The accuracy, reliability, speed and cost of the methods used for malaria diagnosis are key to the diseases’ treatment and eventual eradication. However, improvement in any one of these requirements can lead to deterioration of the rest due to their interdependence. We propose an optical method that provides fast detection of malaria-infected red blood cells (RBCs) at a lower cost. The method is based on the combination of deconvolution, topography and three-dimensional (3D) refractive index reconstruction of the malaria-infected RBCs by use of the transport of intensity equation. Using our method, healthy RBCs were identified by their biconcave shape, quasi-uniform spatial distribution of their refractive indices and quasi-uniform concentration of hemoglobin. The values of these optical and biochemical parameters were found to be in agreement with the values reported in the literature. Results for the malaria-infected RBCs were significantly different from those of the healthy RBCs. The topography of the cells and their optical and biochemical parameters enabled identification of their stages of infection. This work introduces a significant method of analyzing malaria-infected RBCs at a lower cost and without the use of fluorescent labels for the parasites.
Refractive index retrieval is possible using the transport intensity equation (TIE), which presents advantages over interferometric techniques. The TIE method is valid only for paraxial ray assumptions. However, diffraction can nullify these TIE model assumptions. Therefore, the refractive index is problematic for reconstruction in three-dimensions (3D) using a set of defocused images, as diffraction effects become prominent. We propose a method to recover the 3D refractive index by combining TIE and deconvolution. A brightfield (BF) microscope was then constructed to apply the proposed technique. A microsphere was used as a sample with well-known properties. The deconvolution of the BF-images of the sample using the microscope’s 3D point spread function led to significantly reduced diffraction effects. TIE was then applied for each set of three images. Applying TIE without taking into account diffraction failed to reconstruct the 3D refractive index. Taking diffraction into account, the refractive index of the sample was clearly recovered, and the sectioning effect of the microsphere was highlighted, leading to a determination of its size. This work is of great significance in improving the 3D reconstruction of the refractive index using the TIE method.
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 conven
Three-dimensional reconstruction in brightfield microscopy is challenging since a 2D image includes from in-focus and out-of-focus light which removes the details of the specimen’s structures. To overcome this problem, many techniques exist, but these generally require an appropriate model of Point Spread Function (PSF). Here, we propose a new images restoration method based on the application of Multivariate Curve Resolution (MCR) algorithms to a stack of brightfield microscopy images to achieve 3D reconstruction without the need for PSF. The method is based on a statistical reconstruction approach using a self-modelling mixture analysis. The MCR-ALS (ALS for Alternating Least Square) algorithm under non-negativity constraints, Wiener, Richardson–Lucy, and blind deconvolution algorithms were applied to silica microbeads and red blood cells images. The MCR analysis produces restored images that show informative structures which are not noticeable in the initial images, and this demonstrates its capability for the multiplane reconstruction of the amplitude of 3D objects. In comparison with 3D deconvolution methods based on a set of No Reference Images Quality Metrics (NR-IQMs) that are Standard Deviation, ENTROPY Average Gradient, and Auto Correlation, our method presents better values of these metrics, showing that it can be used as an alternative to 3D deconvolution methods.
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