We characterize the backward second-harmonic-generation (SHG) emission in biological samples with a SHG microscope. Contributions from different mechanisms, including direct backward emissions, reflection and back-scattering of the forward emissions, are identified and clarified.

The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS-CoV) in 2003. To detect early SARS-CoV infection, a one-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane M, and spike (S) genes of SARS-CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture-derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS-S, -M, and -N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture-derived SARS-CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS-N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS-S and SARS-M also demonstrated equivalent sensitivity to the commercially available RealArt HPA-Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS-CoV; and there was no cross detection with other coronaviruses and human respiratory tract-associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple-gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS-CoV infection.

The plant hormone abscisic acid (ABA) induces a developmental switch in the aquatic fern Marsilea quadrifolia, causing the formation of aerial type characteristics, including the elongation of petioles and roots, a change in leaf morphology, the expansion of leaf surface area, and the shortening of the internodes. A number of ABA-responsive heterophylly (ABRH) genes are induced early during the transition. Using optically pure isomers of ABA, it was found that both the natural S-(+)-ABA and the unnatural R-(-)-ABA are capable of inducing a heterophyllous switch and regulating ABRH gene expression. When dose responses are compared, the unnatural ABA gives stronger morphogenic effects than the natural ABA at the same concentration, it is effective at lower concentrations, and its optimal concentration is also lower compared with the natural ABA. Deuterium-labelled ABA enantiomers were used to trace the fate of applied ABA and to distinguish the applied compound and its metabolites from the endogenous counterparts. In tissues, the supplied (+)-ABA was metabolized principally to dihydrophaseic acid, while the supplied (-)-ABA was converted at a slower rate to 7'-hydroxy abscisic acid. Treatment with either enantiomer resulted in increased biosynthesis of ABA, as reflected in the accumulation of endogenous dihydrophaseic acid. Taken together, these results suggest two distinct mechanisms of action for (-)-ABA: either (-)-ABA is intrinsically active, or its activity is due to the stimulation of ABA biosynthesis.

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.

Optical second- and third-harmonic generations have attracted a lot of attention in the biomedical imaging research field recently due to their intrinsic sectioning ability and noninvasiveness. Combined with near-infrared excitation sources, their deep-penetration ability makes these imaging modalities suitable for tissue characterization. In this article, we demonstrate a polarization harmonics optical microscopy, or P-HOM, to study the nonlinear optical anisotropy of the nanometer-scaled myosin and actin filaments inside myofibrils. By using tight focusing we can avoid the phase-matching condition due to micron-scaled, high-order structures in skeletal muscle fibers, and obtain the submicron-scaled polarization dependencies of second/third-harmonic generation intensities on the inclination angle between the long axes of the filaments and the polarization direction of the linear polarized fundamental excitation laser light. From these dependencies, detailed information on the tensor elements of the second/third-order nonlinear susceptibilities contributed from the myosin/actin filaments inside myofibrils can thus be analyzed and obtained, reflecting the detailed arrangements and structures of the constructing biomolecules. By acquiring a whole, nonlinearly sectioned image with a submicron spatial resolution, we can also compare the polarization dependency and calculate the nonlinear susceptibilities over a large area of the tissue at the same time-which not only provides statistical information but will be especially useful with complex specimen geometry.

A new optically active (R)-3-ethylmercapto-2-methylpropanol was designed and synthesized by using (D)-2,10-camphorsultam as a chiral auxiliary. Its derivatives with various core structures in the molecules were prepared for investigating mesomorphic properties. Mesophases and the corresponding transition temperatures of the compounds were identified by the polarizing microscopic textures and DSC carlorimetry. Structurally similar compounds were compared to explore the effect of core structures on the formation of mesophases, especially the frustrated phases.

Because it avoids the in-focus photodamage and phototoxicity problem of two-photon-fluorescence excitation, multiharmonic-generation biopsy based on a 1200-1300-nm light source could provide a truly noninvasive and highly penetrative optical sectioning of skin. We study multiharmonic-generation biopsy of fixed mouse skin. Our preliminary study suggests that this technique could provide submicrometer-resolution deep-tissue noninvasive biopsy images in skin without the use of fluorescence and exogenous markers. (C) 2003 Optical Society of America.

Photobleaching as a result of intense laser irradiation has been an important issue in the applications of confocal microscopy. In this study we are employing time-lapsed micro-spectroscopy to compare the effects of photobleaching under single photon and multi-photon excitation. A model is derived to account for photobleaching in both cases and protoplasts from Arabidopsis thaliana are employed as the sample. We have found that multi-photon excitation have impacts on the samples in ways much different from cw illumination in the linear domain.

Highly optically active nonlinear bio-photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second-harmonic generation (SHG) interactions, behaving similarly to man-made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third-harmonic generation, and multiphoton-induced fluorescence. With no energy release during harmonic generation processes, the nonlinear-photonic-crystal-like SHG activity is useful for investigating the dynamics of structure-function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.

A novel multi-modality nonlinear microscopy reveals highly optically-active biophotonic crystal structures in living cells. Numerous biological structures, including stacked membranes and arranged protein structures are highly organized in optical scale and are found to exhibit strong optical activities through second-harmonic-generation (SHG) interactions, behaving similar to man-made photonic crystals. The microscopic technology developed is based on a combination of imaging modalities including not only SHG, but also third-harmonic-generation and multi-photon-fluorescence. With no energy deposition during harmonic generation processes, the demonstrated highly-penetrative yet non-invasive microscopy is useful for investigating the dynamics of structure-function relationship at the molecular and subcellular levels and is ideal for studying living cells that require minimal or no preparation.

