Abstract
A broad range hyper-spectroscopic microscope fed by a supercontinuum laser source and equipped with an almost achromatic optical layout is illustrated with detailed explanations of the design, implementation and data. The real novelty of this instrument, a confocal spectroscopic microscope capable of recording high resolution reflectance data in the VIS-IR spectral range from about 500 nm to 2.5 μm wavelengths, is the possibility of acquiring spectral data at every physical point as defined by lateral coordinates, X and Y, as well as at a depth coordinate, Z, as obtained by the confocal optical sectioning advantage. With this apparatus we collect each single scanning point as a whole spectrum by combining two linear spectral detector arrays, one CCD for the visible range, and one InGaAs infrared array, simultaneously available at the sensor output channel of the home made instrument. This microscope has been developed for biomedical analysis of human skin and other similar applications. Results are shown illustrating the technical performances of the instrument and the capability in extracting information about the composition and the structure of different parts or compartments in biological samples as well as in solid statematter. A complete spectroscopic fingerprinting of samples at microscopic level is shown possible by using statistical analysis on raw data or analytical reflectance models based on Abelés matrix transfer methods.
Highlights
Since the introduction of the basic confocal principle in 1955 [1], Laser Scanning ConfocalMicroscopy (LSCM) has rapidly become an effective and necessary scientific instrument for the observation of small structures in many fields, from material to life science, and is nowadays widely applied by biomedical researchers
The curves are very similar to well-known results [24], for which an annular objective produces an increase of the side lobes, if the central one narrowing promises a better resolution than standard objectives
We present the simple case of the silicon/silicon oxide calibration sample, where the explicit dependence of the local reflectivity on chemically different areas allows precise measurements of the local thickness. This example would be used as a template for a vast range of problems, mainly of a technological kind, for which local composition and local properties are of some value for a deep analysis of the sample properties
Summary
Since the introduction of the basic confocal principle in 1955 [1], Laser Scanning Confocal. Have limited spatial resolution and limited time resolution, due to the slow acquisition time, or require the use of synchrotron radiation as a bright source [15] In this context, Hyperspectral Imaging (HSI), known as Chemical or Spectroscopic Imaging, has emerged as a powerful technique that integrates conventional imaging and spectroscopy [16,17]. We describe a novel confocal microscope aimed at integrating structural and morphological information with detailed spatially resolved spectroscopic properties, powered by a supercontinuum laser source in the visible and near infrared spectral ranges, and able to collect a complete spectroscopic image by the acquisition of a one-shot wide range reflectance spectrum for every image pixel in the three dimensional sets of data typical of a confocal microscope. The spectroscopic analysis of tissues results is under development and will be the argument of our subsequent experimental activity
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