Multiple-objective microscopy with three-dimensional resolution near 100 nm and a long working distance.

The resolution of microscopes is limited by the sizes of their point-spread functions. The invention of confocal, theta, and 4Pi microscopes has permitted the classic Abbe limit to be exceeded. We propose the use of a combination of 4Pi and theta microscopy to decrease resolution by using four illumination objectives and two detection objectives. Using middle numerical aperture, long-working-distance objectives yielded a resolution near 100 nm in the three dimensions, which opens the possibility of exploring large volumes with a high resolution.