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 permi tted the classic Abbe limit to be exceeded. We propose the use of a combina tion of 4Pi and theta microscopy to decrease resolution by using four illum ination objectives and two detection objectives. Using middle numerical ape rture, long-working-distance objectives yielded a resolution near 100 mn in the three dimensions, which opens the possibility of exploring large volum es with a high resolution. (C) 2001 Optical Society of America.