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3.5  Fluorescence Microscopy: The Basics

pass through the sample. The sample beam experiences phase changes due to the different

range of refractive indices inside a cell compared to the culture medium outside the cell,

similar to phase contrast microscopy. The interference pattern formed is a Fourier transform

of the 3D variation of relative phase changes in the sample and therefore the inverse Fourier

transform can render 3D cellular localization information.

Several bespoke digital holographic systems have been developed for studying dense

cultures of swimming cells in particular to permit the investigation of biological physics

features such as hydro-​dynamic coupling effects between cells as well as more physical

biology effects such as signaling features of swimming including bacterial chemotaxis,

which is the method by which bacteria use a biased random walk to swim toward a source

of nutrients and away from potential toxins. Some systems do not require laser illumination

but can function using a relatively cheap LED light source, though they require in general an

expensive camera that can sample at several thousand image frames per second in order to

obtain the time-​resolved information required for fast swimming cells and rapid structural

transitions of flagella or cilia.

3.5  FLUORESCENCE MICROSCOPY: THE BASICS

Fluorescence microscopy is an invaluable biophysical tool for probing biological processes in

vitro, in live cells, and in cellular populations such as tissues. Although there may be poten­

tial issues of phototoxicity as well as impairment of biological processes due to the size of

fluorescent “reporter” tags, fluorescence microscopy is the biophysical tool of choice for

investigating native cellular phenomena in particular, since it provides exceptional detec­

tion contrast for relatively minimal physiological perturbation compared to other biophysical

techniques. It is no surprise that the number of biophysical techniques discussed in this book

is biased toward fluorescence microscopy.

3.5.1  EXCITATION SOURCES

The power of the excitation light from either a broadband or narrow bandwidth source may

first require attenuation to avoid prohibitive photobleaching, and related photodamage, of

the sample. Neutral density (ND) filters are often used to achieve this. These can be either

absorptive or reflective in design, which attenuate uniformly across the VIS light spec­

trum, with the attenuation power of 10^ND where ND is the neutral density value of the filter.

Broadband sources, emitting across the VIS light spectrum, commonly include the mercury

arc lamp, xenon arc lamp, and metal–​halide lamp. These are all used in conjunction with

narrow bandwidth excitation filters (typically 10–​20 nm bandwidth spectral window), which

select specific regions of the light source spectrum to match the absorption peak of particular

fluorophores to be used in a given sample.

Narrow bandwidth sources include laser excitation, with an emission bandwidth of

around a nanometer. Bright LEDs can be used as intermediate bandwidth fluorescence

excitation source (~20–​30 nm spectral width). Broadband lasers, the so-​called white-​light

supercontinuum lasers, are becoming increasingly common as fluorescence excitation

sources in research laboratories due to reductions in cost coupled with improvements in

power output across the VIS light spectrum. These require either spectral excitation filters to

select different colors or a more dynamic method of color selection such as an acousto-​optic

tunable filter (AOTF).

The physics of AOTFs is similar to those of the acousto-​optic deflector (AOD) used, for

example, in many optical tweezers (OT) devices to position laser traps and are discussed in

Chapter 5. Suffice to say here that an AOTF is an optically transparent crystal in which a

standing wave can be generated by the application of radio frequency oscillations across the

crystal surface. These periodic features generate a predictable steady-​state spatial variation of

refractive index in the crystal, which can act in effect as a diffraction grating. The diffraction

angle is a function of light’s wavelength; therefore, different colors are spatially split.

KEY BIOLOGICAL

APPLICATIONS:

NONFLUORESCENCE

MICROSCOPY

Basic cell biology and cell

organelle imaging; Monitoring

cell motility dynamics.