosis, nerve formation reduction, and neural tube distortion in the morphant zebrafish embryos.
In this manuscript, we review the physics and recent developments of the least invasive optical higher harmonic
generation microscopy, with an emphasis on the in vivo molecular imaging applications. Optical higher harmonicgenerations,
including second harmonic generation (SHG) and third harmonic generation (THG), leave no energy
deposition to the interacted matters due to their energy-conservation characteristic, providing the "noninvasiveness"
nature desirable for clinical studies. Combined with their nonlinearity, harmonic generation microscopy provides threedimensional
sectioning capability, offering new insights into live samples. By choosing the lasers working in the high
penetration window, we have recently developed a least-invasive in vivo light microscopy with submicron 3D resolution
and high penetration, utilizing endogenous and resonantly-enhanced multi-harmonic-generation signals in live
specimens, with focused applications on the developmental biology study and clinical virtual biopsy.
Atopic dermatitis (AD) is now very common in people who live in cities, especially for babies and children. Since the
cause of AD is still not completely understood and each person may have his own mixed symptoms that can change over
time, diagnosis of AD can not be done precisely. Unlike some skin diseases, physical biopsy is rarely used in diagnosing
AD on account of its low urgency. Thus, only indirect diagnoses, like asking for a medical history to learn about the
symptoms and to rule out other diseases can be carried out. To gain insight into cellular details of AD for long-term
diagnosing without physical biopsy, a noninvasive in vivo tool with a sub-micron subsurface resolution and high
penetrability has to be used. In this presentation, we show that harmonic optical virtual biopsy can provide the required
noninvasive cellular imaging, and is ideal for future clinical diagnosis of AD. Harmonic optical microscopy has been
demonstrated to have the capability to reveal cellular morphology of human skin from epidermis to dermis layer. Third
harmonic generation (THG), which is sensitive to inhomogeneous interfaces, can show the structures of skins, and can be
used to reveal the morphological changes, for example, the thicken cuticle which is a common symptom of AD. Second
harmonic generation (SHG), which occurs in non-centrosymmetric structures, has excellent contrast in collagen fibers
and can show the pathological changes of dermis layer. Utilizing both THG and SHG, useful information may be given
to facilitate the diagnosis of AD.
Nervous system development is a complicated dynamic process, and many mechanisms remain unknown. By utilizing endogenous second-harmonic-generation as the contrast of polarized nerve fibers and third-harmonic-generation (THG) to reveal morphological changes, we have successfully observed the vertebrate embryonic nervous development from the very beginning based on a 1230-nm light source. The dynamic development of the nerve system within a live zebrafish embryo can be recorded continuously more than 20 hr without fluorescence markers. Since the THG process is not limited by the time of gene expression and differentiation as fluorescence-based techniques are, the observable stages can be advanced to the very beginning of the development process. The complete three-dimensional brain development from a neural plate to a neural tube can be uncovered with a submicron lateral resolution. We have, for the first time, also reported the generation of SHG from myelinated nerve fibers and the outer segment of the photoreceptors with a stacked membrane structure. Our study clearly indicates the fact that higher-harmonics-based optical microscopy has the strong potential to long-term in vivo study of the nervous system, including genetic disorders of the nervous system, axon pathfinding, neural regeneration, neural repair, and neural stem cell development.
Harmonics optical microscopy (HOM) provides a truly “noninvasive” tool for in vivo and long-term study of vertebrate embryonic development. Based on the nonlinear natures, it provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power (~1μm axial resolution) without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamages. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can be used to do functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Zebrafish embryos now have been used to study many vertebrate physiological systems. We have demonstrated an in vivo HOM study of developmental dynamics of several embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.
KEYWORDS: Luminescence, Heart, In vivo imaging, Femtosecond phenomena, 3D image processing, Microscopy, Light sources, Biomedical optics, Absorption, Two photon imaging
Transgenic lines carrying a specific tissue tagged by green-fluorescence-protein (GFP) have been a powerful tool to developmental biology because they encapsulate the expression of endogenous genes. Traditionally with two-photon fluorescence microscopy based on a femtosecond Ti:sapphire laser (with a wavelength between 700-980nm), green fluorescence can be excited by simultaneous absorption of two photons for high-resolution three-dimensional (3D) optical imaging. However for in vivo biological applications, Ti:sapphire-laser based optical technology presents several limitations including finite penetration depth, strong on-focus cell damage, and phototoxicity. For high optical penetration and minimized photodamages, two-photon imaging based on light sources with an optical wavelength located around the biological penetration window (~1300nm) is desired, where unwanted light-tissue interactions including scattering, absorption, and photodamages can all be minimized. Previous experiments around the optical penetration window indicated inefficient green fluorescence excitation of GFP through three-photon absorption. Red fluorescence protein is thus highly desired for future non-invasive in vivo two-photon imaging. Screening from embryos injected with DNA fragment containing a heart-specific regulatory element of zebrafish cardiac myosin light chain 2 gene (cmlc2) fused with HcRed gene, we generate a zebrafish line that has strong two-photon red fluorescence expressed in cardiac cells based on a 1230nm femtosecond light source working in the biological penetration window. Combined with its nonlinearity, high penetration depth, and minimized photodamages, this method provides superb imaging capability compared with the traditional GFP based two-photon microscopy, offering deep insight into the noninvasive in vivo studies of gene expression in vertebrate embryos.
Based on a femtosecond Cr:forsterite laser, harmonics optical microscopy (HOM) provides a truly “noninvasive” tool for in vivo and long-term study of vertebrate embryonic development. Based on optical nonlinearity, HOM provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamage. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can perform functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Here we demonstrate in vivo HOM studies of developmental dynamics of several important embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.
For the purpose of functional third harmonic optical microscopy, it is necessary to find a method to locally enhance third harmonic generation at specific cellular site. We have demonstrated that by matching the third harmonic generation frequency of a Cr:forsterite laser and the surface plasmon resonance frequency of <50-nm silver nanoparticles, localized enhancement of third harmonic intensity of more than 100-folds can be achieved both in phantom and in real biological tissues. This strongly enhanced third harmonic signal can then be applied to specific molecule imaging by attaching the nanoparticles to the target molecule with the advantages of noninvasiveness and deep penetration capability.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.