High-contrast multilayer imaging of biological organisms through dark-field digital refocusing

We have developed an imaging system to extract high contrast images from different layers of biological organisms. Utilizing a digital holographic approach, the system works without scanning through layers of the specimen. In dark-field illumination, scattered light has the main contribution in image formation, but in the case of coherent illumination, this creates a strong speckle noise that reduces the image quality. To remove this restriction, the specimen has been illuminated with various speckle-fields and a hologram has been recorded for each speckle-field. Each hologram has been analyzed separately and the corresponding intensity image has been reconstructed. The final image has been derived by averaging over the reconstructed images. A correlation approach has been utilized to determine the number of speckle-fields required to achieve a desired contrast and image quality. The reconstructed intensity images in different object layers are shown for different sea urchin larvae. Two multimedia files are attached to illustrate the process of digital focusing.