Multispectral and multimodal unstained blood smear images are obtained and evaluated to offer computer‐assisted automated diagnostic evidence for malaria. However, these images suffer from uneven lighting, contrast variability, and local luminosity due to the acquisition system. This limitation significantly impacts the diagnostic process and its overall outcomes. To overcome this limitation, it is crucial to perform normalization on the acquired multispectral images as a preprocessing step for malaria parasite detection. In this study, we propose a novel method for achieving this normalization, aiming to improve the accuracy and reliability of the diagnostic process. This method is based on estimating the Bright reference image, which captures the luminosity, and the contrast variability function from the background region of the image. This is achieved through two distinct resampling methodologies, namely Gaussian random field simulation by variogram analysis and Bootstrap resampling. A method for handling the intensity saturation issue of certain pixels is also proposed, which involves outlier imputation. Both of these proposed approaches for image normalization are demonstrated to outperform existing methods for multispectral and multimodal unstained blood smear images, as measured by the Structural Similarity Index Measure (SSIM), Mean Squared Error (MSE), Zero mean Sum of Absolute Differences (ZSAD), Peak Signal to Noise Ratio (PSNR), and Absolute Mean Brightness Error (AMBE). These methods not only improve the image contrast but also preserve its spectral footprint and natural appearance more accurately. The normalization technique employing Bootstrap resampling significantly reduces the acquisition time for multimodal and multispectral images by 66%. Moreover, the processing time for Bootstrap resampling is less than 4% of the processing time required for Gaussian random field simulation.
