... provides 3D-resolution in the 100 nm range
Ernst Abbe discovered that the focal spot size decreases with the microscope's aperture angle i.e. with the size of the spherical wavefront that is produced by the objective lens. But a regular objective lens, even of the largest aperture, produces just a segment of a spherical wavefront coming from a single direction. As a result the focal spot is longer (z) than wide (x,y) [Fig. 1a]. By contrast, a full spherical wavefront of a solid angle of 4π would lead to a spherical spot and hence to an improvement of spatial resolution in the z-direction.
The idea: Since there are no lenses or mirrors that could provide such a wavefront across a significantly large field of view, the idea behind our 4Pi-microscope [Fig. 2] is to mimic the 'close to ideal' situation by using two opposing objective lenses coherently, so that the two wavefronts add up and join forces [Fig. 1b] [4, 12]. The sketch in Fig. 2 gives an idea about the optical setup - although modern versions are more sophisticated. Allowing the illumination wavefronts to constructively interfere in the sample produces a main focal spot that is sharper in the z-direction by about 3-4 times (4Pi of type A). A similar improvement is obtained if the lenses add their collected fluorescence wavefronts in a common point detector (4Pi of type B). Doing both together is best, of course, and leads to a 5-7-fold improvement of resolution along z (4Pi of type C) [13, 14].
The sidelobe challenge: If the two segments were full spherical halves, the focal spot would be a (nearly) spherical spot, too. But since a considerable solid angle is not provided by the lenses, interference typically spawns off 2 axial side-lobes which, if not taken into account, lead to artefactual images. We deal with this challenge by an appropriate mathematical filter [Fig. 1c] . This filter does not require any information about the object, apart from the height and location of the lobes. Linear filtering is possible if the lobes are significantly less than 50% of the main sharp maximum. This can be reliably fulfilled if multiphoton excitation of the dye is applied [4, 15]. Linear mathematical filtering is fast and a single effective spot is readily achieved [Fig. 1c].
Effective resolution: The resolution is largely given by the extent of the effective 4Pi-spot, which is 3-5 times sharper than the spot of a regular confocal microscope. The variation 3-5 depends on the type of implementation (Type A, B, or C). The improvement is readily observed from the xz-images Fig. 3a and Fig. 3b .
Restoration: By further combining 4Pi-microscopy with non-linear image restoration, a 3D-resolution of ~100 nm is reliably achieved. The combination of 4Pi-microscopy and non-linear image restoration leads to dramatically improved 3D-images [Fig. 3c] [15, 17, 18].
Imaging speed: 4Pi-microscopy has recently been implemented as a fast CCD-based, beam scanning, multifocal multiphoton microscope (MMM) , so that image acquisition time was cut down to about 1 second/ slice. In addition the method was refined for later immersion. As a result, this microscopy technique delivered for the first time 3D-images of live cells in the 100 nm range .
Applications The animated graphic shows a surface reconstructed 3D-image of the GFP-tagged mitochondrial matrix of a live budding yeast cell. Live cell 4Pi-imaging allowed us to study the influence of selected mitochondrial proteins on mitochondrial morphology .
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6 Egner, A., S. Jakobs and S. W. Hell (2002). "Fast 100-nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast." Proc. Natl. Acad. Sci. USA 99: 3370-3375.
12 Hell, S. Europäisches Patent EP 0 491 281 B1 (18.12.1990) "Doppelkonfokales Rastermikroskop".
13 Hell, S. W. and E. H. K. Stelzer (1992). "Properties of a 4Pi-confocal fluorescence microscope." J. Opt. Soc. Am. A 9: 2159-2166.
14 Hell, S. W., S. Lindek, C. Cremer and E. H. K. Stelzer (1994). "Measurement of the 4Pi-confocal point spread function proves 75 nm resolution." Appl. Phys. Lett. 64(11): 1335-1338.
15 Nagorni, M. and S. W. Hell (2001). "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts." J. Opt. Soc. Am. A 18(1): 36-48.
16 Nagorni, M. and S. W. Hell (1998). "4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution." J. Struct. Biol. 123: 236-247.
17 Hell, S. W., M. Schrader and H. T. M. van der Voort (1997). "Far-field fluorescence microscopy with resolution in the 100 nm range." J. Microsc. 187(1): 1-7.
18 Nagorni, M. and S. W. Hell (2001). "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. II. Power and limitation of nonlinear image restoration." J. Opt. Soc. Am. A 18(1): 49-54.
19 Bewersdorf, J., R. Pick and S. W. Hell (1998). "Multifocal Multiphoton Microscopy." Opt. Lett. 23(9): 655-657.