Masakazu Kikawada

Fluorescence enhancement with deep-ultraviolet surface plasmon excitation

We report the experimental demonstration of fluorescence enhancement in fluorescent thin film using surface plasmon excitation in deep-ultraviolet (deep-UV) region. Surface plasmon resonance in deep-UV is excited on aluminum thin film in the Kretschmann-Raether geometry. Considering the oxidation thickness of aluminum, the experimentally measured incident angle dependence of reflectance show good agreement with Fresnel theory. Surface plasmon resonance was excited at the incident angle of 49 degrees for 266 nm p-polarized excitation light on the film of 18 nm-thick aluminum with 6.5 nm-thick alumina. Fluorescence of CdS quantum dots coated on this aluminum film was enhanced to 18-fold in intensity by the surface plasmon excitation.

Enhanced multicolor fluorescence in bioimaging using deep-ultraviolet surface plasmon resonance

Enhanced multicolor fluorescence has been achieved using deep-ultraviolet surface plasmon resonance (DUV-SPR) on an aluminum thin film using the Kretschmann configuration. The film thickness and the incident angle of the light were optimized by calculations using the Fresnel equations. The presence of a surface oxide layer was also considered in the calculations. Experimental measurements showed that DUV-SPR led to a strong enhancement of the fluorescence intensity from both quantum dots and dye-labeled cells.

Surface plasmon-enhanced fluorescence cell imaging in deep-UV region

We present high-sensitivity fluorescent cell imaging results obtained by excitation of the deep-ultraviolet surface plasmon resonance (DUV-SPR). In this study, the excitation conditions for DUV-SPR in the Kretschmann configuration were optimized for cell imaging. The aluminum thickness was analyzed taking into consideration the refractive index of the cells on the aluminum surface. It was found that the optimum aluminum thickness was 21 nm, independent of the refractive index. We demonstrated that the fluorescence from stained cells on the aluminum thin film was enhanced by DUV-SPR. The fluorescence intensity obtained by DUV-SPR excitation was three times higher than that obtained by conventional fluorescence microscopy.

Direct optical measurements of far- and deep-ultraviolet surface plasmon resonance with different refractive indices.

The surface plasmon resonance (SPR) of Al thin films was investigated by varying the refractive index of the environment near the films in the far-ultraviolet (FUV, 120-200 nm) and deep-ultraviolet (DUV, 200-300 nm) regions. An original FUV-DUV spectrometer that adopts an attenuated total reflectance (ATR) system was used. The measurable wavelength range was down to the 180 nm, and the environment near the Al surface could be controlled. The resultant spectra enabled the dispersion relationship of Al-SPR in the FUV and DUV regions to be obtained. In the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on the Al film, the angle and wavelength of the SPR became larger and longer, respectively, compared to those in air. These shifts correspond well with the results of simulations performed using Fresnel equations.

Plasmon-Enhanced Autofluorescence Imaging of Organelles in Label-Free Cells by Deep-Ultraviolet Excitation.

We demonstrate the observation of organelles in label-free cells on an aluminum thin film using deep-ultraviolet surface plasmon resonance (DUV-SPR). In particular, the Kretschmann configuration is used for the excitation of DUV-SPR. MC3T3-E1 cells are directly cultured on the aluminum thin film, and DUV-SPR leads to autofluorescence of in the label-free MC3T3-E1. We found that nucleic acid and mitochondria in these label-free MC3T3-E1 cells quite strongly emit the autofluorescence as a result of DUV-SPR. Yeast cells are also deposited on the aluminum thin film. Tryptophan and mitochondrial nicotinamide adenine dinucleotide (NADH) in the yeast cells are subsequently excited, and their autofluorescence is spectrally analyzed in the UV region. On the basis of these results, we conclude that DUV-SPR constitutes a promising technique for the acquisition of highly sensitive autofluorescence images of various organelles in the cells.

