Biophysics Tools and Techniques for the Physics of Life (Mark C. Leake) (1)

112 4.2 Super-Resolution Microscopy

of a greater number of soft matter materials each with potentially different optical properties.

Not only that, but larger samples encapsulate a greater range of biological processes that may

be manifest over multiple lengths and time scales, making biological interpretation of visible

light microscopy images more challenging.

Experimental biophysical techniques are sometimes optimized toward particular niches

of detection in length and time scale, and so trying to capture several of these in effect in one

go will inevitably present potential technical issues. However, there is good justification for

attempting to monitor biological processes in the whole organism, or at least in a population

of many cells, for example, in a functional tissue, because many of the processes in biology

are not confined to specific niches of length and time scale but instead crossover into sev

eral different length–time regimes via complex feedback loops. In other words, when one

monitors a particular biological process in its native context, it is done so in the milieu of a

whole load of other processes that potentially interact with it. It demands, in effect, a level of

holistic biology investigation. Methods of advanced optical microscopy offer excellent tools

for achieving this objective.

KEY POINT 4.1

The processes of life operate at multiple lengths and time scales that, in general, may

crossover and feedback in complex ways. There is no “unique” level to characterize any

native process, and so ideally each has to be interpreted in the context of all levels. This

is technically demanding, but potentially makes “in vivo” experiments the most rele

vant in terms of genuine biological insight, provided a broad range of length and time

scales can be explored.

4.2 SUPER-RESOLUTION MICROSCOPY

Light microscopy techniques, which can resolve features in a sample better than the

standard optical resolution limit, are called “super-resolution” methods (sometimes written

as superresolution or super resolution). Although super-resolution microscopy is not in the

exclusive domain of cellular biology investigations, there has been a significant number of

pioneering cellular studies since the mid-1990s.

4.2.1 ABBE OPTICAL RESOLUTION LIMIT

Objects that are visualized using scattered/emitted light at a distance greater than ~10

wavelengths are described as being viewed in the far-field regime. Here, the optical diffraction

of light is a significant effect. As a result, the light intensity from point source emitters (e.g.,

approximated by a nanoscale fluorescent dye molecule, or even quantum dots [QDs] and

fluorescent nano-spheres of a few tens of nanometers in diameter) blurs out in space due to

a convolution with the imaging system’s point spread function (PSF). The analytical form of

the PSF, when the highest numerical aperture component on the imaging path has a circular

aperture (normally the objective lens), is that of an Airy ring or Airy disk (Figure 4.1a). This

is the Fraunhofer diffraction pattern given by the squared modulus of the Fourier transform

of the intensity after propagating through a circular aperture. Mathematically, the intensity I

at a diffraction angle a through a circular aperture of radius r given a wavenumber k (= 2π/λm

for wavelength λm of the light propagating through a given optical medium) can be described

by a first-order Bessel function J1:

(4.1)

( ) = ( )

(

)



^



^

2 1

sin