Fluorescent Dye Labels and Stains E-Book

Tarso B. Ledur Kist

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 444

Veröffentlichungsjahr: 2023

Beschreibung

Fluorescent Dye Labels and Stains The only comprehensive database of fluorophores and their physical and photochemical properties Fluorophores are chemical compounds that strongly absorb in the ultraviolet, visible, and/or near-infrared and with bright emission in these ranges. As a result, they are exceptionally valuable as dyes for various analytical processes, capable of labelling and staining particular targets for purposes of fluorescent imaging, sensitive detection, and quantification (exhibiting linear responses over very wide concentration ranges). These compounds are many and varied, and panoramic views of their options, physical properties and their reactions to light excitations can be critical to their successful integration into chemical analysis, pharmaceutical analysis, clinical analysis, microscopies, optical bioimaging, cancer imaging, real-time PCR, flow cytometry, multiplexing in proteomics, life sciences in general, and many other high-tech fields (material sciences, traceability, photovoltaics, quantum computing). Fluorescent Dye Labels and Stains incorporates a comprehensive database of such substances and their characteristics. It provides an introduction to basic theories and foundational terminology, in addition to both the molecular structures and photophysical properties of an enormous range of fluorophores. Assembled over the course of a distinguished career in biochemistry, this database presents valuable information that has never before been available in a single volume. Readers will also find: * Molecular and photochemical information of over 700 fluorophores * A database of parameters, including light excitation ranges, molar absorption coefficients, fluorescence quantum yields, molecular brightness, and many more * Information derived from multiple disciplines, including microscopy, nanoscopy, biochemistry, and molecular biology Fluorescent Dye Labels and Stains is the essential reference for pharmaceutical and biomedical researchers and professionals, academics who study molecular biology or organic chemistry, and any professional whose work includes strong and photostable molecular absorptions and fluorescence.

Leseprobe

Fluorescent Dye Labels and Stains

A Database of Photophysical Properties

Tarso B. Ledur Kist Federal University of Rio Grande do Sul Brazil

This edition first published 2023

© 2023 John Wiley & Sons Ltd

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

The right of Tarso B. Ledur Kist to be identified as the author of this work has been asserted in accordance with law.

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In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Library of Congress Cataloging-in-Publication Data

Names: Ledur Kist, Tarso B., 1962- author. | John Wiley & Sons, publisher.

Title: Fluorescent dye labels and stains : a database of photophysical properties / Tarso B. Ledur Kist.

Description: Hoboken, NJ : John Wiley & Sons, 2023. | Includes bibliographical references and index.

Identifiers: LCCN 2022047668 (print) | LCCN 2022047669 (ebook) | ISBN 9781119835134 (hardback) | ISBN 9781119835141 (pdf) | ISBN 9781119835158 (epub) | ISBN 9781119835165 (ebook)

Subjects: LCSH: Fluorescent labeling. | Fluorescent probes. | Fluorescence microscopy--Technique.

Classification: LCC QH212.F55 L43 2023 (print) | LCC QH212.F55 (ebook) | DDC 570.28/2--dc23/eng/20221117

LC record available at https://lccn.loc.gov/2022047668

LC ebook record available at https://lccn.loc.gov/2022047669

Cover and Author Image: Courtesy of Tarso B. Ledur Kist

Cover Design: Wiley

Set in 9.5/12.5pt STIXTwoText by Integra Software Services Pvt. Ltd, Pondicherry, India

Contents

Cover

Title page

Copyright

Preface

Acronyms

Symbols and Conventions

1 Introduction

2 Basic Definitions and Fundamentals

2.1 Introduction

2.2 Light Sources

2.3 Filtering and Dispersing Light

2.3.1 Absorber Filters

2.3.2 Interference Filters

2.3.3 Polarizers

2.3.4 Prisms

2.3.5 Grating

2.4 Light Detectors

2.5 Light Beams

2.5.1 Radiant Power and Radiance in Space: Divergent and Collimated Beams

2.5.2 Radiant Power and Radiance in Time: Continuous, Modulated, and Pulsed

2.5.3 Spectral Radiant Power (Emission Spectra) of Lamps, LEDs, and Lasers

2.5.4 Light Wavelength, Transmittance, and Absorbance

2.5.5 Spontaneous Decay and Stimulated Emission in Lasers and STED Nanoscopy

2.5.6 Energy, Momentum, Polarization, Spin, and Angular Momentum

2.6 Light Collection Set-Ups

2.6.1 Microscope Objectives

2.6.2 Fluorescence Detection Set-Ups

2.6.3 Fluorescence Imaging Set-Ups

2.7 Fundamentals of Fluorescence

2.7.1 Fluorescence: Fields of Application

2.7.2 Molar Absorption Coefficient

2.7.3 Excitation Spectra

2.7.4 Emission Spectra

2.7.5 Stokes Shift

2.7.6 Fluorescence Quantum Yield

2.7.7 Brightness

2.7.8 Effective Brightness

2.7.9 Fluorescence Mean-Lifetime

2.7.10 Factors Affecting Fluorescence

2.7.10.1 Effect of Microenvironment

2.7.10.2 Influence of Liquid Viscosity on Fluorescence Quantum Yield and Fluorescence Mean-Lifetime

2.7.10.3 Influence of Electric Permittivity and Hydrogen Bonding

2.7.10.4 Effects of Temperature

2.7.10.5 Quenching

2.7.10.6 Self-Quenching

2.7.10.7 Singlet Oxygen Production by Sensitizer Dyes

2.8 Photostability

3 Target-Fluorophore Binding

3.1 Introduction

3.2 Choosing the Right Solvent

3.2.1 Water and PBS

3.2.2 Water Miscible Organic Solvents

3.3 Fluorogenic Reactions

3.3.1 Primary Amines

3.3.1.1 Fluorogenic Reactions of Primary Amines With Homocyclic

o

-Phthaldihaldehydes

3.3.1.2 Fluorogenic Reactions of Primary Amines With Heterocyclic

o

-Dicarboxaldehydes

3.3.1.3 Fluorogenic Reactions of Primary Amines With Other Reagents

3.3.2 Secondary Amines

3.3.3 Thiols

3.3.4 Cyanide

3.3.5

α

-Dicarbonylic Compounds

3.4 Labeling Reactions

3.4.1 Covalent Labeling of Amines

3.4.2 Covalent Labeling of Thiols

3.4.3 Covalent Labeling of Carboxylic Acids

3.4.4 Covalent Labeling of Alcohols

3.4.5 Covalent Labeling of Reducing Saccharides

3.4.6 Others

3.5 Immunofluorescence

4 Classes and Molecular Structures

4.1 Introduction

4.2 Rhodamines

4.2.1 Rhodamines With Absorption Maximum Below 500 nm

4.2.2 Rhodamines With Absorption Maximum Between 500 and 550 nm

4.2.3 Rhodamines With Absorption Maximum Between 550 and 600 nm

4.2.4 Rhodamines With Absorption Maximum Above 600 nm

4.2.5 Rhodamines With a High Net Charge

4.3 HAS-Rhodamines

4.3.1 Carbo-Rhodamines

4.3.2 Silico-Rhodamines

4.3.3 Other HAS-Rhodamines

4.4 Pyronines

4.5 HAS-Pyronines

4.6 Sulforhodamines

4.7 HAS-Sulforhodamines

4.8 Fluoresceins

4.8.1 Non-Halogenated Fluoresceins

4.8.2 Halogenated Fluoresceines

4.8.3 Mercaptofluoresceins

4.8.4 Fluorescein-Analogs

4.9 HAS-Fluoresceins

4.10 Sulfofluoresceins

4.11 Fluorones

4.12 HAS-Fluorones

4.13 Cyanines

4.13.1 Trimethine Cyanines

4.13.2 Pentamethine Cyanines

4.13.3 Heptamethine Cyanines

4.14 Borondipyrromethenes

4.14.1 Small Water-Soluble Borondipyrromethenes

4.14.2 Medium-Sized, Water-Soluble Borondipyrromethenes

4.14.3 Large Water-Soluble Borondipyrromethenes

4.14.4 Other Classes Derived From Borondipyrromethene

4.15 Rhodols

4.15.1 The First Rhodols Synthesized

4.15.2 Rhodols Synthesized More Recently