Enhanced photoelectron emission from aluminum thin film by surface plasmon resonance under deep-ultraviolet excitation

We report photoelectron emission enhancement of aluminum thin films by surface plasmon excitation in the deep-ultraviolet region. Deep-ultraviolet light with a wavelength of 266 nm has enough energy to cause electron emission from aluminum and excite surface plasmons on aluminum. We applied the Kretschmann configuration to excite surface plasmons. The enhancement factor of the emission current is found to depend on the enhanced electric field intensity excited by surface plasmons. The maximum emission efficiency of photoelectrons is 2.9 nA mW−1 for an aluminum thickness of 19 nm with an alumina thickness of 4 nm. The characteristics of the dependence of the photoelectron emission efficiency on the applied bias between the anode and cathode are investigated.

Development of far- and deep-ultraviolet surface plasmon resonance (SPR) sensor using aluminum thin film

We investigated the surface plasmon resonance (SPR) of aluminum (Al) thin films with varying refractive index of the environment near the films in the far‒ultraviolet (FUV, ≤ 200 nm) and deep‒ultraviolet (DUV, ≤ 300 nm) regions. By using our original FUV‒DUV spectrometer which adopts an attenuated total reflectance (ATR) system, the measurable wavelength range was down to the 180 nm, and the environment near the Al surface could be controlled. In addition, this spectrometer was equipped with a variable incident angle apparatus, which enabled us to measure the FUV‒DUV reflectance spectra (170–450 nm) with various incident angles ranging from 45° to 85°. Based on the obtained spectra, the dispersion relation of Al‒SPR in the FUV and DUV regions was obtained. In the presence of various liquids (HFIP, water, alcohols etc.) on the Al film, the angle and wavelength of the SPR became larger and longer, respectively, compared with those in the air (i.e., with no materials on the film). These shifts correspond well with the results of simulations performed according to the Fresnel equations, and can be used in the application of SPR sensors. FUV‒DUV‒SPR sensors (in particular, FUV‒SPR sensors) with tunable incident light wavelength have three experimental advantages compared with conventional visible‒SPR sensors, as discussed based on the Fresnel equations, i.e., higher sensitivity, more narrowly limited surface measurement, and better material selectivity.

Sensitive imaging of organelles in label-free cells by surface plasmon resonance in deep-ultraviolet region(Conference Presentation)

In this research, we demonstrate the enhanced autofluorescence and high-sensitivity bioimaging of intracellular organelles using DUV-SPR. The Kretschmann configuration is used for excitation of DUV-SPR. We used an aluminum thickness of 24 nm. The alumina surface was estimated to be 6 nm by comparison between the experimental and calculated results. Reflectance after culturing of cells was measured. DUV-SPR is excited at an incident angle of 52° after the biological samples are cultured. MC3T3-E1 cells as Label-free cells are directly cultured on an aluminum and glass surfaces, and they were cultured on the both substrates in an incubator. Autofluorescence spectra excited of the label-free MC3T3-E1 cells was measured by 266-nm exictation. The autofluorescence intensity for the aluminum is higher than that for the glass. In the autofluorescence spectra, MC3T3-E1 cells exhibited two fluorescence peaks, which were located around 330 and 500 nm. These 330 and 500 nm emissions indicate aromatic amino acid and mitochondria, respectively. Both of the ehnahcement factors were 8 times. We also observed autofluorescence of aromatic amino acid and mitochondrial NADH in the label-free MC3T3-E1 cells cultured on the aluminum and glass surfaces. In the autofluorescence image with DUV-SPR, organelles can be clearly observed in the MC3T3-E1 cells. On the other hand, the autofluorescence intensity is very weak in the image without DUV-SPR. Accordingly, DUV-SPR can facilitate the observation of proteins, DNA in nucleus, and other structures that cannot be excited by visible light. DUV-SPR is shown to be a powerful technique for acquiring high-sensitivity label-free observation of biological samples.