Digital holographic microscopy and focusing methods based on image sharpness.

Digital holographic microscope allows imaging of opaque and transparent specimens without staining. A digitally recorded hologram must be reconstructed numerically at the actual depth of the object to obtain a focused image. We have developed a high-resolution digital holographic microscope for imaging amplitude and phase objects with autofocusing capability. If the actual depth of an object is not known a priori, it is estimated by comparing the sharpness of several reconstructions at different distances, which is very demanding in means of computational power when the recorded hologram is large. In this paper, we present 11 different sharpness metrics for estimating the actual focus depths of objects. The speed performance of focusing is discussed, and a scaling technique is introduced where the speed of autofocusing increases on the order of square of the scale ratio. We measured the performance of scaling on computer-generated holograms and on recorded holograms of a biological sample. We show that simulations are in good agreement with the experimental results.

Real-time, auto-focusing digital holographic microscope using graphics processors.

The most significant advantage of holographic imaging is that one does not need to do focusing alignment for the scene or objects while capturing their images. To focus on a particular object recorded in a digital hologram, a post-processing on the recorded image must be performed. This post-processing, so called the reconstruction, is essentially the calculation of wave propagation in free space. If the object's optical distance to the recording plane is not known a priori, focusing methods are used to estimate this distance. However, these operations can be quite time consuming as the hologram sizes increase. When there is a time constraint on these procedures and the image resolution is high, traditional central processing units (CPUs) can no longer satisfy the desired reconstruction speeds. Then, especially for real-time operations, additional hardware accelerators are required for reconstructing high resolution holograms. To this extend, today's commercial graphic cards offer a viable solution, as the holograms can be reconstructed tens of times faster with a graphics processing unit than with the state-of-the-art CPUs. Here we present an auto-focusing megapixel-resolution digital holographic microscope (DHM) that uses a graphics processing unit (GPU) as the calculation engine. The computational power of the GPU allows the DHM to work in real-time such that the reconstruction distance is estimated unsupervised, and the post-processing of the holograms are made completely transparent to the user. We compare DHM with GPU and CPU and present experimental results showing a maximum of 70 focused reconstructions per second (frps) with 1024 × 1024 pixel holograms.