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 image r 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 evalua te 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 experimen tal 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.
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. It is now extended to the microscopy area because of its various advantages.
Several works illustrate the importance of multispectral microscopy. Zoueu et al. [
Several multispectral imaging technologies, in spite of their high cost, are used in the sequential or parallel acquisition of images of the same scene using different wavelengths and generate an optical spectrum associated to any pixel in the image [
In such a system, the spectral information reconstruction requires images of acceptable quality (sharp and of the same scale). But, using a large spectral band induces the variation of focal plan position of the imager due to the phenomenon of chromatic aberration which affects the quality of the images. This is a major problem met in multispectral imaging. To reduce distortion due to chromatic aberration, reflex objective can be used such as Cassgrain objective [
In the present work, 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. By calibration, we mean:
- the determination of the wavelength used to fix its focal plan where the phenomenon of chromatic aberration is more reduced,
- the correction of the extracted spectra from the images making them in agreement with the standards spectra,
- the determination of modulation transfer function which is an important tool in an imager’s characterization,
- the evaluation of its capacity to recognize the specimen’s properties.
For this purpose, we used it to make measurements on samples with well-known properties such as homogeneous filters and organic polymer’s microspheres (latex) with diameter
improved version of the LED multispectral microscope built by Brydegaard et al. [
The light sources S1, S2 and S3 are controlled via a data acquisition (DAQ) connected to a computer (PC). All are controlledby a LabView (Lab View 8.6, licence no. M 74 × 11278) operation code. Each light source is a set of quasi-monochromatic Light Emitting Diodes (LEDs) with the following spectral band: 375 nm, 400 nm, 435 nm, 470 nm, 525 nm, 590 nm, 625 nm, 660 nm, 700 nm, 750 nm, 810 nm, 850 nm, 940 nm. Before any measurement, we need to align all the optics. All The measurements are done in the darkness to minimize back ground light.
Measurements are made on two types of samples:
-Filters, used as homogeneous samples whose standard spectra are extracted from the spectrometer (Compact Spectra Suite Ocean Optics USB4000).
-Organic polymer’s microspheres (latex) with diameter
The microspheres’ samples are prepared on slides made of the highest purity, corrosion-resistant glass with dimensions 75 mm × 25 mm × 1 mm. The measure in each mode (transmission, reflection or scattering) needs three types of images acquired in the same acquisition conditions:
-The image
-The image
-The image
To make the measurement, we first select a wavelength of illumination called reference wavelength. One illuminates the sample at this wavelength and we make sure that the image is well contrasted before running the measurement. The measurement is an automated acquisition of thirteen images from the same scene and each image corresponds to a wavelength of illumination. The wavelengths selection as reference is part of the goals of this study.
In the transmission mode, we active the geometry “brightfield” as indicated on the interface presented by Lab view operation code. The signal
The corrected image
In the measurement of the reflectance, the alignement of optics in the microscope and fixation of its focal plan at the reference wavelength being done during the measures in transmission mode, we active the geometry ”reflection” inthe Lab view code interface in order to run the automated acquisition of the thirteen images.The signal
In scattering mode, the geometry “darkfield” is activated to run the automated acquisition of the thirteen images. The signal
The measure on a specimen in a mode consists to run an automated acquisition of a set of thirteen images from the same scene with each image corresponding to one wavelength of illumination. But before running the automated acquisition, we choose a wavelength of illumination called reference wavelength. We illuminate the sample at this wavelength and we appreciate the contrast of the image before doing the measurement. The contrast of the image reveals that the image is in the focal plan (of the multi spectral microscope) corresponding to that reference wavelength. But, the focal plan of an optical system depends on the wavelength of illumination (light) which passes through the system. Since the acquisition is made on a set of the thirteen quasi-monochromatic sources, then the other twelve images are not in focal plan defined by the chosen reference wavelength. This constitutes a difficulty metin multi spectral imaging system using a large spectral band. The variation of focal plan position is due to the phenomenon of chromatic aberration depending on the refractive index variation of the optical system in function of the wavelength for the light passing through it. This phenomenon rises up the diffraction effects and blurs the images inducing shadow around them. To get the best reference wavelength for all the three modes, we used each of the thirteen wavelengths as a reference wavelength followed by the acquisition of the set of the thirteen images in transmission mode. Then, we proceed with two types of images comparison. Each wavelength chosen as a reference defines a focal plan. In each set of the thirteen images acquired after the choice of the reference, there’s only one image which is well contrasted. Indeed, this image is acquired with light source used to fix the focal plan. Then, we have thirteen contrasted images, each one corresponding to a reference wavelength. First, we compare these thirteen contrasted images to appreciate that presenting more reduced shadow. This comparison highlights that the reference wavelengths 590 nm, 625 nm, 660 nm and 700 nm are the ones whose corresponding images present more reduced shadow spreading on 7 pixels, 8,06 pixels, 7,07 pixels and 7 pixels respectively (
The first comparison shows that fixing the focal plan with the reference 590 nm or 700 nm, the microsphere’s image observed at the wavelength 590 nm or 700 nm respectively, present more reduced shadow (
The second comparison reveals that only with the reference 700 nm, the microsphere’s images acquired using the set of the thirteen wavelengths present in average more reduced shadow (
comparison allow to select 700 nm as the best reference wavelength to fix the focal plan of the multi spectral microscope.
