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.
References
[1]
Minsky, M. Memoir on inventing the confocal scanning microscope. Scanning 1988, 10, 128–138.
[2]
Sun, Y.; Wallrabe, H.; Seo, S.-A.; Periasamy, A. Fret microscopy in 2010: The legacy of Theodor F？rster on the 100th anniversary of his birth. ChemPhysChem 2011, 12, 462–474.
Booth, M.; Juskaitis, R.; Wilson, T. Spectral confocal reflection microscopy using a white light source. J. Eur. Opt. Soc., Rapid Publ. 2008, 3, doi:10.2971/jeos.2008.08026.
[7]
Chiu, L.D.; Su, L.; Reichelt, S.; Amos, W.B. Use of a white light supercontinuum laser for confocal interference-reflection microscopy. J. Microsc. 2012, 246, 153–159.
[8]
Bini, J.; Spain, J.; Nehal, K.; Hazelwood, V.; DiMarzio, C.; Rajadhyaksha, M. Confocal mosaicing microscopy of human skin ex vivo: Spectral analysis for digital staining to simulate histology-like appearance. J. Biomed. Opt. 2011, 16, doi:10.1117/1.3596742.
Kunstar, A.; Leijten, J.; van Leuveren, S.; Hilderink, J.; Otto, C.; van Blitterswijk, C.A.; Karperien, M.; van Apeldoorn, A.A. Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy. J. Biomed. Opt. 2012, 17, doi:10.1117/1.JBO.17.11.116012.
Bellisola, G.; Sorio, C. Infrared spectroscopy and microscopy in cancer research and diagnosis. Am. J. Cancer Res. 2012, 2, 1–21.
[13]
Colagar, A.; Chaichi, M.; Khadjvand, T. Fourier transform infrared microspectroscopy as a diagnostic tool for distinguishing between normal and malignant human gastric tissue. J. Biosci. 2011, 36, 669–677.
[14]
Diem, M.; Miljkovi？, M.; Bird, B.; Chernenko, T.; Schubert, J.; Marcsisin, E.; Mazur, A.; Kingston, E.; Zuser, E.; Papamarkakis, K.; et al. Applications of infrared and Raman microspectroscopy of cells and tissue in medical diagnostics: Present status and future promises. Spectroscopy 2012, 27, 463–496.
[15]
Holman, H.-Y.N.; Martin, M.C.; Blakely, E.A.; Bjornstad, K.; McKinney, W.R. IR spectroscopic characteristics of cell cycle and cell death probed by synchrotron radiation based Fourier transform IR spectromicroscopy. Biopolymers 2000, 57, 329–335.
Selci, S.; Bertani, F.R.; Ferrari, L. Supercontinuum ultra wide range confocal microscope for reflectance spectroscopy of living matter and material science surfaces. AIP Adv. 2011, 1, doi:10.1063/1.3631661.
[19]
Born, M.; Wolf, E. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light; Cambridge University Press: Cambridge, UK, 1999.
[20]
Carr, G.L. Resolution limits for infrared microspectroscopy explored with synchrotron radiation. Rev. Sci. Instrum. 2001, 72, 1613–1619.
[21]
Fujiyoshi, S.; Fujiwara, M.; Kim, C.; Matsushita, M.; van Oijen, A.M.; Schmidt, J. Single-component reflecting objective for low-temperature spectroscopy in the entire visible region. Appl. Phys. Lett. 2007, 91, 051125:1–051125:3.
[22]
Mattson, E.C.; Miriam, U.; Binod, M.; Zahrasadat, A.; Carol, J.H. Multi-beam synchrotron FTIR chemical imaging: Impacts of Schwarzschild objective and spatial oversampling on spatial resolution. J. Phys. 2013, 425, doi:10.1088/1742-6596/425/14/142001.
[23]
Mattson, E.C.; Nasse, M.J.; Rak, M.; Gough, K.M.; Hirschmugl, C.J. Restoration and spectral recovery of mid-infrared chemical images. Anal.Chem. 2012, 84, 6173–6180.
[24]
Sheppard, C.J.R.; Wilson, T. Image formation in confocal scanning microscopes. Optik 1980, 4, 331–342.
[25]
Pawley, J.B. Handbook of Biological Confocal Microscopy; SpringerScience+Business Media, LLC: New York, NY, USA, 2006.
[26]
Palik, E.D. Handbook of Optical Constants of Solids; Academic Press: San Diego, CA, USA, 1998.
[27]
Itzkan, I.; Qiu, L.; Fang, H.; Zaman, M.M.; Vitkin, E.; Ghiran, I.C.; Salahuddin, S.; Modell, M.; Andersson, C.; Kimerer, L.M.; et al. Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels. Proc. Natl. Acad. Sci. USA 2007, 104, 17255–17260.
[28]
Amigo, J.M.; Cruz, J.; Bautista, M.; Maspoch, S.; Coello, J.; Blanco, M. Study of pharmaceutical samples by NIR chemical-image and multivariate analysis. TrAC Trends Anal. Chem. 2008, 27, 696–713.
[29]
Kong, W.; Zhang, C.; Liu, F.; Nie, P.; He, Y. Rice seed cultivar identification using near-infrared hyperspectral imaging and multivariate data analysis. Sensors 2013, 13, 8916–8927.
[30]
Zhang, X.; Tauler, R. Application of multivariate curve resolution alternating least squares (MCR-ALS) to remote sensing hyperspectral imaging. Anal. Chim. Acta 2013, 762, 25–38.
[31]
Salman, A.; Shufan, E.; Zeiri, L.; Huleihel, M. Detection and identification of cancerous murine fibroblasts, transformed by murine sarcoma virus in culture, using Raman spectroscopy and advanced statistical methods. BBA-Gen. Subjects 2013, 1830, 2720–2727.
