Dogan, Hulya; Dogan, Ramazan Ozgur; Ay, Ilyas; Sezen, Sena F.

Depth of focus (DOF) is defined as the axial range in which the specimen stage moves without losing focus while the imaging apparatus remains stable. It may not be possible to capture an image that includes the entire specimen in focus due to the narrow DOF in microscopic systems. Extended depth of focus (EDOF) is used to overcome this limitation in microscopic systems. Although the researchers have developed so many EDOF microscope approaches, this research field still has some crucial shortcomings such as high computational costs, complexity and execution time, requiring additional equipment, low precise characterization of curves, and edges in images, varying performance depending on the specimen and microscope, using only gray levels of input images to acquire the pixel’s focus values. In order to minimize these shortcomings and comprehensively analyze the performance of EDOF approaches, a novel multi-focus image data set is generated, and a deep learning-based EDOF microscope approach is proposed in this study. When compared with the state-of-art EDOF approaches, our study provides various crucial contributions such as the first EDOF approach based on unsupervised deep learning, providing more accurate and specimen-free EDOF, generating a novel multi-focus image data, not requiring any pre- or post-processing technique and acquiring the pixel’s focus degrees using deep features. In order to evaluate the effectiveness of the suggested approach, 20 different EDOF approaches are applied to a multi-focus image data set containing 9 image collections (4 synthetic and 5 microscope image collections) in total. Performance analysis metrics with and without requiring a reference image are preferred to identify which EDOF microscope approach can extract more essential details from the multi-focus images for the synthetic and microscope image collections, which are Root Mean Square Error (RMSE), Peak Signal Noise Ratio (PSNR), Universal Quality Index (UQI), Correlation Coefficient (CC), Perception-based Image Quality Evaluator (PIQE), Blind/Referenceless Image Spatial Quality Evaluator (BRISQUE), Extension of Universal Quality Index for N Images (UQIN), and Naturalness Image Quality Evaluator (NIQE). Objective and subjective analysis of this study demonstrates that unsupervised deep learning model is more efficient to transmit crucial details from multi-focus images. Moreover, the suggested EDOF microscope approach with highest PSNR, UQI, CC, UQIN and lowest RMSE, PIQE, BRISQUE, NIQE produces higher performance than the state-of-art approaches.

Dogu, Fatma Tuana; Dogan, Hulya; Dogan, Ramazan Ozgur; Ay, Ilyas; Sezen, Sena F.; Seyman, M.

During microscopic examination process, experts firstly determine the most appropriate focusing point by precisely moving the microscope stage in the Z axis. This manual focusing process may cause variability in results depending on hand-eye coordination and individual experience. Therefore, the development of automatic focusing offers superior quality and consistent imaging. Many researchers have studied automatic focusing systems and proposed many focus functions to extract focus information from multi-focus images. Nevertheless, these focusing systems still consist of several significant limitations such as requiring a supplementary material, longer running time, producing different performance depending on the sample and magnification objective. In this study, an efficient technique based on deep learning is proposed to automatically determine the focused image during microscopic examination processes to minimize these limitations in the literature. This technique takes a sequence of images with the same field of view and different focuses as input and gives the image with maximum focusing as output. The proposed technique is compared with other automatic focusing techniques on the literature. In this study, novel multi-focus image sequences obtained from liver, intestine, heart, kidney, and lung samples is prepared to evaluate the performance of automatic focusing techniques. These sequences are obtained by scanning with 10× and 40× magnification objectives. Running time, accuracy, number of local maximum points, range, and noise levels are used as evaluation criteria in this study. According to evaluation criteria results obtained in this study, it has been proven that the proposed technique provides better performance than other automatic focusing techniques.