Biophotonics_Concepts_to_Applications

and higher photon excitations are also possible if the density of the excitation
photons is high enough to ensure a sufficient level offluorophore excitation.
In two-photon microscopy the photon concentration must be approximately a
million times that required for an equivalent number of single-photon absorptions.
This is accomplished with high-power mode-locked pulsed lasers, which typically
are used to achieve the high photon concentrations. Such lasers generate a signif-
icant amount of power during pulse peaks, but have an average power that is low
enough not to damage the specimen. Example lasers used for multiphoton micro-
scopy configurations emit short pulses of around 100 fs (10−^13 s) with a repetition
rate of 80–100 MHz.

Example 8.7Because the energy of a photon is inversely proportional to its

wavelength, the wavelengths of the two photons should be about twice that

required for single-photon excitation. What two-photon wavelengths are

needed to excite an ultraviolet-absorbingfluorophore in the 320-nm region?

Solution: The two photons need to have a wavelength of 640 nm (red light).

This red light will result in secondaryfluorescence emission of longer (blue or

green) wavelengths in the blue or green spectral region.

Example 8.8What are some optical sources that can be used for multiphoton

microscopy?

Solution: Some common lasers and their selected emission wavelengths that

are applicable to multiphoton microscopy include the following:

• Ti-sapphire: 100 fs pulses of 1.5 W at 1050 nm


• Ytterbium-dopedfiber lasers: 100 fs pulses of 2 W at 1050 nm


• Nd:YAG (neodymium: yttrium aluminum garnet) laser: 50 ns pulses
  1064 nm

S 0

S 1

S 0

S 1

Excitation

Excitation photons

photon

One-photon excitation Tw o -photon excitation

Fluorescence

emission

Fluorescence

emission

Vibratio nal

relaxation

Vibratio nal

relaxation

Fig. 8.14 Examples of one-photon excitation and two-photon-excitation

252 8 Microscopy