| Multifocal
Multiphoton Microscopy... |
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time-multiplexing leads to superior 3D-imaging. |
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| 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. |
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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. |
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  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. |
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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. |
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The
setup:
In a multifocal multiphoton microscope [19] 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.