Nonlinear Optical Imaging Research Articles

Selective Detection and Quantitation of Organic Molecule Crystallization by Second Harmonic Generation Microscopy

Second order nonlinear optical imaging of chiral crystals (SONICC) was applied to selectively detect crystal formation at early stages and characterize the kinetics of nucleation and growth. SONICC relies on second harmonic generation (SHG), a nonlinear optical effect that only arises from noncentosymmetric ordered domain structures, which include crystals of chiral molecules. The model systems studied include pharmaceutically relevant compounds: griseofulvin and chlorpropamide. SONICC demonstrates low detection limits producing an 8 order of magnitude improvement relative to macroscopic average techniques and 5 order of magnitude improvement relative to optical microscopy. SONICC was also applied to examine the kinetics of crystallization in amorphous griseofulvin. The results show that SONICC enables simultaneous monitoring of individual crystal growth, nucleation rate, and macroscopic crystallization kinetics.

Targeting Non‐Fluorescent Molecules by Nonlinear Optical Imaging

Targeting Non‐Fluorescent Molecules by Nonlinear Optical Imaging

Ionization penalty in nonlinear optical bioimaging

The noninvasiveness of nonlinear optical imaging techniques is quantified in terms of the number of free electron generated in the laser-tissue interaction region per photon emitted into the nonlinear optical signal. For a broad variety of biomarker dyes and bioactivity reporter proteins, this ratio is shown to approach a critical value of unity for field intensities above 1 TW/cm2. Closed-form analytical expressions for the ionization penalty function and the critical pulse repetition rate are derived for few-cycle laser pulses.

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

Nonlinear optical imaging of lyotropic cholesteric liquid crystals

We use nonlinear optical microscopy combining Second Harmonic Generation (SHG) microscopy and Two-Photon Excited Fluorescence (2PEF) signals to characterize collagen lyotropic liquid crystals. We show that SHG signals provide highly contrasted images of the three-dimensional texture of cholesteric patterns with submicrometer lateral resolution. Moreover, simultaneous recording of the 2PEF signal enables in situ quantitative mapping of the molecular concentration and its correlation with the observed textures. We apply this technique to the characterization of biomimetic textures obtained in concentrated collagen liquid solutions. We successfully image biologically relevant organizations that are similar to the collagen organization found as a stabilized state in compact bones.

Dyes for biological second harmonic generation imaging

Nonlinear optical imaging has revolutionized microscopy for the life sciences. Second harmonic generation (SHG), the younger sibling of two-photon excited fluorescence (2PF), is a technique that can produce high resolution images from deep inside biological tissues. Second harmonic light is generated by the coherent scattering of an ensemble of aligned chromophores in a focused, pulsed laser beam. SHG is only generated at the focal spot, reducing the background signal, and requires ordered chromophores, so is highly structure-specific. In contrast to two-photon fluorescence, the physical process that creates the signal does not require the formation of excited states, allowing elimination of harmful photochemistry. While the SHG of native proteins and biopolymers is well known, the use of exogenous dyes can provide SHG contrast from areas without a sufficiently high intrinsic quadratic hyperpolarizability, β. Dyes for SHG primarily target lipid bilayers; a trait that, combined with sensitivity to transmembrane potential, allows monitoring of action potentials in a variety of excitable cells, most importantly mammalian neurons. This article summarizes the principles of SHG imaging and explores approaches for maximizing the SHG signal from a biological specimen. We survey methods of optimizing the optical set-up, enhancing the β of the dye and achieving biological compatibility. In conclusion, we examine novel applications of SHG imaging and highlight promising directions for the development of the field.

