KEY WORDSfocal range; optical microscopy; digital image processing; image fusion; complex wavelet transform ABSTRACT Microscopy imaging often suffers from limited depth-of-field. However, the specimen can be "optically sectioned" by moving the object along the optical axis. Then different areas appear in focus in different images. Extended depth-of-field is a fusion algorithm that combines those images into one single sharp composite. One promising method is based on the wavelet transform. Here, we show how the wavelet-based image fusion technique can be improved and easily extended to multichannel data. First, we propose the use of complex-valued wavelet bases, which seem to outperform traditional real-valued wavelet transforms. Second, we introduce a way to apply this technique for multichannel images that suppresses artifacts and does not introduce false colors, an important requirement for multichannel optical microscopy imaging. We evaluate our method on simulated image stacks and give results relevant to biological imaging. Microsc. Res. Tech. 65:33-42, 2004. © 2004 Wiley-Liss, Inc.
INTRODUCTIONLimited depth-of-field is a common problem in biological imaging with conventional light microscopy. Often, the specimen's profile covers more than the attainable depth-of-field. Portions of the object's surface outside the optical plane appear defocused in the acquired image plane. This becomes worse as the magnification M increases because the numerical aperture NA increases, too: