Practical Fluorescence Spectroscopy

Zygmunt (Karol) Gryczynski, Ignacy Gryczynski

• Publisher: CRC Press
• Year: 2019
• Print ISBN: 9781439821695
• eBook ISBN: 9781498752954
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

Presenting a detailed, hands-on approach to fluorescence spectroscopy, this book describes experiments that cover basic spectroscopy and advanced aspects of fluorescence spectroscopy. It emphasizes practical guidance, providing background on fundamental concepts as well as guidance on how to handle artifacts, avoid common errors, and interpret data. Nearly 150 experiments from biophysics, biochemistry, and the biomedical sciences demonstrate how methods are applied in practical applications. The result is a hands-on guide to the most important aspects of fluorescence spectroscopy, from steady-state fluorescence to advanced time-resolved fluorescence.

• Provides a complete overview of nearly 150 experiments using fluorescence spectroscopy, from basic to advanced applications
• Presents laboratory methods using a variety of instrumental setups with detailed discussion of data analysis and interpretations
• Covers steady-state phenomena, time-resolved phenomena, and advanced methods
• Spans biophysical, biochemical, and biomedical applications
• Describes related concepts, theory, and mathematical background as well as commercially available instruments used for measurements

CHAPTER 1

Theory of Light and Light Interaction with Matter

CONVENTIONALLY, WE CALL “LIGHT” a small range of a broad spectrum of electromagnetic radiation that corresponds to the visible spectral range (400–700 nm) as shown in Figure 1.1. Light is electromagnetic radiation which presents properties of both a wave and a particle. A “particle” of light is called a photon. The dual nature of light is often a source of confusion, but experiments confirming both interpretations exist. For example, experiments involving single-photon double-slit interference and the photoelectric effect have shown both the wave and particle nature of light, respectively. A photon is a quanta or the smallest possible amount/part of an electromagnetic energy/wave. Since we almost always refer to multiple photons, a photon can be referred to as a quantum of electromagnetic radiation. Both definitions will be used throughout this text. For example, a single chromophore (molecule) absorbs or emits a photon of a certain energy. It is important to remember that when discussing photons or electromagnetic waves, we are talking about light; both representations are equivalent, but one is often much easier applied to a particular scenario. For example, it is more natural to discuss a metal interacting with an oscillating electric wave rather than a stream of “particles” (photons) that never physically collide with the metal surface.

FIGURE 1.1 Electromagnetic radiation. The expanded range of 400–700 nm represents visible light.

1-1 BASICS OF LIGHT

This section is meant as a review of the basics of light as electromagnetic waves or photons. More importantly, it should demonstrate that there are many different ways of looking at light from a mathematical perspective, but they all lead to the same interpretation. Various uses of light are presented across many different fields, especially biology, chemistry, physics, and engineering. Within each field, and indeed specializations within each individual field, the nomenclature and properties of interest vary. For example, in biology, spectra are frequently given in a wavelength scale (nanometers) but in the semiconductor field, they are typically presented on a scale in terms of energy (electron volts). However, any spectrum may be presented in wavelength or energy without a loss of information. By the end of this chapter, the reader should have a fluid understanding of the relationships between different notations.

When looking at the light, one most readily thinks about its brightness and color. The brightness of the light is determined by the intensity (energy), passing through a certain surface area orthogonal to the direction of light propagation per unit of time (we also call it photon flux). The intensity of light is typically denoted as I. A simple way of quantifying the brightness of the light is to think about how many photons hit a detector (e.g., human retina, charge-coupled device, photodiode) per unit time. The more photons that hit a detector, the brighter (i.e., more intense) the light source is. From the wave point of view, the intensity of the electromagnetic radiation is proportional to the square of the electric field amplitude. Color is related to the period (duration of time of one cycle of a wave) of the electromagnetic radiation; equivalently, color (wavelength) is related to the frequency or energy of the light wave.

Figure 1-1.1 shows a typical schematic of a light wave. The electromagnetic field is depicted as orthogonal electric and magnetic fields E and B, respectively. It should be noted that E and B are rarely drawn to scale as the amplitude of B is roughly 1/c the size of that of E. In Figure 1-1.1, the wave is traveling in the $\stackrel{⌢}{z}$-direction, the electric field points in the $\widehat{x}$-direction, and the magnetic field points in the $\stackrel{⌢}{y}$-direction. It is important to notice that the electric field, magnetic field, and the direction of propagation are all orthogonal (akin to the “right-hand rule”). The electric field of light is given as:

FIGURE 1-1.1 A light wave and its comparison with a typical chromophore.

$\mathit{E}\text{(}x,t\text{)}=E_0\widehat{\mathit{e}}\text{sin(}kx+\omega t+\phi \text{)}$

(1-1.1)

E₀ is the maximum intensity of the electric field or the amplitude of the electric vector and $\widehat{e}$ is the directional unit vector. This directional unit vector indicated allows for the direction of the electric field to point in any arbitrary direction. In the example in Figure 1-1.1, $\widehat{e}=\widehat{x}$; one may also see E₀, a constant vector, in place of $E_0\widehat{x}$. In this case, $E_0=|{\mathit{E}}_0|$, and $\widehat{e}=\frac{E_0}{|E_0|}$. Thus, Equation 1-1.1 becomes:

$\mathit{E}\text{(}x,t\text{)}={\mathit{E}}_0\text{sin(}kx+\omega t+\phi \text{)}$

(1-1.2)

Thus, the intensity of this light ray is:

$I=\frac{cn{\epsilon }_0}{2}{|\mathit{E}|}^2=\frac{cn{\epsilon }_0}{2}{\mathit{E}}_0^2$

(1-1.3)

where c is the speed of light, n is the refractive index of the medium the light is traveling in, and ${\epsilon }_0$ is the permittivity of vacuum. Light...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Authors
• CHAPTER 1 ■ Theory of Light and Light Interaction with Matter
• CHAPTER 2 ■ Experimental Basics
• CHAPTER 3 ■ Steady State Experiments—Transmission/Absorption
• CHAPTER 4 ■ Fluorescence—Steady-State Phenomena
• CHAPTER 5 ■ Steady-State Fluorescence: Applications
• CHAPTER 6 ■ Steady-State Fluorescence Polarization: Anisotropy
• CHAPTER 7 ■ Fluorescence: Time-Resolved Phenomena
• CHAPTER 8 ■ Advanced Experiments
• INDEX