Neutral push-pull chromophores for nonlinear optical imaging of cell membranes

A new class of push-pull molecules was recently identified, based on pyridine dicarboxamide as an electron acceptor group and bearing a fluorenethynyl pi-conjugated bridge. The molecules present good second and third order nonlinear properties, with a static quadratic hyperpolarisability mubeta (at 1.907 microm) of 320 x 10(-48) esu (mubeta(0) = 249 x 10(-48) esu) and a maximum two-photon absorption cross-section of 1146 GM. Starting from this generic structure, we designed a series of eight amphiphilic nonlinear probes, varying the length of the lipophilic tail and the nature of the polar head, and tested their cell membrane affinity by nonlinear optical imaging. A good membrane affinity was observed with probes bearing short alkyl chains and carbohydrate moieties as the polar head, emphasizing the importance of the lipophilic-hydrophilic balance, as well as the role of the polar head. This original approach based on carbohydrate head groups is an important advancement in the design of membrane probes, since these highly versatile functional groups confer adequate hydrophilicity and yet conserve overall molecular neutrality.

Innovative TCSPC–SHG microscopy imaging to monitor matrix collagen neo-synthetized in bioscaffolds

We propose an innovative invasiveless technique in the field of nonlinear optical imaging to facilitate monitoring of cell/scaffold combinations for tissue repair. By using a near infrared (NIR) femtosecond excitation, we were able to introduce a new index based on decay time response for fluorescence (F) and Second Harmonic Generation (SHG) obtained with Time Correlated Single Photon Counting (TCSPC) microscopy to monitor structural information on the state of the matrix collagen. Some human Mesenchymal Stem Cells (hMSCs) seeded in 3D scaffolds were tested with different culture times (from D7 to D56) to analyze the effect of Tumor Growth Factor beta 1 (TGF-β1) on type-2 collagen expression in the matrix. After 14 days in the presence of TGF-β1, our results showed an increase in the expression of type-2 collagen synthesized by hMSCs, and a change in collagen conformation, as an indication of its ability to be detected as a harmonophore by TCSPC-SHG without the need for an exogenous probe.

Multimodal nonlinear optical imaging of atherosclerotic plaque development in myocardial infarction-prone rabbits

Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits.

Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.

Second-order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 mum) with a minimal background due to the surrounding lipid mesophase ( approximately 1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.

Enabling tools for engineering collagenous tissues integrating bioreactors, intravital imaging, and biomechanical modeling

Many investigators have engineered diverse connective tissues having good mechanical properties, yet few tools enable a global understanding of the associated formation of collagen fibers, the primary determinant of connective tissue stiffness. Toward this end, we developed a biomechanical model for collagenous tissues grown on polymer scaffolds that accounts for the kinetics of polymer degradation as well as the synthesis and degradation of multiple families of collagen fibers in response to cyclic strains imparted in a bioreactor. The model predicted well both overall thickness and stress-stretch relationships for tubular engineered vessels cultured for 8 weeks, and suggested that a steady state had not yet been reached. To facilitate future refinements of the model, we also developed bioreactors that enable intravital nonlinear optical microscopic imaging. Using these tools, we found that collagen fiber alignment was driven strongly by nondegraded polymer fibers at early times during culture, with subsequent mechano-stimulated dispersal of fiber orientations as polymer fibers degraded. In summary, mathematical models of growth and remodeling of engineered tissues cultured on polymeric scaffolds can predict evolving tissue morphology and mechanics after long periods of culture, and related empirical observations promise to further our understanding of collagen matrix development in vitro.

Efficient Near-IR Hyperthermia and Intense Nonlinear Optical Imaging Contrast on the Gold Nanorod-in-Shell Nanostructures