4.15.3 Rhodol Analogs

4.16 HAS-Rhodols

4.17 Rosamines

4.18 HAS-Rosamines

4.18.1 Silico-Rosamines

4.18.2 Phospha-Rosamines

4.18.3 Other HAS-Rosamines

4.19 Rosols

4.20 HAS-Rosols

4.21 Pyrodols and Pyrodones

4.22 Trianguleniums

4.23 Acridines

4.23.1 Simple Acridines

4.23.2 Acridones

4.24 Merocyanines

4.25 Phenoxazines

4.26 Coumarins

4.26.1 7-Hydroxy Coumarins

4.26.2 Small 7-Amino Coumarins

4.26.3 More Elaborated 7-Amino Coumarins

4.27 Sulforhodols

4.28 Pyrenes

4.29 Quinolines

4.30 Benzothiazoles

4.31 Chromones

4.32 Naphthalimides

4.33 Indoles

4.34 Naphthalenes

4.35 Squaraines

4.36 Pteridines

4.37 Isoquinolines

4.38 Benzene Derivatives

4.39 Other Single Structures

4.39.1 Small Structures

4.39.2 Medium-Sized Structures

4.39.3 Large Structures (Na > 80)

4.40 Hybrid Structures

4.40.1 Hybrid Structures: Fusion of Two Existing Dyes

4.40.2 Hybrid Structures: Single Bond Connected Dyes

4.40.3 Hybrid Structures: Polymethine Bridged Dyes

4.41 Non-Disclosed Structures

4.42 Fluorescent Structures Other Than Small-Molecule Organic Dyes

5 Scattergrams of the Photophysical Properties

5.1 Introduction

5.2 Photophysical Properties Along the Spectrum

5.2.1 Molecular Sizes vs. λ

a,max

5.2.2 Molar Absorption Coefficients vs. λ

a,max

5.2.3 Fluorescence Quantum Yield vs. λ

a,max

5.2.4 Brightness vs. λ

a,max

5.2.5 Stokes Shift vs. λ

a,max

5.2.6 Stokes Shift vs. Brightness

5.2.7 Fluorescence Mean-Lifetime vs. λ

a,max

5.2.8 Fluorescence Mean-Lifetime vs. Brightness

5.3 Fluorophore Charges

6 Band Shapes and Excitation and Emission Ranges

6.1 Introduction

6.2 Typical Absorption and Emission Spectra of Some Classes

6.3 Coarse Prediction of Excitation and Emission Ranges

7 Measuring Photostability and Mitigating Photobleaching

7.1 Introduction

7.2 Measuring Photostability

7.3 Mitigating Photobleaching

Appendix A A1. Short Name, Name, Class, Molecular Formula, and References

Appendix B B1. Ranked by Excitation Maximum

Appendix C C1. Ranked by Emission Maximum

Appendix D D1. Ranked by Stokes Shifts

Appendix E E1. Ranked by Brightness

Appendix F F1. Ranked by Fluorescence Mean-Lifetime

Appendix G G1. Ranked by Molecular Net Charge

Index

End User License Agreement

List of Tables

APPENDIX 01

Table A.1 The information provided...

Table A.2 Fluorescent dyes of...

APPENDIX 02

Table B.1 Photophysical properties of...

APPENDIX 03

Table C.1 Photophysical properties of...

APPENDIX 04

Table D.1 Photophysical properties of...

APPENDIX 05

Table E.1 Photophysical properties of...

APPENDIX 06

Table F1 Photophysical properties of fluorescent...

APPENDIX 07

Table G.1 Photophysical properties of...

CHAPTER 03

Table 3.1 The specifications of...

Table 3.2 Laboratory water purity...

Table 3.3 General properties of...

Table 3.5 A protocol for...

Table 3.4 Table comparing the...

Table 3.7 Protocol for the...

Table 3.8 Protocol for the...

Table 3.9 Common functional groups...

Table 3.10 Protocol for the...

Table 3.11 Protocol for the...

CHAPTER 04

Table 4.1 Structures of the...

CHAPTER 05

Table 5.1 Shapes used to...

Table 5.2 The minimum number...

Table 5.3 Average Stokes shift...

CHAPTER 06

Table 6.1 Approximate (≈) relationships...

Table 6.2 The average values...

CHAPTER 07

Table 7.1 Some relative photobleaching...

List of Illustrations

CHAPTER 02

Figure 2.1 (A) Comparison of two...

Figure 2.2 Photograph of a dichroic...

Figure 2.3 Polarizers in action. Photograph...

Figure 2.4 (A) Photograph of a...

Figure 2.5 The most commonly used...

