Multifocal Multiphoton Microscopy (MMM)... ... time-multiplexing leads to superior 3D-imaging.
The growing need for visualizing fast cellular events has initiated the development of a number of real-time 3D-imaging modes, with the most common being those based on scanning confocal and multiphoton fluorescence microscopy. In these microscopes the acquisition time is readily reduced by increasing the scanning speed, however, this approach is not as light-efficient as the parallel application of multiple foci. Unfortunately, the use of multiple foci is challenged by the concomitant deterioration of the 3D-imaging capability of these microscopes. The problem is that interference between neighboring focal fields reinforce each other in specific planes located above and beneath the focal plane. This focal overlap is of particular concern in popular Nipkow disk scanning confocal microscopy whose sectioning ability is additionally compromised by cross-talk between detection pinholes. Hence, the conflict between the density of the foci, i.e. the degree of parallelization, and the axial sectioning has remained a classical problem in 3D-microscopy.
Parallelize without compromise: Exploiting specific properties of pulsed-mode multiphoton excitation, we and others have now succeeded in resolving this conflict [22, 23]. The basic idea is that the laser pulses of neighboring foci are temporally separated by at least one pulse duration, so that interference is avoided [24, 25]. We refer to this method as time-multiplexing (TMX). Moreover, with a high degree of time multiplicity, the interfocal distance can be reduced to such an extent that lateral scanning becomes obsolete. In this case axial scanning is sufficient to record a 3D-image.
The setup: In a multifocal multiphoton microscope  a mode-locked Ti:Sa laser beam (l= 800 nm) was split into 25-40 beamlets by means of a rotating array of microlenses arranged on a Nipkow-type spiral. The beamlets passed through the optics of an inverted microscope and were sharply focused in the sample. The fluorescence was imaged by the same optics onto a camera. For typical pulse duration of 130 fs, a delay of 250 fs is sufficient to ensure that the excitation fields of neighboring beamlets are separated in time. Such a delay is achieved by forcing a subset of the beamlets through 150 mm of glass. This was realized by two rigidly mounted co-rotating glass disks with suitable arrays of holes ensuring that adjacent foci illuminate the sample at different time points. The Figure shows the setup of a time-multiplexed multifocal multiphoton microscopy. The inset in (a) sketches a temporal delay mask placed behind the microlenses, ensuring that neighboring foci pass the focal plane in the sample at different time points. L1,2, M and DM denote lenses, a flip mirror and a dichroic mirror, respectively.
Pollen grains point up superior imaging: The gain in axial sectioning is demonstrated by comparing volume rendered 3D-images of fluorescent pollen grains of about 30 µm in diameter. The comparison clearly reveals that TMX eliminates cross-talk and leads to superior parallelized 3D-imaging.
Prospects of TMX: In summary, temporal separation of the pulses of adjacent foci in a multifocal multiphoton microscope resolves the conflict between parallelization and axial sectioning in 3D-fluorescence microscopy. This concept gains additional relevance because it is readily extended to other forms of non-linear microscopy, such as higher harmonics and CARS microscopy. Most strikingly, by matching the number of delays with the number of foci, time-multiplexing will allow one to reduce the interfocal distance to such an extent that in-plane scanning is redundant and 3D-imaging accomplished by moving the beams along the optic axis only.
22 Andresen, V., A. Egner and S. W. Hell (2001). "Time-multiplexed multifocal multiphoton microscope." Opt. Lett. 26(2): 75-77.
23 Fittinghoff, D.N. and J. A.Squier (2000)."Time-decorrelated multifocal array for multiphoton microscopy and micromachining." Opt. Lett. 25(16), 1213-1215.
24 Egner, A. and S. W. Hell (2000). "Time multiplexing and parallelization in multifocal multiphoton microscopy." J. Opt. Soc. Am. A 17(7): 1192-1201.
25 Buist, A.H., M. Müller, J. Squier, and G. J. Brakenhoff (1998). "Real time two-photon absorption microscopy using multipoint excitation." J. Microsc. 192(2), 217-219.
19 Bewersdorf, J., R. Pick and S. W. Hell (1998). "Multifocal Multiphoton Microscopy." Opt. Lett. 23(9): 655-657.