As a novel modality of optical microscopy, second-harmonic generation (SHG) provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that well-formed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.
We present the first experimental comparison between optical second harmonic generation images and atomic force
microscope images in a matrix of nano-scaled collagen fibrils. Substantial variation of forward and backward propagated
second harmonic generation radiation is observed in a single collagen fibril and is nicely correlated with the accurately
determined thickness from an atomic force microscope. Contradicting to conventional nonlinear optical theory, our result
indicates a linear relationship between fibril thickness and forward / backward second harmonic generation ratio. This is
the first demonstration of estimating fibril thickness with nanometer precision by a noninvasive optical method.
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.
We demonstrate a compact and self-starting fiber-delivered femtosecond Cr:forsterite laser for nonlinear light microscopy. A semiconductor saturable absorber mirror provides the self-starting mechanism and maintains long-term stability in the laser cavity. Four double-chirped mirrors are employed to reduce the size of the cavity and to compensate for group velocity dispersion. Delivered by a large-mode-area photonic crystal fiber, the generated laser pulses can be compressed down to be with a nearly transform-limited pulse width with 2.2-nJ fiber-output pulse energy. Based on this fiber-delivered Cr:forsterite laser source, a compact and reliable two-photon fluorescence microscopy system can thus be realized.
Traditional biopsy requires the removal, fixation, and staining of tissues from the human body. Its procedure is invasive and painful. Therefore, a novel method of optical biopsy is desired which can perform in vivo examination and is noninvasive, highly penetrative, with no energy deposition and damage, without invasive pharmaceutical injection, and with three-dimensional (3D) imaging capability and sub-micron spatial resolution. Two-photon fluorescence microscopy (TPFM) is previously applied for biopsy of skin due to its high lateral resolution, low out-of-focus damage, and intrinsic 3D section capability. However, for future clinical applications without surgery, current 700-850 nm based laser scanning technology still presents several limitations including low penetration depth, in-focus cell damages, multi-photon phototoxicity due to high optical intensity in the 800 nm wavelength region, and toxicity if exogenous fluorescence markers were required. Here we demonstrate a novel noninvasive optical biopsy method called harmonics optical biopsy (HOB), which combines both second harmonic generation imaging and third harmonic generation imaging. Due to virtual transition nature of harmonic generations and based on light sources with an optical wavelength located around the biological penetration window (~1300nm), our HOB can serve as a truly non-invasive biopsy tool with sub-micron three-dimensional spatial resolution without any energy deposition and exogenous contrast agents. From preliminary experiment result, our HOB can reconstruct 3D cellular and subcellular images from skin surface through dermis. Besides, by utilizing backward propagating detection geometry, we will show that this technique is ideal for non-invasive clinical biopsy of human skin diseases and even useful for the early diagnosis of skin cancer symptom such as the angiogenesis.
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.
Since the first demonstration in 1990, two-photon fluorescence microscopy (TPFM) has made a great impact on biomedical researches. With its high penetration ability, low out-of-focus photodamage, and intrinsic three-dimensional (3D) sectioning capability, TPFM has been widely applied to various medical diagnosis and genome researches. Recently, single-mode optical fibers were introduced into the TPFM systems for remote optical pulse delivery. Fiber-based TPFM has advantages including isolating the vibration from laser and electronic devices, flexible system design, and low cross-talks. It is also the first step toward an all-fiber based two-photon endoscope. However, due to serious temporal broadening when conventional Ti:sapphire based femtosecond pulses propagate through the fiber, the two-photon excitation efficiency of the fiber-optic TPFM is much lower than the conventional one. The temporal broadening effect mainly comes from group velocity dispersion (GVD) and self-phase modulation (SPM), which also leads to significant spectral broadening. To reduce the temporal broadening effect, here we present a hollow-core photonic-bandgap fiber based TPFM. By replacing the conventional single-mode fiber with the hollow core photonic bandgap fiber, the GVD and SPM effects can be greatly reduced for high intensity, ultra-short pulse delivery. Femtosecond Ti:sapphire pulses passing through the fiber with negligible GVD and SPM effects is demonstrated in this paper. Much improvement of two-photon fluorescence excitation efficiency is thus achieved with the hollow-core photonic-bandgap fiber based TPFM.
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.