Figure 2.6 Schematic drawing of (A...

Figure 2.7 Radiant power (P), expressed...

Figure 2.8 Sources with a continuous...

Figure 2.9 Four objectives with angular...

Figure 2.10 Amount of light collected...

Figure 2.11 The mechanism by which...

Figure 2.12 Optical set-ups for...

Figure 2.13 The two most common...

Figure 2.14 This figure shows only...

Figure 2.15 The Jablonski diagram illustrating...

Figure 2.16 (A) Excitation spectrum of...

Figure 2.17 (A) A laser line...

Figure 2.18 Measurement of the fluorescence...

Figure 2.19 Effect of temperature on...

CHAPTER 03

Figure 3.1 The molecular structure of...

Figure 3.2 Evaporation rate of water...

Figure 3.3 Fluorogenic derivatization reaction of...

Figure 3.4 Fluorogenic reactions of primary...

Figure 3.5 Capillary electrophoresis separation of...

Figure 3.6 Fluorogenic reaction of NDA...

Figure 3.7 Fluorogenic reaction of primary...

Figure 3.8 Fluorogenic derivatization reaction of...

Figure 3.9 Fluorogenic reaction of primary...

Figure 3.10 Derivatization reaction of primary...

Figure 3.11 Absorption spectra of the...

Figure 3.12 Derivatization reaction of primary...

Figure 3.13 Reaction of glyoxylic acid...

Figure 3.15 Three common labeling (or...

Figure 3.14 Some labeling derivatization reactions...

Figure 3.16 Fluorescent labeling reactions of...

Figure 3.17 Fluorescent labeling reactions of...

Figure 3.18 Fluorescent labeling reactions of...

Figure 3.19 Molecular structures of the...

CHAPTER 04

Figure 4.1 Core structures of the...

Figure 4.2 Atom numbering commonly used...

Figure 4.3 The xanthene structure and...

Figure 4.4 Structures of rhodamines with...

Figure 4.5 Structures of the rhodamines...

Figure 4.6 Structures of the rhodamines...

Figure 4.7 Rhodamines that have an...

Figure 4.8 Rhodamines that have a...

Figure 4.9 Structures and photophysical properties...

Figure 4.10 Structures and photophysical properties...

Figure 4.11 Structures and photophysical properties...

Figure 4.12 Molecular structure and some...

Figure 4.13 Structures and photophysical properties...

Figure 4.14 Structures and some photophysical...

Figure 4.15 The structures of Sulfo...

Figure 4.16 Some ionic forms of...

Figure 4.17 Detailed mechanism of lactonization...

Figure 4.18 Structures and some photophysical...

Figure 4.19 Structures and some photophysical...

Figure 4.20 Structures and some photophysical...

Figure 4.21 Structures and some photophysical...

Figure 4.22 Structures and some photophysical...

Figure 4.23 Structures and some photophysical...

Figure 4.24 Structures and some photophysical...

Figure 4.25 Molecular structures and some...

Figure 4.26 Molecular structures and some...

Figure 4.27 Molecular structures and some...

Figure 4.28 Molecular structures and photophysical...

Figure 4.29 Molecular structures and photophysical...

Figure 4.30 Molecular structures and photophysical...

Figure 4.31 Core structure of borondipyrromethene...

Figure 4.32 Molecular structures and photophysical...

Figure 4.33 Molecular structures and the...

Figure 4.34 Molecular structures and the...

Figure 4.35 Molecular structures and photophysical...

Figure 4.36 Molecular structure and photophysical...

Figure 4.37 Molecular structures and some...

Figure 4.38 Structures and photophysical properties...

Figure 4.39 Molecular structures of some...

Figure 4.40 Molecular structures and photophysical...

Figure 4.41 Molecular structure and photophysical...

Figure 4.42 Molecular structure and photophysical...

Figure 4.43 Molecular structure and photophysical...

Figure 4.44 Molecular structure and photophysical...

Figure 4.45 Molecular structure and photophysical...

Figure 4.46 Structures of acridines which...

Figure 4.47 Structures of acridones which...

Figure 4.48 Molecular structures and photophysical...

Figure 4.49 Structures and photophysical properties...

Figure 4.50 The molecular structure and...

Figure 4.51 The molecular structure and...

Figure 4.52 The molecular structure and...

Figure 4.53 Molecular structure and some...

