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DOI: 10.1201/9781003336433-3

3

Making Light Work

in Biology

Basic, Foundational Detection and Imaging

Techniques Involving Ultraviolet, Visible,

and Infrared Electromagnetic Radiation

Interactions with Biological Matter

I don’t suppose you happen to know

Why the sky is blue?…

Look for yourself. You can see it’s true.

—​John Ciardi (American poet 1916–​1986)

General Idea: Here, we discuss the broad range of experimental biophysical techniques that pri­

marily act through the detection of biological components using relatively routine techniques,

which utilize basic optical photon absorption and emission processes and light microscopy, and

similar methods that extend into the ultraviolet (UV) and infrared (IR) parts of the electromag­

netic spectrum. These include methods to detect and image cells and populations of several

cells, as well as subcellular structures, down the single-​molecule level both for in vitro and in

vivo samples. Although the techniques are ubiquitous in modern biophysics labs, they are still

robust, have great utility in addressing biological questions, and have core physics concepts at

their heart that need to be understood.

3.1  INTRODUCTION

Light microscopy, invented over 300 years ago, has revolutionized our understanding of bio­

logical processes. In its modern form, it involves much more than just the magnification of

images in biological samples. There are invaluable techniques that have been developed to

increase the image contrast. Fluorescence microscopy, in particular, is a very useful tool for

probing biological processes. It results in high signal-​to-​noise ratios (SNRs) for determining

the localization of biological molecules tagged with a fluorescent dye but does so in a way

that is relatively noninvasive. This minimal perturbation to the native biology makes it a tool

of choice in many biophysical investigations.

There has been enormous development of visible (VIS) light microscopy tools, which

address biological questions at the level of single cells in particular, due in part to a bidir­

ectional development in the operating range of sample length scales over recent years. Top-​

down improvements in in vivo light microscopy technologies have reduced the scale of spatial

resolution down to the level of single cells, while bottom-​up optimization of many emerging