measurement-scattering-nonlinearities-from-single-plasmonic

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Osaka University

Nonlinear plasmonics has attracted a lot of interests due to its wide applications. Recently, we demonstrated saturation and reverse saturation of scattering from a single plasmonic nanoparticle, which exhibits extremely narrow side lobes and central peaks in scattering images [ACS Photonics 1(1), 32 (2014)]. It is desirable to extract the reversed saturated part to further enhance optical resolution. However, such separation is not possible with conventional confocal microscope. Here we combine reverse saturable scattering and saturated excitation (SAX) microscopy. With quantitative analyses of amplitude and phase of SAX signals, unexpectedly high-order nonlinearities are revealed. Our result provides greatly reduced width in point spread function of scattering-based optical microscopy. It will find applications in not only nonlinear material analysis, but also high-resolution biomedical microscopy.

Point spread function analysis with saturable and reverse saturable scattering.

A unique case of carcinosarcoma of the prostate detected in a 71-year-old man is presented. Pelvic exenteration was performed, and the resected prostatic mass was found to consist of two histologically distinct elements; adenocarcinoma and sarcoma with focal osteosarcomatous element. The patient is still alive with neither metastasis nor recurrence. This is the 42nd case of carcinosarcoma of the prostate to be reported in the literature.

Prostatic carcinosarcoma: a case report and review of literature.

To investigate the relationship between variations of the pelvic artery arrangement and the age at erectile dysfunction (ED) onset, as some men develop ED while relatively young, while others maintain erectile function into old age despite having cardiovascular diseases, thus congenital factors might be involved.

Variations of the internal pudendal artery as a congenital contributing factor to age at onset of erectile dysfunction in Japanese.

We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and experimental investigations show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensity and contributes to improve the spatial resolution in three dimensions beyond the diffraction limit of light.

High-resolution confocal microscopy by saturated excitation of fluorescence.

To clarify the physiological effects of aldosterone on human penile corpus cavernosum (hPCC) tissue, as aldosterone has a wider physiological action than just the maintenance of electrolyte balance, and there are mineralocorticoid receptors, i.e. aldosterone receptors, in hPCC tissue.

Effect of aldosterone on isolated human penile corpus cavernosum tissue.

We demonstrate high-resolution fluorescence imaging in biological samples by saturated excitation (SAX) microscopy. In this technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity to induce strong nonlinear fluorescence responses in the center of laser focus, which contributes the improvement of the spatial resolution in three dimensions. Using SAX microscopy, we observed stained microtubules in HeLa cells with improved spatial resolution. We also measured the relation of the fluorescence and excitation intensity with several kinds of fluorescence dyes and, in the results, confirmed that SAX microscopy has the potential to observe any kind of fluorescence samples in current usage.

Beyond the diffraction-limit biological imaging by saturated excitation microscopy.

• To examine the feasibility of three-dimensional (3D) CT cavernosography in the diagnosis of corporal veno-occlusive dysfunction.

Three-dimensional CT cavernosography: reconsidering venous ligation surgery on the basis of the modern technology.

• To evaluate virtual cavernoscopy as a diagnostic tool in erectile dysfunction.

Virtual cavernoscopy: a novel diagnostic tool for use in the corpus cavernosal lumen in patients with erectile dysfunction.

We report the use of fluorescent nanodiamonds (FNDs) as a photostable fluorescent probe for high resolution saturated excitation (SAX) microscopy. We confirmed that FNDs show a nonlinear fluorescence response under saturated excitation conditions generated by intense excitation light. Using FNDs, we quantified the spatial resolution improvement inherent in SAX microscopy, and experimentally demonstrated the scalability of the spatial resolution of SAX microscopy. The photostability of the FNDs allowed us to perform nanoparticle imaging of a multicolor-stained macrophage cell with a spatial resolution beyond the diffraction limit.

SAX microscopy with fluorescent nanodiamond probes for high-resolution fluorescence imaging.

Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinearity in the fluorescence response induced by the SAX. Calculation of the optical transfer functions and the edge responses for SAX microscopy revealed the contrast improvement of high-spatial frequency components in the sample structure and the effective reduction of background signals from the out-of-focus planes. Experimental observations of the edge response and x-z cross-sectional images of stained HeLa cells agreed well with theoretical investigations. We applied SAX microscopy to the imaging of three-dimensional cultured cell clusters and confirmed the resolution improvement at a depth of 40 μm. This study shows the potential of SAX microscopy for super-resolution imaging of deep parts of biological specimens.

Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters.

We show that scattering from a single gold nanoparticle is saturable for the first time. Wavelength-dependent study reveals that the saturation behavior is governed by depletion of surface plasmon resonance, not the thermal effect. We observed interesting flattening of the point spread function of scattering from a single nanoparticle due to saturation. By extracting the saturated part of scattering via temporal modulation, we achieve λ/8 point spread function in far-field imaging with unambiguous separation of adjacent particles.

Measurement of a saturated emission of optical radiation from gold nanoparticles: application to an ultrahigh resolution microscope.

Saturated excitation (SAX) microscopy utilizes the nonlinear relation between fluorescence emission and excitation under saturated excitation to improve the spatial resolution of confocal microscopy. In this study, we theoretically and experimentally investigate the saturation of fluorescence excitation under modulated excitation to optimize the excitation conditions for SAX microscopy. Calculation of the relationships between fluorescence and excitation intensity with different modulation frequencies reveals that the lifetime of the triplet state of the fluorescent probe strongly affects the strength of the demodulated fluorescence signals. We also find that photobleaching shows little dependence on the modulation frequency. These investigations allow us to determine the optimum excitation conditions, that is, the conditions providing sufficient fluorescence saturation without strong photobleaching. For a sample stained with ATTO Rho6G phalloidin, we estimate the optimal excitation conditions, which are produced with 50 kHz excitation modulation and a 50 μsec pixel dwell time, and successfully perform three-dimensional imaging with sub-diffraction resolution.

Saturated excitation microscopy with optimized excitation modulation.

We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions.

In this review, we introduce the principles of spatial resolution improvement in super-resolution microscopies that were recently developed. These super-resolution techniques utilize the interaction of light and fluorescent probes in order to break the diffraction barrier that limits spatial resolution. The imaging property of each super-resolution technique is also compared with the corresponding conventional one. Typical applications of the super-resolution techniques in biological research are also introduced.

Introduction to super-resolution microscopy.

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.

Article	Year
Point spread function analysis with saturable and reverse saturable scattering. Optics express\| PubMed ID: 25401635	2014
Prostatic carcinosarcoma: a case report and review of literature. International journal of urology : official journal of the Japanese Urological Association\| PubMed ID: 12588610	2003
Variations of the internal pudendal artery as a congenital contributing factor to age at onset of erectile dysfunction in Japanese. BJU international\| PubMed ID: 18005213	2008
High-resolution confocal microscopy by saturated excitation of fluorescence. Physical review letters\| PubMed ID: 18233334	2007
Effect of aldosterone on isolated human penile corpus cavernosum tissue. BJU international\| PubMed ID: 18294301	2008
Beyond the diffraction-limit biological imaging by saturated excitation microscopy. Journal of biomedical optics\| PubMed ID: 19021372	2008
Three-dimensional CT cavernosography: reconsidering venous ligation surgery on the basis of the modern technology. BJU international\| PubMed ID: 20868388	2011
Virtual cavernoscopy: a novel diagnostic tool for use in the corpus cavernosal lumen in patients with erectile dysfunction. BJU international\| PubMed ID: 21332903	2011
SAX microscopy with fluorescent nanodiamond probes for high-resolution fluorescence imaging. Biomedical optics express\| PubMed ID: 21750771	2011
Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters. Journal of biomedical optics\| PubMed ID: 24296998	2013
Measurement of a saturated emission of optical radiation from gold nanoparticles: application to an ultrahigh resolution microscope. Physical review letters\| PubMed ID: 24483931	2014
Saturated excitation microscopy with optimized excitation modulation. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 24488765	2014
Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions. Interface focus\| PubMed ID: 24511385	2013
Introduction to super-resolution microscopy. Microscopy (Oxford, England)\| PubMed ID: 24671128	2014
A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy. Nature methods\| PubMed ID: 25894946	2015
Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging. Journal of biomedical optics\| PubMed ID: 26238663	2015

Masahito Yamanaka

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