Figure 4.54 The most common fluorescent...

Figure 4.55 Molecular structures and some...

Figure 4.56 Molecular structure of some...

Figure 4.57 Molecular structures of few...

Figure 4.58 Examples of fluorescent derivatives...

Figure 4.59 The structures of indoles...

Figure 4.60 Molecular structures of naphthalene...

Figure 4.61 Molecular structures of squaraine...

Figure 4.62 Molecular structures and photophysical...

Figure 4.63 Molecular structures and some...

Figure 4.64 Molecular structures of benzene...

Figure 4.65 Molecular structures of...

Figure 4.66 Molecular structures of...

Figure 4.67 Molecular structures of...

Figure 4.68 Molecular structures of fluorescent...

Figure 4.69 Molecular structures of fluorescent...

Figure 4.70 Molecular structure of a...

CHAPTER 05

Figure 5.1 Scattergram of dye sizes...

Figure 5.2 Scattergram of molar (decadic...

Figure 5.3 Scattergram of fluorescence quantum...

Figure 5.4 Scattergram of molecular fluorescence...

Figure 5.5 Stokes shifts of the...

Figure 5.6 Stokes shifts of the...

Figure 5.7 Scattergram of fluorescence mean...

Figure 5.8 Scattergram of fluorescence mean...

Figure 5.9 Scattergram showing the number...

Figure 5.10 Scattergram showing the number...

CHAPTER 06

Figure 6.1 Normalized absorption and emission...

Figure 6.2 Typical normalized absorption and...

CHAPTER 07

Figure 7.1 Possible irradiation conditions using...

Figure 7.2 Photobleaching of a series...

Figure 7.3 Photobleaching of a series...

Figure 7.4 Examples of dyes with...

Figure 7.5 A squaraine (blue) with...

Guide

Cover

Title page

Copyright

Table of Contents

Preface

Acronyms

Symbols and Conventions

Begin Reading

Appendix A A1. Short Name, Name, Class, Molecular Formula, and References

Appendix B B1. Ranked by Excitation Maximum

Appendix C C1. Ranked by Emission Maximum

Appendix D D1. Ranked by Stokes Shifts

Appendix E E1. Ranked by Brightness

Appendix F F1. Ranked by Fluorescence Mean-Lifetime

Appendix G G1. Ranked by Molecular Net Charge

Index

End User License Agreement

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Preface

This book presents the molecular structures and photophysical properties of over seven hundred fluorescent dyes with medium to high brightness in aqueous solutions. It has been written to be a reliable and useful source of information for users of fluorescent dye labels and stains in general. The specific aqueous solution used to measure the photophysical parameters of each of the seven hundred dyes is clearly provided in this succinct database.

The photophysical parameters presented in this database include: wavelengths of absorption and/or excitation maximum, wavelengths of emission maximum, Stokes shifts, excitation ranges, emission ranges, molar absorption coefficients, fluorescence quantum yields, brightnesses, fluorescence mean-lifetimes, sizes (represented by the number of atoms of the structures), and charges in aqueous solutions at neutral pH. Additional interesting properties of some of the fluorescent dyes have also been included, such as photostability and pKa (when present at around neutral pH). Scattergrams are systematically used to give panoramic views of the photophysical data that allow users to find desired photophysical properties within a given spectral range.

Previously, this data was only available from varied sources that contained partial lists, were missing data, and, in many cases, omitted descriptions of the liquids in which parameters were measured. It is known that almost all fluorescent dyes that exhibit high molecular fluorescence brightness in aqueous solutions are also bright in organic solvents; however, the opposite is not true. Most dyes that exhibit a high brightness in organic solvents exhibit a lower and, in many cases, a very low brightness in aqueous solutions. Even worse, the great majority of fluorescent dyes available are not soluble enough in aqueous solutions to have their photophysical parameters measured. Hence, the importance of this database, where only dyes whose photophysical parameters were measured in plain water, phosphate-buffered saline, or aqueous buffers (without organic modifiers) are included. Some dyes are no longer commercially available, but are nonetheless kept in the database for completeness and historic record purposes.

The author began recording the spectral and photophysical data of fluorescent dyes in his Master’s degree, and has continued throughout his whole academic career. Measurements were collected in practice and from the literature, and now the time has come to share this collection with the whole community in the form of the current printed database. Probably not all bright water-soluble fluorescent dyes are here, as some may have gone unnoticed. Therefore, the author apologizes to the developers of these missing fluorophores for this.

