A Two-Photon Laser-Scanning Confocal Fluorescence Microscope

Steve M. Potter and Scott E. Fraser

Confocal intro

Confocal microscopy has revolutionized biologists’ ability to
observe microscopic structures within thick (tens or hundreds of microns) specimens. With
normal fluorescence microscopy, one cannot resolve deep structures within a specimen
because of light emitted and scattered by the out-of-focus tissue. By placing small
apertures in the light path at points confocal to the focal point within the specimen,
almost all of the out-of-focus fluorescence is blocked, allowing detection of just the
point of interest. By scanning the light source, a laser beam, across the tissue in the X
and Y directions, a high-resolution image of a thin slice of the tissue can be constructed
digitally within seconds. By making a series of such optical `slices’ through the
thickness (Z-direction) of the specimen, its three-dimensional representation can be
generated and manipulated with image-processing software.

Problems with confocal

Because we are interested in observing living specimens, often at
several stages during development, we run into some serious problems with normal (visible light) confocal
fluorescence microscopy. One of these is photobleaching of the fluorescent label
(chromophore). Because the small confocal aperture blocks most of the light emitted by
the tissue, including light coming from the plane of focus, the exciting laser must be
very bright to allow an adequate signal-to-noise ratio. This bright light causes
fluorescent dyes to fade within minutes of continuous scanning. Thus, the fluorescence
signal weakens as subsequent scans are made, either to produce a 3D image, or to observe a
single slice at several time points. In addition to photobleaching, phototoxicity is also
a problem. Excited fluorescent dye molecules generate toxic free-radicals. Thus, one
must limit the scanning time or light intensity if one hopes to keep the specimen alive.

The solution: Two-Photon Microscopy!

In 1994, we constructed a multiphoton microscope that greatly reduces
both of these problems. (download the paper) This device depends on the 2-photon effect, by which a
chromophore is excited not by a single photon of visible light, but by two lower-energy
(infrared) photons that are absorbed contemporaneously (within 1 femtosecond).
Fluorescently-labeled specimens are illuminated by an exotic (=$$) titanium:sapphire laser
that produces very short (less than 200 fs) pulses of infrared light–with a very large
peak amplitude (50 kW)–at a rate of 76 MHz.

Fluorescence from the two-photon effect depends on the square of the
incident light intensity, which in turn decreases approximately as the square of the
distance from the focus. Because of this highly nonlinear (~fourth power) behavior, only
those dye molecules very near the focus of the beam are excited. The tissue above and
below the plane of focus is merely subjected to infrared light that causes neither
photobleaching nor phototoxicity. Although the peak amplitude of the IR pulses is large,
the mean power of the beam is only a few tens of milliwatts, not enough to cause
substantial heating of the specimen.

The 2-photon laser-scanning microscope surpassed our expectations
in every respect. Its usefulness with a variety of living specimens and a variety of
chromophores has been demonstrated (see sample images). Multiphoton laser-scanning microscopy is now the standard for imaging living specimens, both in vivo and in vitro.

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