Chapter 4 – Making Light Work Harder in Biology  143

4.5.5  SECOND-​HARMONIC IMAGING

While conventional light microscopes obtain image contrast largely from the spatial differences

either in optical density or refractive index in the sample, second-​harmonic imaging (SHI)

microscopy utilizes the generation of second harmonics from the incident light in the sample.

Second-​harmonic generation (SHG), or frequency doubling, involves two photons of the same

frequency interacting to generate a single photon with twice the frequency and half the wave­

length (an example of sum frequency generation). Biological matter capable of SHG requires

periodic structural features with chiral molecular components, which result in birefringence,

an optical feature in which the refractive index of a medium depends upon the wavelength of

incident light (see Chapter 3). Good examples of this are the extracellular matrix protein col­

lagen, well-​ordered myosin protein filaments in muscle tissue, microtubules from the cytoskel­

eton, and structurally ordered features of cell membranes. SHI can also be used in monitoring

the formation of crystals (see Chapter 7) for use in x-​ray crystal diffraction, which can be used

for determining the structure of biomolecules to an atomic level precision (see Chapter 5).

SHI microscopy offers many advantages for in vivo imaging. It is a label-​free method

and so does not impair biological function due to the presence of a potentially bulky

fluorophore probe. Also, since it requires no fluorescence excitation, there is less likelihood

from phototoxicity effects due to free radical formation. Typically, SHI microscopy is utilized

with NIR incident light and so has much reduced scattering effects compared to visible light

methods and can be used to reconstruct 3D images of deep tissue samples. Similarly, third-​

harmonic imaging microscopy, in which three incident photons interact with the sample to

generate a single photon of one-​third the original wavelength, has been utilized in some in

vivo investigations (see Friedl, 2007).

4.5.6  LIGHT SHEET MICROSCOPY

Light sheet microscopy, also known as selective plane illumination microscopy, is a prom­

ising biophysical tool that bridges the length scales between single-​cell imaging and multicel­

lular sample imaging (see Swoger et al., 2014, for a modern review). It evolved from confocal

theta microscopy and involves orthogonal-​plane fluorescence optical sectioning; illuminating a

sample from the side typically via a thin sheet of light was generated using a cylindrical lens onto

a single plane of a transparent tissue sample, which has been fluorescently labeled. Since just one

plane of the sample is illuminated, there is minimal out-​of-​plane fluorescence emission contam­

ination of images, permitting high-​contrast 3D reconstruction of several diverse in vivo features.

The development of live fruit fly embryos has been investigated with this technique

(Huisken et al., 2004), as well as tracking of nuclei in live zebrafish embryos (Keller et al.,

2008), growing roots tissue in developing plants, developing gut tissue, and monitoring down

to subcellular levels in functional salivary glands to imaging depths of ~200 μm (Ritter et al.,

2010). Variants of this technique have now been applied to monitor single cells.

A recent tissue decolorization method has been used on live mice in combination with

light sheet fluorescence microscopy (Tainaka et al., 2014). This involves using a specific

chemical treatment involving aminoalcohol, which results in removing the normal pink

color associated with oxygen-​carrying heme chemical groups in the hemoglobin of the red

blood cells, thereby decolorizing any tissue that contains blood. Decolorization thus reduces

the absorption of excitation light and improves its depth of penetration in live tissues and its

consequent signal-​to-​noise ratio, facilitating single-​cell resolution while performing whole-​

body imaging. This approach shows significant promise in determining the functional

interactions between cells in living tissue, to the so-​called cellular circuits of organisms.

4.5.7  OPTICAL COHERENCE TOMOGRAPHY

Optical coherence tomography (OCT) is based on low-​coherence interferometry. It uses the

long coherence length of light sources to act as a coherence rejection filter to reduce the