The extensive data collection necessary to make this book a reality would not have been possible without the help of so many bright students and colleagues, as well as the professionalism of many companies (suppliers of fluorescent dye labels, stains, and probes). The author would also like to thank Prof A.Z. Khoury, Prof D. Lüdcke, and Prof A. Manz for their many stimulating conversations; Prof J.N. Picada for the suggestion to write this book; and both Dr W. Nietfeld and Prof H. Lehrach from the Max Planck Institute in Berlin, where many photophysical parameters were measured by the author during a post-Doctorate leave.

Finally, this book is dedicated to all Organic Chemists from all over the world for their talented work that has brought so many interesting fluorescent structures to our daily lives and impressive developments across so many fields. Moreover, these high-tech products are now being developed and synthesized using increasingly sustainable and environmentally-friendly methods.

April 2022

Tarso B. Ledur Kist

Porto Alegre

Acronyms

A

Acetate buffer

ACN

Acetonitrile; ethanenitrile

ADC

Antracene-2,3-dicarboxaldehyde

APD

Avalanche photodiode

B

Borate buffer

BODIPY

4,4-Difluoro-4-bora-3a,4a-diaza-

s

-indacene

C

Carbonate buffer

CCD

Charged coupled device

CBQCA

3-(4-Carboxybenxoyl)quinoline-2-carboxaldehyde

DMF

Dimethylformamide;

N

,

N

-dimethylmethanamide

DMSO

Dimethylsulfoxide; dimethyl(oxido)sulfur

FRET

Förster resonant energy transfer

HAS

Heteroatom-substituted

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

IA

Iodoacetamido

ITC

Isothyocianate

ME

Maleimido

NDA

Naphthalene-2,3-dicarboxaldehyde

NHS

N

-Hydroxysuccinimidyl ester

NIR

Near-infrared

NMP

N

-Methyl-2-pyrrolidinone

OPA

o

-Phthaldialdehyde

P

Phosphate buffer

PBS

Phosphate-buffered saline (usually: 8.0 g/L NaCl, 0.2 g/L KCl, 1.42 g/L Na

2

HPO

4

, and 0.24 g/L KH

2

PO

4

)

PD

Photodiode

PC

Propylene carbonate; 4-methyl-1,3-dioxolan-2-one

PMT

Photomultiplier tube

RT

Room temperature

SE

N

-Hydroxysuccinimidyl ester

THF

Tetrahydrofuran; 1,4-epoxybutane

Tris

Tris(hydroxymethyl)aminomethane; 2-amino-2-(hydroxymethyl)propane-1,3-diol

UV

Ultraviolet

UVA

UV radiation in the 315 and 400 nm range. Informally also known as “black light”

Vis

Visible

2 Basic Definitions and Fundamentals

2.1 Introduction

This chapter presents some basic tools (light sources, optical components, and light detectors), concepts, and definitions that are important to the field of fluorescence. The definitions of radiant power, radiance, and spectral radiance, for example, are highlighted as they have a precise meaning. The use of the term “intensity” is discouraged as it may refer nonspecifically to photon flux, radiant power, radiance, or spectral radiance [1].

The technologies used to produce, manipulate, and detect electromagnetic radiation, especially light (the visible part of the spectrum), have developed enormously during the last few decades. In the first sections of this chapter, the most important tools such as filters, gratings, and polarizers, which are used to manipulate light in the field of fluorescence imaging and detection, are briefly presented and discussed. The available options of light sources, as well as their pros and cons, are also presented en passant. Light detectors are concisely presented in a single section, despite the vastness of the field and the many developments in this area. The interconnected topics of light beams, radiant power, radiance, spectral radiance (emission spectra), microscope objectives, and both imaging and detection set-ups are concisely presented. Finally, the most important definitions related to the field of fluorescent dyes, such as the absorption spectra, excitation spectra, emission spectra, Stokes shift, molar absorption coefficients, fluorescence quantum yields, molecular brightness, and fluorescence mean-lifetimes, are presented.