New gold nanorod (Au NR)-in-shell nanostructures were developed to be more efficacious than Au NRs in near-IR (NIR) hyperthermia and nonlinear optical imaging contrast. Au NR-in-shell nanostructures are composed of an intact Au NR in a Au/Ag nanoshell. These nanostructures have a broad, intense absorption band that extends from 400 nm to 900 nm in the NIR. They are more efficient and efficacious than Au NRs with respect to in vitro hypothermia performance. Au NR-in-shell-labeled cancer cells were destroyed using continuous-wave NIR radiation with 50% less laser power than needed for Au NRs. Noticeably, the area of the destroyed cells was significantly larger than the size of the laser irradiation beam; in contrast, the destroyed area was usually restricted to the size of the laser beam spot when Au NRs were used. With their extraordinarily broad and strong surface plasmon resonance band, Au NR-in-shell nanostructures efficiently augmented several multiphoton nonlinear processes as well. The multiphoton emission spectrum covered almost the entire visible wavelength. The yield of the multiphoton signals of Au NR-in-shell nanostructures was on average 55 times larger than that of Au NRs. In vitro images of cancer cells targeted by Au NR-in-shell nanostructures revealed a stronger multiphoton contrast than those targeted by Au NRs.

Nonlinear optical imaging of individual carbon nanotubes with four-wave-mixing microscopy.

Dual color four-wave-mixing (FWM) microscopy is used to spatially resolve the third-order optical response from individual carbon nanotubes. Good signal-to-noise is obtained from single-walled carbon nanotubes (SWNT) sitting on substrates, when the excitation beams are resonant with electronic transitions of the nanotube, by detecting the FWM response at the anti-Stokes frequency. Whereas the coherent anti-Stokes (CAS) signal is sensitive to both electronic and vibrational resonances of the material, it is shown that the signal from individual SWNTs is dominated by the electronic response. The CAS signal is strongly polarization dependent, with the highest signals found parallel with the enhanced electronic polarizibility along the long axis of the SWNT.

In vivo fluorescence imaging with high-resolution microlenses

Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

High resolution three-dimensional reconstruction of the collagenous matrix of the human optic nerve head

Glaucoma is the second most common cause of blindness worldwide, leading to irreversible loss of vision. Prior studies indicate that ocular pressure-induced displacement of the lamina cribrosa (LC) may be responsible for retinal ganglion cell axon damage inside the neural canal. We present a novel approach to imaging the entire lamina cribrosa and the scleral canal at high lateral and axial resolution by using a combination of array tomography and nonlinear optical imaging of serial ultrathin orthogonal sections to detect second harmonic generated (SHG) signals from collagen. The resulting images can be analyzed individually or combined to form a three-dimensional reconstruction of the lamina. Due to the specificity of SHG generated from collagen the density and distribution of collagen inside the scleral canal can be objectively quantified with a high degree of accuracy. The reconstruction shows a non-uniform distribution of collagen along both the longitudinal and orthogonal axes. Mapping the collagen density by geographic region reveals significant differences in collagen content that result in “thin spots” with low collagen density as well as areas of very high collagen content. This suggests a non-uniform mechanical stiffness across the lamina that may account for increased axon damage observed in glaucoma patients. The inferior temporal region of the ONH in particular is marked by low collagen density, which corresponds with clinical observations identifying this region as being more susceptible to damage during the onset of glaucoma. Further application of this technique will help characterize the relationship of age, race and gender on the morphology of the LC.

In Vitro and In Vivo Nonlinear Optical Imaging of Silicon Nanowires

Understanding of cellular interactions with a nanostructure requires tracking directly the nanostructure. Current investigation is challenged by the lack of a strong, intrinsic signal from the nanostructure. We demonstrate intensive four-wave mixing and third-harmonic generation signals from dimension-controllable silicon nanowires as small as 5 nm in diameter. The nonlinear optical signals observed from the nanowires are highly photostable with an intensity level of 10 times larger than that observed from silver nanoparticles of comparable sizes. This intrinsic optical signal enabled intravital imaging of nanowires circulating in the peripheral blood of a mouse and mapping of nanowires accumulated in the liver and spleen, opening up further opportunities to investigate in vivo cellular response to nanomaterials as a function of size, aspect ratio, and surface chemistry.

Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues

An extremely compact all-fiber-optic scanning endomicroscopy system was developed for two-photon fluorescence (TPF) and second harmonic generation (SHG) imaging of biological samples. A conventional double-clad fiber (DCF) was employed in the endomicroscope for single-mode femtosecond pulse delivery, multimode nonlinear optical signals collection and fast two-dimensional scanning. A single photonic bandgap fiber (PBF) with negative group velocity dispersion at two-photon excitation wavelength (i.e. approximately 810 nm) was used for pulse prechirping in replacement of a bulky grating/lens-based pulse stretcher. The combined use of DCF and PBF in the endomicroscopy system made the endomicroscope basically a plug-and-play unit. The excellent imaging ability of the extremely compact all-fiber-optic nonlinear optical endomicroscopy system was demonstrated by SHG imaging of rat tail tendon and depth-resolved TPF imaging of epithelial tissues stained with acridine orange. The preliminary results suggested the promising potential of this extremely compact all-fiber-optic endomicroscopy system for real-time assessment of both epithelial and stromal structures in luminal organs.

Improving the Photostability of Membrane Probes through Fluorination

High photostability is a desired property for fluorescent probes, especially when long recording time is necessary. It becomes more crucial for long wavelength fluorescent dyes because they generally have lower fluorescence quantum yields and are more vulnerable to chemical and photochemical attacks. We have developed a series of optical probes with high membrane affinities that are able to report on their local membrane environment and act as sensors of membrane voltage. These dyes have hemicyanine chromophores with donor-bridge-acceptor structures. Following our recent success in developing near-infrared fluorescent membrane probes, we further tried to improve the performance of these dyes through fluorination. Synthetic pathways to hemicyanines with fluorine substitution at various positions have been developed and the performances of these fluorinated dyes have been evaluated. It was found that the effect of fluorination depends critically on the site of fluorination: donor, acceptor, or bridge. Fluorination on the vinylene bridge appears to give optimal outcomes: improvement in photostability, red-shifts in absorption and emission spectra, and increase in absorption cross section. On the other hand, fluorination on the pyridinium acceptor results in an undesirable outcome: the dye is much less stable than the nonfluorinated counterpart in PBS buffer. Fluorinated membrane dyes generally show response kinetics and voltage sensitivities similar to their nonfluorinated counterparts when tested in a voltage-clamped hemispherical lipid bilayer (HLB) apparatus and in nonlinear optical imaging. (Supported by NIH grants EB001963 and U54RR022232)

Associate Professor of Chemistry

Second-order nonlinear optical imaging of chiral crystals (SONICC) enables sensitive and selective detection of sub-diffraction limited protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. Under low magnificaiton (large field of view) applications, SONICC can detect individual crystals as small as 100 nm in diameter, which is conservatively 6 orders of magnitude lower that achievalbe using current methods for crystal detection in commercial screening platforms. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets using a crystallization micro-array, the SHG intensities rivaled those of fluorescence but with superb selectivity for crystalline regions. Furthermore, SONICC is directly compatibility with virtually all common protein crystallization platforms without modification.

Imaging Gold Nanorods by Plasmon-Resonance-Enhanced Four Wave Mixing

The current work investigates the four-wave mixing (FWM) signal from gold nanorods (NRs) using two synchronized lasers and its potential applications in bioimaging. Using the lightning rod model, we show that the strongest FWM occurs when the pump laser wavelength is tuned to be resonant with the longitudinal plasmon resonance wavelength of NR. The calculation is experimentally demonstrated by comparing the intensities of FWM from NRs with different plasmon resonance wavelengths. The FWM signal is further found to be enhanced by aggregation of NRs and is strongly dependent on pulse width. The FWM intensity from NRs is ∼39 times stronger than the coherent anti-Stokes Raman scattering intensity from melamine beads. This plasmon-resonance-enhanced FWM signal enables NRs to be used as a nonlinear optical (NLO) imaging probe.