Page 244 - Fundamentals of Light Microscopy and Electronic Imaging

TWO-PHOTON AND MULTIPHOTON LASER SCANNING MICROSCOPY 227

where h is Planck’s constant and
is the frequency of vibration, the same energy can be
provided by two photons having half the frequency (twice the wavelength) of a single
excitatory photon if these wavelengths are absorbed by the fluorochrome simul-
taneously. Thus, illumination with intense 800 nm light can induce fluorescence by a
2-photon mechanism in a fluorophore that is normally excited by 400 nm light in a sin-
gle photon mechanism. The same 800 nm wavelength could be used to induce fluores-
cence by a 3-photon mechanism in a different fluorochrome that absorbs 267 nm light.
Fluorescence from 2-photon or multiphoton excitation is not commonly observed,
because 2 or more photons must be absorbed by the fluorochrome simultaneously, a
condition requiring very intense illumination. However, the application of pulsed
infrared lasers in microscopy has led to the development of the 2-photon laser-scanning
microscope (TPLSM). Like the CLSM, the TPLSM uses a laser, a raster scanning mech-
anism, and an objective lens for point scanning of the specimen. But TPLSM has unique
characteristics and offers several distinct advantages:

• Excitation wavelengths in the near IR are provided by a pulsed IR laser such as a
titanium:sapphire laser. Excitation occurs in the near UV, causing fluorescence
emission in the blue portion of the visual spectrum.
• Fluorochromes in the focal plane are differentially excited because with 2P excita-
tion the efficiency of fluorescence excitation depends on the square of the light
intensity, not directly on intensity as is the case for 1P excitation. Thus, by properly
adjusting the amplitude of laser excitation, it is possible to differentially excite fluo-
rochromes within the focal plane. The laser power, pulse duration, pulse frequency,
and wavelength are critical variables for determining 2P excitation. Figure 12-13
shows the difference between 2P- and 1P-induced fluorescence in a transparent
cuvette containing a fluorescent dye.
• Differential fluorescence excitation in the focal plane of the specimen is the hall-
mark feature of TPLSM and contrasts with CLSM, where fluorescence emission
occurs across the entire thickness of a specimen excited by the scanning laser beam.
Thus, in TPLSM, where there is no photon signal from out-of-focal-plane sources,
the S/N in the image is improved. Because the fluorescence emission occurs only in
the focal plane, the rate of photobleaching is also considerably reduced for the
specimen as a whole.
• The use of near-IR wavelengths for excitation permits examination of relatively
thick specimens. This is because cells and tissues absorb near-IR light poorly, so
cellular photodamage is minimized, and because light scattering within the speci-
men is reduced at longer wavelengths. The depth of penetration can be up to 0.5
mm for some tissues, 10 times the penetration depth of a CSLM.
• A confocal detector pinhole is not required, because there is no out-of-focal-plane fluo-
rescence that must be excluded. Nevertheless, emitted fluorescent wavelengths can be
collected in the usual way and delivered by the galvanometer mirrors to the pinhole
and detector as in CSLM, but the efficiency of detection is significantly reduced. This
method is called descanned detection. A more efficient method of detection involves
placing the detector near the specimen and outside the normal fluorescence pathway
(external detection). Several other detection methods are also possible.

Despite the potential advantages, there are still practical limitations and problems
that remain to be solved. Since the titanium:sapphire laser presently used for TPSLM