2.2 Light Sources

The number of available light sources is constantly growing and there is an increasing diversity of light source options for fluorescence excitation. Therefore, there is a suitable light source for every application. Tungsten filament lamps (or incandescent lamps) are the oldest and simplest. They emit a continuous spectral radiant power (in both time and frequency) represented by the Planckian curve. Indeed, quantum theory emerged when M. Planck tried to find the equation to model this phenomenon from first principles. The best theory at that time (J.W. Maxwell’s Theory of Electromagnetism) could not provide a good model to explain the phenomenon called black body radiation, and this led to the discovery of the granular nature of light. This was an important fact that contributed to the development of quantum mechanics during its early days, the first three decades of the twentieth century. According to quantum theory, electromagnetic radiation is best modeled as packets of energy (photons) traveling in space, instead of a continuous plane wave with an arbitrary amplitude. Moreover, these photons are characterized by the frequency which defines the energy and linear momentum they carry through space, in addition to their states of spin, polarization, and angular momentum (Section 2.5.6).

Halogen lamps are a special case of filament lamps, as they are intense light sources (high radiant power) with longer operation lifetimes than common tungsten lamps. In these lamps, a halogen gas is added to a quartz reinforced bulb containing the metal filament. The halogen gas continuously removes the metal deposited on the inner bulb’s surface and deposits it back onto the hot filament, thereby increasing the lifespan of filament lamps when they are operated at higher temperatures and high radiant power outputs.

Electrical discharge lamps are another important source of light. In these lamps an electrical discharge passes through a gas and produces light. They use a true gas, at low or high pressure, and sometimes a mixture of gas and metal (e.g., sodium) vapors. Deuterium lamps are an important and widely used source of UV radiation in spectrophotometers, spectrofluorometers, imaging systems, and detection systems in analytical instruments (e.g., chromatography and electrophoresis).

However, all the above-mentioned sources are noisy, bulky, hot, and not efficient at converting electric energy into luminous energy, since a large fraction of the electric energy input is converted into undesired thermal energy (heat, infrared radiation, and microwave radiation). Moreover, the light produced by most of the previous sources has low radiance and is difficult to collimate (into a beam) in space and modulate in time; i.e., it is hard to produce light beams that are both narrow and/or pulsed in time. Pulsed light sources are very important for sensitive detection in many fields. For example, lock-in amplifiers are widely used to extract good signals from noisy baselines. The LEDs (Light Emitting Devices) that have been developed in the last few decades are an interesting alternative as they are very compact, more efficient at converting electric energy into luminous energy, exhibit a much longer lifetime, have a more stable luminous power output (radiant power), and the light they produce is easier to collimate and modulate in time.

Finally, lasers (an acronym for Light Amplification by Stimulated Emission of Radiation that became a noun) are a very special light source as their fundamental principle of light production is stimulated emission, not spontaneous decay as in all previous examples. Stimulated emission produces light beams with special properties. These beams are monochromatic, collimated, polarized, have a high radiance (L) (or radiant power per unit area, see Section 2.5.1), and are coherent (Section 2.5.5).

2.3 Filtering and Dispersing Light

2.3.1 Absorber Filters

Absorber filters or color filters are designed to perform the following tasks: i) to work as bandpass filters; i.e., they let a band of the spectrum pass through while absorbing the neighboring ranges. They are characterized by the transmittance (in percentage) at the center of the band and by the band width (in nm or Hz), which is defined as the width of the band at half height of the band; ii) to work as shortpass filters (they let shorter wavelengths pass through) or, equivalently, highpass filters (they let higher frequencies pass through). Shortpass (or highpass) filters are characterized by a cut-off wavelength (or frequency). They are transparent to wavelengths shorter than the cut-off and opaque to wavelengths above it; iii) longpass filters are the opposite of shortpass filters as they let longer wavelengths pass through. Equivalently, they are lowpass filters as they let lower frequencies pass through. They are also characterized by a cut-off frequency, but the transparent and opaque regions are reversed when compared to shortpass filters (ii).

The working mechanism of these filters relies on the selective absorption of light by the chromophores (pigments) that are usually dissolved in the filter materials (homogeneous solid solutions). A good color filter does not scatter light and does not fluoresce upon illumination, otherwise it would decrease the signal-to-noise ratio of detection systems or the contrast of the images in imaging systems. However, the reflection of ~4% of light at the air–solid interfaces is, in general, unavoidable and must be properly handled to prevent it from entering the detection or imaging sensing area. Inorganic pigments are used more often than organic pigments as they are less prone to photobleaching (Section 2.8