# Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/4Pi Microscope

A 4Pi Microscope is a laser scanning fluorescence microscope with an improved optical axis|axial resolution. The typical value of 500-700 nm can be improved to 100-150 nm which corresponds to an almost spherical focal spot with 5-7 times less volume than that of standard confocal microscopy.

## Working principle

Scheme of a 4Pi microscope

.

The improvement in resolution is achieved by using two opposing objective lenses which both are focused to the same geometrical location. Also the difference in optical paths lengths through each of the two objective lenses is carefully aligned to be minimal. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and also the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The solid angle $\Omega$ that is used for illumination and detection is increased and approaches the ideal case. In this case the sample is illuminated and detected from all sides simultaneously.

The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a beam splitter (BS) and directed by mirrors towards the two opposing objective lenses. At the common focal point superposition of both focused light beams occurs. Excited molecules at this position emit fluorescence light which is collected by both objective lenses, combined by the same beam splitter and deflected by a dichroic mirror (DM) onto a detector. There superposition of both emitted light pathways can take place again.

In the ideal case each objective lens can collect light from a solid angle of $\Omega=2\pi$. So, with two objective lenses one can collect from every direction (solid angle $\Omega=4\pi$). The name of this type of microscopy is derived from the maximal possible solid angle for excitation and detection. Practically one can achieve only aperture angles of about 140° for an objective lens, which corresponds to $\Omega$ about $1.3\pi$.

It can be operated in three different ways: In a 4Pi microscope of type A, the coherent superposition of excitation light is used to generate the increased resolution. The emission light is either detected from one side only or in an incoherent superposition from both sides. In a 4Pi microscope of type B, only the emission light is interfering. When operated in the type C mode, both excitation and emission light are allowed to interfere leading to the highest possible resolution increase (~7 fold along the optic axis as compared to confocal microscopy).

In a real 4Pi microscope light is not applied from all sides, leading to so-called side lobes in the point spread function. Typically (but not inevitably) two-photon excitation microscopy is used in a 4Pi microscope in combination with an emission pinhole to lower these side lobes to a tolerable level.

## History

In 1971, Christoph Cremer and Thomas Cremer proposed the creation of a perfect hologram, i.e. that carries the whole field information of the emission of a point source in all directions, a so-called $4\pi$ hologram.[1]

The first description of a practicable system of 4Pi microscopy, i.e. the setup with two opposing, interfering lenses, was invented by Stefan Hell in 1991[2]. He could demonstrate it experimentally also in 1994[3].

In the following years the number of applications for this microscope have grown. Parallel excitation and detection with 64 spots in the sample simultaneously combined with the improved spatial resolution resulted in the successful recording of the dynamics of mitochondria in yeast cells with a 4Pi microscope in 2002[4].

A commercial version was launched by microscope manufacturer Leica Microsystems in 2004[5].

Up to now, the best quality in a 4Pi microscope was reached in conjunction with the STED principle[6]. Using a 4Pi microscope with appropriate excitation and de-excitation beams it was possible to create a uniformly 50 nm sized spot which corresponds to a decreased focal volume compared to confocal microscopy by a factor of 150-200.

## References

1. Considerations on a laser-scanning-microscope with high resolution and depth of field: C. Cremer and T. Cremer in M1CROSCOPICA ACTA VOL. 81 NUMBER 1 September, pp. 31—44 (1978)
Basic design of a confocal laser scanning fluorescence microscope & principle of a confocal laser scanning 4Pi fluorescence microscope, 1978
2. European patent EP 0491289
3. S.W. Hell, E.H.K. Stelzer, S. Lindek, C. Cremer (1994). "Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy". Optics Letters 19: 222-224. doi:10.1364/OL.19.000222.
4. A. Egner, S. Jakobs, S.W. Hell (2002). "Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast". PNAS 99: 3370-3375. doi:10.1073/pnas.052545099.
5. Review article 4Pi microscopy
6. R. Schmidt, C.A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, S.W. Hell (2008). "Spherical nanosized focal spot unravels the interior of cells". Nature Methods 5: 539-544. doi:10.1038/nmeth.1214.