Chapter 4 – Making Light Work Harder in Biology  123

this therefore reveals spatial features that would normally be smaller than the optical reso­

lution limit. In practice, the fringe pattern is rotated in the focal plane at multiple orientations

(three orientations separated by 120° is typical) to obtain resolution enhancement across the

full lateral plane. The actual pattern itself is removed from the imaging by filtering in fre­

quency space; however, unavoidable artifacts of the pattern lines do occur, which can result

in embarrassing overinterpretation of cellular data if careful controls are not performed.

The spatial resolution enhancement in standard SIM relies on a linear increase in spa­

tial frequency due to the sum of spatial frequencies from the sample and pattern illumin­

ation. The latter is diffraction-​limited and so the maximum possible enhancement factor for

spatial resolution is 2. But, if the rate of fluorescence emission is nonlinear with excitation

intensity (e.g., approaching very high intensities close to photon absorption saturation of

the fluorophore), then the effective illumination pattern may contain harmonics with spatial

frequencies that are integer multiples of the fundamental spatial frequency from the pattern

illumination and can therefore generate greater enhancement in spatial resolution. This has

been utilized in nonlinear SIM techniques called “saturated pattern excitation microscopy”

and SSIM, which can generate a spatial resolution of a few tens of nanometers. The laser exci­

tation intensities required are high, and therefore, sample photodamage is an issue, and the

imaging speeds are currently still low at a maximum of tens of frames per second.

KEY POINT 4.3

Most super-​resolution techniques suffer issues of cellular photodamage to differing

extents. For example, PALM/​STORM uses harmful UV light of several thousand acti­

vation cycles, and photoblinking methods also use very high-​excitation intensities of

visible light, STED using a damaging high-​intensity depletion beam. Caution should

be applied when interpreting any study, which purports to perform “live-​cell” studies

super-​resolution techniques. However, a key here is the use of appropriate biological

control experiments—​any live-​cell imaging requires a large number of careful control

experiments.

4.2.12  NEAR-​FIELD EXCITATION

Optical effects that occur over distances less than a few wavelengths are described as near-​

field, which means that the light does not encounter significant diffraction effects and so the

optical resolution is better than that suggested by the Abbe diffraction limit. This is utilized

in scanning near-​field optical microscopy (SNOM or NSOM) (Hecht et al., 2000). This often

involves scanning a thin optical fiber across a fluorescently labeled sample with excitation

and emission light conveyed via the same fiber. The vertical distance from sample to fiber

tip is kept constant at less than a wavelength of the emitted light. The lateral spatial reso­

lution is limited by the diameter of the optical fiber itself (~20 nm), but the axial resolution is

limited by scanning reliability (~5 nm). Scanning is generally slow (several seconds to acquire

an image), and imaging is limited to topographically accessible features on the sample (i.e.,

surfaces).

However, samples can also be imaged in SNOM using other modes beyond simply cap­

turing reflected and/​or emitted light. For example, many of SNOM’s applications use trans­

mission mode with the illumination external to the fiber. These are either oblique above the

sample for reflection, or from underneath a thin transparent sample for transmission. The

fiber then collects the transmitted/​reflected light after it interacts with the sample.

An important point to consider is the long time taken to acquire data using SNOM. SNOM

takes several tens of minutes to acquire a single image at high pixel density, an order of mag­

nitude longer than alternative scanning probe methods such as atomic force microscopy

(AFM) for the equivalent sample area (see Chapter 6). The high spatial resolution of ~20 nm

that results is a great advantage with the technique, though the poor time resolution is a sig­

nificant drawback with regard to monitoring the dynamic biological processes. Fluorescent