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. Thus induced two-photon absorption and the accompanied multi-photon absorption/ionization not only cause photo-bleaching but also cell damage in the vicinity of the focal point. In this paper, we study the wavelength dependent cell damage induced by high intensity femtosecond near infrared lasers. The study was performed with a Ti:sapphire laser and a Cr:forsterite laser. With a longer output wavelength from a Cr:forsterite laser, multi-photon absorption and auto-fluorescence were found to be significantly suppressed and the destructive plasma formation was found to be greatly reduced. Sustained multi-photon spectra can be observed in most plant specimens with a tightly focused Cr:forsterite laser beam under long term irradiation with more than 100 mW laser average power. In contrast, multi-photon absorption induced destructive plasma formation were frequently observed with a tightly focused Ti:sapphire laser beam within seconds with more than 10 mW laser average power.

We report our study of wavelength dependent on-focus cell damage in multi-photon confocal microscopy with femtosecond IR laser sources. Experiments were performed in selected plant tissues. Two different laser sources were selected: a femtosecond Ti:sapphire laser and a femtosecond Cr:forsterite laser. We selected Cr:forsterite laser as the comparison laser source due to its emission wavelength (1230 nm in this study) longer than the three-photon absorption threshold of protein. Similar to previous studies, with more than 10 mW micro-irradiation at 800 nm from a femtosecond Ti:sapphire laser, multi-photon absorption induced cell damages due to plasma generation, accompanied with strong luminescence, can be frequently observed in our studies. The dosages (illumination time) required to produced plasma generation (characterized by strong luminescence) were found to decrease with increased illumination intensity. In contrast, with a longer output wavelength from a Cr:forsterite laser at 1230 nm, multi-photon absorption and auto-fluorescence were found to be significantly suppressed and the cell damage was found to be greatly reduced. Sustained multi-photon spectra can be observed in most plant specimens even with a tightly focused Cr:forsterite laser beam, with similar or higher intensity enough to produce plasma generation at 800 nm wavelength (with over 100 mW average power), under long term irradiation.

We demonstrate a novel multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser at 1230 nm. By acquiring the whole nonlinear spectrum in the visible and near-NIR region, this novel technique allows a combination of different imaging modalities, including second-harmonic generation, third-harmonic generation, and multiple-photon fluorescence. Combined with the selected excitation wavelength, which is located in the IR transparency window, this microscopic technique can provide high penetration depth with reduced damage and is ideal for studying living cells.

Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption and scattering coefficient of biological specimen in the near infrared (NIR) range. Because of the need of high peak power for efficiently exciting two-photon fluorescence, the relationship between cell damage and peak power has become an interesting and much debated topic in the applications of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes have impacts on cell physiology and viability in ways much different from low illumination in the linear domain. In this article, we discuss some of the issues in two-photon fluorescence microscopy, including the degree of transparency of the specimen, a comparison of single- and two-photon excited fluorescence spectra, and the cell damage under high intensity illumination, using plant cells as a model.

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.

We report the experimental results of our using irregularly shaped diamond microparticles as handles for laser tweezers. Because of their irregular optical shape, control of the rotation of diamond microparticles can easily be achieved in a gradient force optical trap by use of a fixed linearly polarized beam with a fundamental Gaussian mode. By changing the laser focal plane upon a diamond particle near the liquid surface or interfaces, one can fully manipulate both the direction and speed of the rotation. The ability to manipulate a diamond-particle-tagged biological specimen by optical tweezers is discussed. The application of these particles as handles for optical tweezers is demonstrated by optical manipulation of biological cells. Independent movement of linear translation and rotation, with controllable rotation directions and speeds, is successfully achieved.

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.

The non-linear nature of multi-photon fluorescence excitation, SHG and THG restricts the signal detecting volume to the vicinity of the focal point. As a result, the technology has intrinsic optical sectioning capability. The use of multi-photon fluorescence excitation also allows micro-fluorometry at high spatial resolution. Under high intensity illumination, biological specimen not only emits fluorescence, but also generates harmonic emissions. Conventional ultra-fast Ti-sapphire laser allows efficient excitation of most biologically important fluorescent probes and SHG in the deep blue range. In contrast, the use of ultra-fast Cr-forsterite laser makes possible simultaneous detection of two- and three-photon fluorescence, SHG and THG

The intrinsic confined photo-interacting volume in multi-photon fluorescence microscopy provides the possibility of obtaining fluorescence spectrum from specific cellular structure in a tissue. In this article, we demonstrated that it is feasible to obtain useful two-photon pumped fluorescence spectrum from cell wall and single chloroplast. The difference in fluorescence spectra obtained with single- and two-photon excitation indicates that a significant shift in fluorescence maximum may occur due to the non-linear nature of excitation. Therefore, in order to properly interpret two-photon fluorescence micrographs, it is important to characterize the fluorescence spectrum of the specimen and the commonly used fluorescence probes. The fluorescence spectra will in turn be useful in the selection of filter sets in multi-photon fluorescence microscopy.