Optical metrology with wide field of view for 3D mapping of tissues

Traditional optical metrology techniques face significant challenges when applied to biological specimens, largely due to the specimens' inherent transparency. This often results in inadequate imaging and compromised measurement accuracy. To circumvent these challenges, confocal microscopy has become the preferred method for metrological analysis of such samples. However, it is important to note that confocal microscopy is fundamentally a point -based technique, which inherently limits its field of view (FOV). In this paper, we explore the application of a depth-from-focus method for the 3D mapping of biological tissues in a wide FOV. Although this technique is well-established for reflective and absorbent samples, its use has been limited with transparent, and specifically biological, samples. The lack of critical surface texture in transparent samples hinders the acquisition of the necessary imaging detail when using traditional transillumination or epi-illumination systems. Additionally, conventional high-resolution depth-from-focus systems typically employ microscope objectives that yield a narrow depth of field however limiting the FOV due to high magnification. We address these challenges by introducing two key innovations. First, we implement a ring LED illumination system to enhance the clarity of the sample surface image. Second, we substitute the microscope objective with a photographic lens paired with a larger sensor to expand the FOV. These adaptations, along with a developed focus estimation algorithm, enable high-resolution 3D mapping of biological tissues, overcoming the limitations of existing methods.