138 4.5  Light Microscopy of Deep or Thick Samples

z-​stacks through the sample using confocal microscopy; this means generating multiple images

through the sample at different focal heights, so in effect optically sectioning the sample.

Since the height parameter z is known for each image in the stack, deconvolution

algorithms (discussed in Chapter 7) can attempt to reconstruct the true positions of the

fluorophores in the sample providing the 3D PSF is known. The 3D PSF can be estimated

separately by immobilizing the sparse population of purified fluorophore onto a glass micro­

scope coverslip and then imaging these at different incremental heights from the focal plane

to generate a 3D look-​up table for the PSF, which can be interpolated for arbitrary value of z

during the in vivo sample imaging.

The main issues with this approach are the slowness of imaging and the lack of sample

homogeneity. The slowness of the often intensive computational component of conventional

deconvolution microscopy in general prevents real-​time imaging of fast dynamic biological

processes from being monitored. However, data can of course be acquired using fast confocal

Nipkow disk approaches and deconvolved offline later.

A significant improvement in imaging speed can be made using a relatively new tech­

nique of light-​field microscopy (see Levoy et al., 2009). It employs an array of microlenses to

produce an image of the sample, instead of requiring scanning of the sample relative to the

confocal volume of the focused laser beam. This results in a reduced effective spatial reso­

lution, but with a much enhanced angular resolution, that can then be combined with decon­

volution analysis offline to render more detailed in-​depth information in only a single image

frame (Broxton et al., 2013), thus with a time resolution that is limited only by the camera

exposure time. It has been applied to investigating the dynamic neurological behavior of the

small flatworm model organism of Caenorhabditis elegans (see Section 7.3).

4.5.2  ADAPTIVE OPTICS FOR CORRECTING OPTICAL INHOMOGENEITY

However, deconvolution analysis in itself does not overcome the problems associated with

the degradation of image quality with deep tissue light microscopy due to heterogeneity in

the refraction index. This results from imaging through multiple layers of cells, which causes

local variations in phase across the wave front through the sample, with consequent interfer­

ence effects distorting the final image, which are difficult to predict and correct analytically

and which can be rate limiting in terms of acquiring images of sufficient quality to be mean­

ingful in terms of biological interpretation.

Variations in refractive index across the spatial extent of a biological sample can introduce

optical aberrations, especially for relatively thick tissue samples. Such optical aberrations

reduce both the image contrast and effective optical resolution. They thus set a limit for prac­

tical imaging depths in real tissues. Adaptive optics (AO) is a technology that can correct for

much of this image distortion.

In AO, a reference light beam is first transmitted through the sample to estimate the local

variations of phase due to the refractive index variation throughout the sample. The phase

variations can be empirically estimated and expressed as a 2D matrix. These values can then

be inputted into a 2D phase modulator in a separate experiment. Phase modulators can take

the form either of a deformable mirror, microlens array, or an SLM. These components can

all modulate the phase of the incident light wave front before it reaches the sample to then

correct for the phase distortion as the light passes through the sample (Figure 4.3a).

The end result is a reflattened, corrected wave front emerging from the sample (for a

recent review, see Booth, 2014). AO has been applied to image to tissue depths of up to sev­

eral hundred microns. It is also compatible with several different forms of light microscopy

imaging techniques.

4.5.3  PTYCHOGRAPHY METHODS FOR NUMERICAL FOCUSING

Another emerging light microscopy technique that shows promise for imaging at tissue depths

in excess of 100 μm is ptychography (also referred to as Fourier ptychography). Ptychography