142 4.5  Light Microscopy of Deep or Thick Samples

wave; the scattered E-​field is proportional to the second derivative of p, hence 1/​λ²

dependence, and the scattered intensity is proportional to the square of the scattered

E-​field, hence 1/​λ⁴dependence.

Another advantage of two-​photon microscopy is the greater localization precision of

the excitation volume. The effective cross-​section for two-​photon absorption is very small

compared to the single-​photon absorption process due to the narrow time window required

for the absorption process. This requires a high incident flux of photons from a focused

laser source with the two-​photon absorption only probably very close to the center of the

focal volume. This means that the effective focal excitation volume is an order of magnitude

smaller than that of single-​photon confocal microscopy, or ~0.1 fl. Thus, the excitation is sig­

nificantly more localized, resulting in far less contamination in the images from out of focal

plane emissions.

The spectral emission peak of QDs is temperature sensitive since the population of high-​

energy excitons is governed by the Boltzmann factor, which is temperature sensitive, but this

sensitivity is significantly more for two-​photon laser excitation compared to the standard

one-​photon excitation process (see Chapter 3), and this has been exploited in using QDs as

nanother-​mometers (see Maestro et al., 2010). This is manifest as a drop in QD brightness

when measuring over a wavelength window close to the peak of a factor of ~2 when changing

the local temperature from 30°C to 50°C and thus potentially is a good probe for investigating

temperature changes relevant to biological samples, which has been tested as a proof-​of-​

principle to measure the local temperatures inside human cancer cells.

A significant disadvantage with 2PE microscopy is that the incident light intensity needs

to be so high that photodamage/​phototoxicity becomes problematic; this can be seen

clearly by using death sensors, in the form of individual muscle cells whose speed of con­

traction is inversely proportional to the extent of their photodamage. Also, the technique

requires raster scanning technology before images can be reconstructed, and so it is slower

than camera detector pixel array–​based imaging, which requires no scanning, limiting the

range of dynamic biological processes that can be investigated. However, tissue depths of

up to 1.6 mm have been imaged using 2PE, with investigation of gray brain matter in mice

(Kobat, 2011).

The limit beyond this depth using 2PE is again due to scatter resulting from unavoidable

single-​photon scattering. To counteract this, researchers have developed three-​photon exci­

tation microscopy, such that the wavelength of the incident photons is three times that of the

required for the equivalent single-​photon absorption event, which reduces the single-​photon

scattering even more. 3PE has enabled brain imaging in mice to be extended to ~2.5 mm

depth (Horton et al., 2013).

KEY POINT 4.5

MPE fluorescence microscopy is emerging as a tool of choice for imaging deep into

tissues, resulting in significantly reduced back scatter and a spatially more confined

excitation volume compared to single-​photon fluorescence microscopy and is espe­

cially powerful when combined with AO to correct for optical inhomogeneity in thick

tissues.

Developments in optogenetics technologies (Chapter 7) have also benefited from MPE

microscopy. Optogenetics is a method that uses light to control nerve tissue by genetically

inserting lightsensitive proteins into nerve cells that open or close ion channels in response

to the absorption of light at specific wavelengths. In combining this approach with 2PE, it is

now possible to control the operation of multiple specific nerve fibers relatively deep in living

tissue (Prakash et al., 2012).