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3.4  Nonfluorescence Microscopy

with a higher-​intensity cell perimeter, while positive defocusing generates the inverse of this,

due to light interference at the image plane between undeviated and transmitted light beams

whose optical path length (OPL), which is the product of the geometrical path length of the

light beam with the index of refraction in that optical media, depends upon the extent of

defocus. The contrast at the cell perimeter is a function of the radius of curvature of the cell,

and for cells of a micron length scale a defocus value of a few hundred nanometers generates

optimum perimeter contrast.

Another approach to improve contrast is to tag a biomolecule with a reporter probe

designed to generate a high local signal upon excitation with light, for example, a probe

coated in a high atomic number metal such as gold generates a high scatter signal for cer­

tain wavelengths of VIS light. Here, photon scattering is elastic and so the wavelength of

scattered light is the same as the incident light; thus, any scattered light not from the labeled

biomolecules must be blocked. These include back reflections from the glass microscope

coverslip/​slide. The regions of the sample not containing tagged biomolecules appear dark on

the camera detector, hence the name dark-​field microscopy. In transmitted light dark field, a

modified condenser lens blocks out the central aperture resulting in highly oblique illumin­

ation on the sample; nonscattered light will emerge at too steep an angle to be captured by

the objective lens, whereas light diffracted by the sample will be forward scattered at small

angles and can be captured.

A similar approach can be used with reflected light, in general using a laser source (hence

laser dark field) in which an oblique angled laser beam incident on the sample emerging from

the objective lens is either transmitted through the coverslip in the absence of any sample, or

back scattered by the sample back into the objective lens (Figure 3.2c). An additional enhance­

ment of contrast can be achieved by the generation of surface plasmons, whose intensity is

a function of the particle size (a few tens of nanometers) and the laser wavelength. This can

generate very high SNRs on in vitro samples facilitating extremely high time resolutions of

~10⁻⁶ s.

Dark-​field microscopy has more limited use with living samples than bright-​field micros­

copy because the relatively large size of a cell compared to the scatter signal either from a

native unlabeled biomolecule or from a biomolecule that has been labeled using a dark-​field

probe (e.g., a gold-​coated bead of tens of nanometers in diameter) can result in significant

scatter from the cell body itself, which can swamp the probe signal. The scatter signal from

unlabeled biomolecules can be prohibitively small, but using a scatter label can also present

technical challenges; it is not easy to specifically label biomolecules inside living cells with,

for example, a gold nanoscale bead without nonspecific labeling of other cellular structures.

For certain cell types (e.g., prokaryotes), it is also difficult to introduce such a large scatter

probe while still keeping the cell intact, limiting its application to accessible surface features.

The practical lower size limit to detect a reproducible, measurable signal from the scattered

light is a few tens of nanometers; the size of the probe is large compared to single biomolecules

implying some steric hindrance effects with impairment of normal biological operation.

Note that there are advanced new techniques such as interferometric scattering microscopy,

which use interferometric methods of scattered light detection that can be used to detect the

scattered signal directly from unlabeled biomolecules themselves (see Chapter 4).

3.4.2  CONTRAST ENHANCEMENT USING OPTICAL INTERFERENCE

Optical interference techniques can be used to modify a light microscope to increase image

contrast. Phase contrast microscopy utilizes differences in refractive index inside a biological

sample. Optically, transparent cells and tissues exhibit a range of refractive indices nt as a

function of spatial localization across the tissue, 1.35–​1.45 being typical for cellular material,

which compares with that of water, nw ~ 1.33. The result is that the phase of light propagating

through a region of sample, which has a length parallel to the optic axis Δz of a few microns,

will be retarded. Such objects are examples of phase objects. The retardation for many cells

is roughly around a quarter of a wavelength λ relative to that of light passing through the

largely aqueous environment in between cells (Figure 3.2d). It is trivial to derive the following