The images acquired from different filters (filter 1, filter 2 and filter 3) with the microscopeled to the extraction of their spectra in transmission and reflection modes.
with the spectra obtained from the same filters with the spectrometer Compact Spectra Suite. We can see in
The thirteen wavelengths ranging from 375 nm to 940 nm give thirteen spectral channels (p).
The transfer function
The Equation (4) does not take into account the imperfection of the optics in the multi spectral device. That induces discrepancy between the constructed spectra and the standard spectra. Chromatic aberration, eventual fluorescence of the specimen and camera shot noise also induce this discrepancy [
We can see easilythe contribution for the LED’s non-monochromaticity to the discrepancy.
From spectra presented in
Filters | Reflectance extracted from multi spectral device | Reflectance extracted from standard spectrometer |
---|---|---|
1 and 3 | Lower than 1%, 5% | Quasi-constant close to 50% |
2 | Included between 1%, 5% and 10% | Included between 45% and 53% |
3 | Higher than 10% | Included between 55% and 85% |
The modulation transfer function (FTM) is an important tool allowing to quantify an image quality. It expresses the imager’s capacity to transmit information from a scene in spatial frequency domain and determines its resolution. In this work, the FTM of our multi spectral imager in transmission mode is evaluated through the edge method [
- acquisition of an image of a tilted edge,
- determination of the edge spread function ESF,
- differentiation of the ESF to obtain the line spread function LSF,
- calculation of the Fourier Transform of the LSF to obtain the FTM.
For each illumination source, we evaluated the modulation transfer function following the lines and columns of the imager (Camera pixel size: 2.2 mm × 2.2 mm) called respectively horizontal FTM and vertical FTM (
The decrement of FTM for high spatial frequencies shows that our device behaves like a low-pass filter and transmits more details in low spatial frequency band. This decrement depends on the illumination wavelength used. Using the wavelength 375 nm, the decrement of the corresponding FTM (in low spatial frequency band) is the most quick but, as we use the wavelength 700 nm, the decrement of the corresponding FTM is less quick. This confirms that images acquired with 700 nm as illumination wavelength are more contrasted whereas the images acquired with 375 nm contain less detail.
After the determination of correction coefficients, the multi spectral microscope is used to make measurements on organic polymer’s microspheres of diameter
If a sample of three objects of colors red, green and blue is illuminated using red-light, only the red object will be observed. If it is illuminated using green light, only the green object will be seen. If it’s illuminated by blue light, only the blue object will be observed. So, each wavelength reveals specific information about the specimen.
We apply K-means to an image by dividing its intensity dispatching in five clusters. This method puts the different parts of the microsphere into different clusters according to their similar spectra. We obtained the configuration in
That is due to the microsphere’s sphericity. Figures 8(a)-(b) present standardized spectra of a microsphere.
A low cost multi mode multi spectral microscope using a camera and a set of monochromatic Light Emitting Diodes (LEDs) ranging from 375 nm to 940 nm as illumination sources was built. The microscope allows the automated acquisition of thirteen images from the same scene of a sample illuminated by each source, and the extraction of spectra corresponding to its three modes transmission, reflection and scattering. The goal of this work is to characterize it and valid its use. So, in transmission mode we determined its modulation transfer function which is an important characteristic tool of an imager. The wavelength 700 nm was chosen to be the reference for the image acquisition. At this wavelength, the acquired images are well contrasted and diffraction effects and chromatic aberration are reduced. Measurements made with filters suggested correction coefficients in transmission mode and reflection mode. By applying PCA to the microsphere’s images, the multi spectral device
has confirmed its homogeneity. Through K-means method, the experimental instrument recognized the sphericity of the microsphere. This led to the extraction of the standard transmittance and reflectance spectra. Therefore, this microscope is used as a routine instrument.
Marcel A. Agnero,Jérémie T. Zoueu,Kouakou Konan, (2016) Characterization of a Multimodal and Multispectral Led Imager: Application to Organic Polymer’s Microspheres with Diameter Φ = 10.2 μm. Optics and Photonics Journal,06,171-183. doi: 10.4236/opj.2016.67019