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3.5  Fluorescence Microscopy: The Basics

A fluorophore either is chemically attached to the FISH probe via one or more bases via a

fluorescent secondary antibody or is a chemical tag that binds to the probe. FISH can isolate

the position of individual genes and can be used clinically in probing a range of disorders in

a developing fetus in the womb by testing extracts of amniotic fluid.

Fluorophores can also use covalent chemical conjugation to attach to specific biological

molecules. For example, a common strategy is to use the reactive sulfhydryl (–​SH) side group

of cysteine amino acids in proteins to conjugate to a fluorophore a dye molecule to generate a

new thiol covalent bond (–​S–​R where R is the chemical group of the dye molecule). Proteins

that contain several cysteine amino acid groups can lead to problems due to multiple labeling,

and genetic modifications are often performed to knock out some of these additional cysteine

residues. In addition, proteins that contain no native cysteines can be genetically modified to

introduce an additional foreign cysteine. This general technique is called “site-​specific cyst­

eine mutagenesis.”

Other less specific chemical conjugation methods exist. These methods target more general

reactive chemical groups, including amino (–​NH2) and carboxyl (–​COOH) groups, especially

those present in the substituent group of amino acids. The principal issue is that the binding

target is not specific and so dye molecules can bind to several different parts of a biomol­

ecule, which makes interpretation of imaging data more challenging, in addition to potentially

affecting biological function of the molecule in unpredictable ways. Click chemistry is an alter­

native method of conjugation. The “click” is meant to convey a convenience of simply snapping

objects together. The most utilized type of click chemistry relies on strong covalent bonds

formed between a reacting azide and a carbon–​carbon alkyne triple bond. The main challenge

is to introduce the foreign chemical group, either the azide or alkyne, into the biomolecule.

With DNA, a variety of foreign chemical conjugating groups can be introduced using oligo

inserts. These are short ~10 bp sections of DNA whose sequence is designed to insert at spe­

cific regions of the large DNA molecule used under in vitro investigation, and each oligo insert

can be manufactured to be bound to a specific chemical group for conjugation (e.g., azide and

biotin). All chemical conjugation techniques are discussed in more detail in Chapter 7.

Organic dye fluorophores may also be conjugated to small latex beads that have a typical

diameter of a few hundred nanometers, small microspheres or large nanospheres depending

on which way you look at it, which may then be conjugated to a biological substructure to

yield a brighter probe that permits faster imaging. The biggest disadvantage is the size of these

sphere probes, which is large enough to impair the normal biological processes in some way.

Most organic dye fluorophores emit VIS light. However, VIS light results in greater elastic

scattering from unlabeled surrounding tissue, manifested as noise in fluorescence micros­

copy. Progress in developing fluorophores that are excited at longer wavelengths, some even

in the IR region, is of assistance here.

3.5.6  FLASH/​REASH PROBES

Improvements in minimizing biological process impairments are offered through the use

of the green fluorescein arsenical helix (FlAsH) binder and the pink organic dye resorufin

(ReAsH). This technology utilizes a genetically encoded arrangement of four cysteine amino

acid residues in a specific protein under study inside a cell. Cells are incubated with membrane-​

permeable FlAsH/​ReAsH reagents, which can then bind to the four cysteine residues inside the

cell, which convert the dyes into a fluorescent form. FlAsH/​ReAsH reagents have an effective

diameter of 1–​2 nm with a binding site in a protein requiring as few as six amino acids (four for

the cysteine residues plus two more to generate the 3D shape for a binding pocket) and there­

fore exhibit minimal steric hindrance effects. But it is technically difficult to introduce the

reagents, and the nonspecificity of binding combined with cellular toxicity has limited its use.

3.5.7  SEMICONDUCTORS, METAL-​BASED FLUOROPHORES, AND NANODIAMONDS

Other fluorescent probes that have been applied to populations of cells include

semiconductor-​based fluorescent nanocrystals that generate quantized excitation energy