The ingenious technique of 4Pi microscope employs juxtaposed dual objectives to produce excitation light at a common focal plane. The resulting constructive and destructive interference reduces the possible axial resolution to approximately 100 nanometers (from the typical 400 to 700 nanometers observed in confocal microscopy). Furthermore, the point-spread function of a 4Pi microscope is almost 1.5-fold sharper in the lateral dimension and 7-fold sharper in the axial direction compared to a confocal instrument. Images are scanned pixel-by-pixel and can be used with multiphoton excitation coupled to detection with high-performance CCD camera systems.
Hell, S. W. and Nagorni, M.
4Pi confocal microscopy with alternate interference. Optics Letters 23: 1567-1569 (1998). One of the original papers on 4Pi confocal microscopy, which describes destructive interference of the converging excitation wavefronts. The authors discuss linear lobe deconvolution and nonlinear image restoration of raw data, and demonstrate imaging of microtubules with an axial resolution approaching 100 nanometers.
Hell, S. W., Schader, M. and Van der Voort, H. T. M.
Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range. Journal of Microscopy 187: 1-7 (1997). Introduction of the 4Pi microscopy concept to conduct three-dimensional microscopy with a nearly isotropic resolution (in effect, equal lateral and axial resolutions) that reduces the point-spread function size to 10 to 20 percent of the diffraction-limited size.
Hell, S. W. and Stelzer, E. H. K.
Properties of a 4Pi confocal fluorescence microscope. Journal of the Optical Society of America A9: 2159-2166 (1992). The authors discuss theoretical and practical aspects of point-spread function engineering by 4Pi confocal microscopy. In addition, a functional instrument was demonstrated to produce an axial resolution of approximately 110 nanometers.
Schrader, M., Hell, S. W. and van der Voort, H. T. M.
Three-dimensional super-resolution with a 4pi-confocal microscope using image restoration. Journal of Applied Physics 84: 4033-4042 (1998). A research report describing the combination of two-photon excitation in 4Pi fluorescence microscopy with post-acquisition image restoration to yield a fundamental improvement of three-dimensional resolution in transparent, fluorescent specimens.
Lang, M. C., Staudt, T., Engelhardt, J. and Hell, S. W.
4Pi microscopy with negligible sidelobes. New Journal of Physics 10: 043041-13(2008). In 4Pi microscopy, the coherent addition of wavefronts from opposing objectives requires mathematical deconvolution to remove sidelobes. The authors demonstrate a technique to decrease the effective point-spread function size with negligible lobes and a resulting gain in axial resolution.
Bahlmann, K., Jakobs, S. and Hell, S. W.
4Pi-confocal microscopy of live cells. Ultramicroscopy 87: 155-164 (2001). The first demonstration of 4Pi confocal microscopy to image living cells. Water immersion objectives were employed to examine the membrane of live E. coli cells to produce 4.3-fold better axial resolution when compared to the highest resolution obtained with confocal microscopy.
Hell, S. W. and Stelzer, E. H. K.
Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation. Optics Communications 93: 277-282 (1992). A theoretical paper that suggests a dramatic increase in resolution using multiphoton excitation coupled to opposed objectives. The authors claim (correctly at the time) that the proposed microscope yields the highest resolution ever achieved in far-field microscopy.
Egner, A., Verrier, S., Goroshkov, A., Soling, H. and Hell, S. W.
4Pi-microscopy of the Golgi apparatus in live mammalian cells. Journal of Structural Biology 147: 70-76 (2004). A detailed study of live-cell imaging using 4Pi confocal fluorescence microscopy. The authors demonstrate superresolution three-dimensional imaging of the Golgi apparatus in a living cell at approximately 100 nanometer resolution.
Egner, A., Jakobs, S. and Hell, S. W.
Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proceedings of the National Academy of Sciences (USA) 99: 3370-3375 (2002). Using a technique that is described as beam-scanning multifocal multiphoton 4Pi-confocal microscopy, the authors achieve fast fluorescence imaging of live cells with axial superresolution (approximately 100 nanometers).
Gugel, H., Bewersdorf, J., Jakobs, S., Engelhardt, J., Storz, R. and Hell, S. W.
Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. Biophysical Journal 87: 4146-4152 (2004). A description of "type C" 4Pi microscopy to achieve 7-fold sharper optical sections (compared to confocal microscopy) in live-cell imaging.