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.


  • Potter, S. M. (1996) “Vital imaging: Two photons are better than one.” Curr. Biol. 6: 1595-1598. (Invited) Download paper
  • Potter, S. M., Pine, J. and Fraser, S. E. (1996) ‘Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.” Scanning Microscopy Supplement 10: 189-199. (Invited) Download
  • Potter, S. M., Wang, C. M., Garrity, P. A. and Fraser, S. E. (1996) “Intravital imaging of green fluorescent protein using 2-photon laser-scanning microscopy.” Gene 173: 25-31. Download
  • Cross-view 3D Stereo Time-lapse movie

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