144 4.5  Light Microscopy of Deep or Thick Samples

detection of multiple scattering events. In conventional interference techniques, for example,

single-​wavelength laser interferometry, interference occurs over a distance of a few meters. In

OCT, a less coherent light source, for example, an LED, might be used, which exhibits shorter

coherence lengths over a few tens of microns, which is useful for biophysics in corresponding

approximately to the length scale of a few layers of cells in a tissue.

The incident light in an OCT system is normally divided into two beams to form a sample

path and a reference path. A confocal light volume is typically used as a mode of illumination

onto a sample. After both beams are scattered from the confocal volume, they are recombined

and imaged onto one or more photodiode detectors. The two beams will generate an inter­

ference pattern on the detector if the optical path length from both beams is less than the

coherence length of the light source.

In conventional light microscopy, the majority of scattered light generates background

noise. This is especially prevalent with deep tissue imaging due to multiple scattering events

through several layers of cells. However, in OCT, multiple scatter events can be rejected on

the basis of them having a longer optical path length than the optical coherence length, since

these events do not form an interference pattern. Thus, an accepted scattered photon will

have arrived typically from just a single back reflection event from a cellular structure.

This rejection of multiple scatter noise permits a 3D tomographic image to be reconstructed

down to a depth of a several tens of microns. OCT is now a standard technique in biomedical

imaging for ophthalmology, for example, to generate 3D details of the retina of the eye but is

emerging as a useful biophysical tool in research labs for imaging deep tissues and bacterial

biofilms.

A variant of OCT is angle-​resolved low-​coherence interferometry (a/​LCI). This is a

relatively new light-​scatting tool, which can obtain information about the size of cellular

structures, including organelles such as cell nuclei. It combines the depth resolution of OCT

with angle-​resolved elastic light-​scattering measurements (see section in the following text)

to obtain in-​depth information on the shape and optical properties of cellular organelles.

In a/​LCI, the light scattered by a sample at different angles is mixed with a reference beam

to produce an inference pattern. This pattern can then be analyzed to generate the spatial

distribution of scattering objects in the sample using inverse light-​scattering analysis based

on Mie scattering theory, which assumes spherical scattering objects (or the equivalent T-​

matrix theory, which is computationally more expensive but can be applied to nonspherical

particles). Since the interference pattern is a measure of differences in optical path length

of the scale of less than the wavelength of light, this approach can generate very precise

estimates of the size and shape of intracellular-​scattering objects like nuclei. This biophysical

technology also shows promise as a clinical tool for detecting cancerous cells.

4.5.8  REMOVING THE DEEP TISSUE BARRIER

Arguably, the simplest approach to overcoming the issues of optical heterogeneity and signal

and the attenuation effect of excitation and signal intensity of light when imaging through

relatively thick sections of biological tissue is to remove that barrier of thick tissue. For

example, this approach has been used in experiments on nerve cells in the brains of living

rodents and primates using an optogenetics approach. To excite the proteins in individual

nerve cells using light, it is often easiest to remove a small section of the bone from the

skull. Superficial areas of the brain (i.e., relatively close to the skull), which include the cere­

bral cortex responsible for voluntary control of muscles, can then be activated by light using

either an optical fiber or LED directly mounted to the skull of the animal, so that the light

does not have to propagate through the bony tissue. Other methods transect out a portion

of bone from the skull but replace it using a zirconium dioxide substrate (also known as zir­

conia), which is mechanically strong but optically transparent. Areas of the brain far from

the surface can, in principle, be accessed using implanted optical fibers to deliver and receive

light as appropriate.

Similar tissue resection methods can be applied for imaging several types of biological

tissues that are close to the surface. Optical fibers can also be used more generally to access

KEY BIOLOGICAL

APPLICATIONS: DEEP

IMAGING

Monitoring processes at a

depth of at least several cells, for

example, in tissues and biofilms.