Abstract
Multispectral microscopy enables information enhancement in the study of specimens because of the large spectral band used in this technique. A low cost multimode multispectral microscope using a camera and a set of quasi-monochromatic Light Emitting Diodes (LEDs) ranging from ultraviolet to near-infrared wavelengths as illumination sources was constructed. But the use of a large spectral band provided by non-monochromatic sources induces variation of focal plan of the imager due to chromatic aberration which rises up the diffraction effects and blurs the images causing shadow around them. It results in discrepancies between standard spectra and extracted spectra with microscope. So we need to calibrate that instrument to be a standard one. We proceed with two types of images comparison to choose the reference wavelength for image acquisition where diffraction effect is more reduced. At each wavelength chosen as a reference, one image is well contrasted. First, we compare the thirteen well contrasted images to identify that presenting more reduced shadow. In second time, we determine the mean of the shadow size over the images from each set. The correction of the discrepancies required measurements on filters using a standard spectrometer and the microscope in transmission mode and reflection mode. To evaluate the capacity of our device to transmit information in frequency domain, its modulation transfer function is evaluated. Multivariate analysis is used to test its capacity to recognize properties of well-known sample. The wavelength 700 nm was chosen to be the reference for the image acquisition, because at this wavelength the images are well contrasted. The measurement made on the filters suggested correction coefficients in transmission mode and reflection mode. The experimental instrument recognized the microsphere’s properties and led to the extraction of the standard transmittance and reflectance spectra. Therefore, this microscope is used as a conventional instrument.
Highlights
Multispectral microscopy establishes a link between spatial and spectral information resulting from light-matter interaction
A low cost multimode multispectral microscope with Cassgrain objective using a camera and a set of monochromatic Light Emitting Diodes (LEDs) as illumination sources was constructed and we present a new approach to calibrate that instrument to be a standard one
Before running the automated acquisition, we choose a wavelength of illumination called reference wavelength
Summary
Multispectral microscopy establishes a link between spatial and spectral information resulting from light-matter interaction. It can be described as spectroscopy and microscopy coupler. This technique is well established in remote sensing and satellite imagery areas. Several works illustrate the importance of multispectral microscopy. Zoueu et al [1] have shown that using multispectral imaging allows detecting malaria without using fluorescent labels for parasites as it is the case of thick and thin blood smears. Multispectral imaging is an important tool in the delimitation of reserved forests [2] and in agriculture framework for rapid detection of fruit diseases [3] [4]
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