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DOI: 10.1201/9781003336433-4

4

Making Light Work

Harder in Biology

Advanced, Frontier UV–​VIS–​IR Spectroscopy

and Microscopy for Detection and Imaging

The dream of every cell is to become two cells.

—​Francois Jacob (Nobel laureate Prize for Physiology or Medicine,

1965, From Monod (1971))

General Idea: The use of visible light and “near” visible light in the form of ultraviolet and

infrared to detect/​sense, characterize, and image biological material is invaluable. Here, we

discuss advanced biophysical techniques that use visible and near visible light, including

microscopy methods, which beat the optical resolution limit, nonlinear visible and near visible

light tools, and light methods to probe deeper into biological material than basic microscopy

permits.

4.1  INTRODUCTION

The United Nations Educational, Scientific and Cultural Organization announced that 2015

was the International Year of Light, highlighting the enormous achievements of light science

and its applications. It is no surprise that there are several biophysical tools developed that

use light directly to facilitate detection, sensing, and imaging of biological material. Many

of these go far beyond the basic methods of light microscopy and optical spectroscopy we

discussed in Chapter 3.

For example, up until the end of the twentieth century, light microscopy was still

constrained by the optical resolution limit set by the diffraction of light. However, now

we have a plethora of the so-​called super-​resolution techniques that can probe biological

samples using advanced fluorescence microscopy to a spatial precision that is better than

this optical resolution limit. An illustration of the key importance of these methods was

marked by the award of a Nobel Prize in 2014 to Eric Betzig, Stephan Hell, and William

Moerner for the development of “super-​resolved” fluorescence microscopy. The fact that

they won their Nobel Prize in Chemistry is indicative of the pervasive interdisciplinary

nature of these tools.

There are other advanced methods of optical spectroscopy and light microscopy that have

been developed, which can tackle very complex questions in biology, methods that can use

light to probe deep into tissues and label-​free tools that do not require a potentially invasive

fluorescent probe but instead utilize advanced optical technologies to extract key signatures

from native biological material.

The macroscopic length scale of whole organisms presents several challenges for light

microscopy. The most significant of these is that of sample heterogeneity, since the larger the

sample, the more heterogeneous it is likely to be, with a greater likelihood of being composed