Confocal microspectroscopy

Chapter 1 Introduction

1.1 Confocal microspectroscopy

1.1-1 History of microscopy

Microscopy has been widely used and there has been a great effort to improve the resolution and precision. Electron microscopy, in particular, can detect very small objects down to a few nanometers, but the cost is high and the convenience is relative low. Optical microscopy, though its spatial resolution is usually determined by diffraction limit of light, now becomes essential tool for characterization of nanostructures. Advances in digital imaging and analysis have also enabled microscopists to acquire quantitative measurements quickly and efficiently [1]. With the help of techniques, such as dark-field, phase contrast, fluorescence, and confocal, the specimen contrast is improved. The following sections would give further discussions.

1.1-2 Light scattering and absorption microscopy to evaluate electronic structure

To identify the electronic structure of molecules, absorption spectroscopy is usually the first choice because of the specificity and the quantitative character. However, for

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nanoparticles (NPs), it is hard to measure the absorption spectra and to distinguish each one based upon Lambert-Beer Law; the short path length limits the interaction between the illumination light and the NPs, also the illumination beam is too large compared to their sizes resulting from the diffraction limit of light.

Recently, photothermal microspectroscopy is developed to measure single noble metallic NPs, based on the change of refractive index due to changes in temperature and density of the sample [2]. Furthermore, Photothermal Heterodyne Imaging (PHI) allows for the unprecedented detection of gold NPs down to 1.4 nm in diameter [3]. Because heating is necessary for PHI technique, it is not suitable for nonmetallic objects. On the other hand, light scattering microspectroscopy can be applied to not only nonfluorescent but also nonmetallic NPs. Combining dark-field technique which detects only scattered light from sample performs high contrast and S/N ratio by black background. From a viewpoint of electronic information, scattering relies on the same basic optical response to the absorption process, so the scattering

spectroscopy is an alternative measurement to evaluate the electronic structure.

1.1-3 Confocal microscopy using supercontinuum

Confocal microscopy has been developed to be a powerful tool for a few decades for the high contrast and single-point measurement. Especially, the combination of the visualization

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of 3-D structure and spectra improves the application in biological and medical science.

Point light source illuminates at the focal plane, detecting object in a point, so that the light source and the detected spot are confocal. The pointed detection then is imaged at the pinhole. Confocal microscopy is originated in three mutually confocal points and the key technique is the spatial filter. With an aperture, the out-of-focus light would be blocked and permits only the well-defined point forming image, hence the contrast is increased.

Furthermore, scanning with a pair of mirrors (Galvano mirror) constructs the three-dimensional image of the sample easily [4].

Confocal laser scanning microscopy (CLSM) has attracted much more attentions because laser shows high energy density with high degree of spatial and temporal coherence, which increases the resolution and signal intensity. W. Denk et al succeeded in two-photon laser scanning fluorescent measurement by CLSM [5]. With a colliding-pulse-mode-locked (CPM) dye laser producing ultrashort pulses, the probability of two-photon molecular excitation becomes appreciable [5]. Brakenhoff et al. demonstrated that the section inherent to two-photon imaging could be improved by the introduction of confocal aperture with amplified Ti: Sapphire laser [6]. The use of femtosecond laser can shield sample from heat generation and damage; however, the tuning range is limited and the available excitation

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range does not efficiently excite fluorophores designed for single-photon excitation [7].

Supercontinuum is the formation of spectra broadening by an intense laser propagating through a nonlinear media. The characteristics of supercontinuum are the huge bandwidth, spatial coherence, and high brightness. The huge bandwidth provides a broad range of spectroscopic transitions in which many species can be detected simultaneously. The high spectral brightness and spatial coherence give high spectral resolutions [8].

Stefano and his coworker presented a new approach of reflectance laser scanning confocal system in which the spectroscopic imaging capabilities are achieved with the help of wavelength-tunable source [9]. The use of supercontinuum confocal microscope in combination with fluorescence for spectrally resolved imaging offers a great analysis of the details of living cells [10].

1.1-4 Confocal light scattering using supercontinuum

CLSM combined with fluorescence has been widely used for the study of living tissue, especially in imaging [11]. The application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize well-defined lipid bilayer models was reported two decades ago [12]. However, fluorescence spectroscopy is limited to fluorescent materials,

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quenching, and environmental factors, which have to be controlled during the analyses to obtain the reproducible measurements [13].

Scattering, on the other hand, is free from the limitations of fluorescence. Additionally, combining confocal system and supercontinuum give an effective technique for nanostructure.

Lindfors et al. have demonstrated that confocal microscopy using supercontinuum reveals spectroscopy and imaging of single Au NPs down to 10 nm [14]. Since noble metallic NPs exhibit strong scattering in visible region, it recently has been developed as label-free plasmonic biosensors [15]. Due to the outstanding results obtained by confocal light scattering [14, 15], we are able to explore electronic spectral properties of nanostructure with a high spatial resolution.