Fundamentals of Bioimaging - Bioengineering: A Conceptual Approach - page 209

Cells and their components are mainly transparent and the myriad of molecules
within them are indistinguishable from each other in a normal microscope [ 1 ]. But if
the protein of interest lights up in bright green, it is easy to detect and distinguish
from all other molecules in the cell. Many different imaging modalities—or types of
imaging methods—fi ll different scientifi c and/or clinical niches. Every modality
has limitations: a particular method can be low in quality, slow to acquire images,
expensive, or not suitable for all patients. Bioimaging had its breakthrough in 1994
when Martin Chalfi e from Colombia University in the US expressed a protein
from a jellyfi sh in E. coli and the round worm C. elegance [ 1 ]. The protein was GFP
(Green Fluorescent Protein) which is fl uorescent and emits green light when excited
by blue light. The result was overwhelming with bright green bacteria and worms,
but the real power of bioimaging was demonstrated when scientists realized that
GFP by means of genetic engineering can be fused to natural proteins in animals,
plants or microorganisms. Now, not the whole organism but rather a particular
protein will light up, so its distribution within the cell is visualized. Moreover, GFP
was fused to promoters so it could tell where and when expression was directed.
The art of bioimaging is to develop probes that can be specifi cally attached to
certain molecules and that emit light in distinct colors so that several probes can
be used in the same sample in order to monitor more than one biological process
at a time . GFP has been modifi ed to emit cyan, blue and yellow light and other proteins
add red to the palette. A classical method to label proteins or other molecules is to raise
antibodies against them and couple these to a fl uorescent probe. We have seen that
already on one of the author's images from fl uorescent microscope, where the macro-
phage's enzyme COX-2 is envisioned with fl uorescently labeled antibody produced on
this enzyme ( Fig. 5.3a ). Due to the size of the antibodies this method will, however,
often render the molecule of interest biologically inactive and is accordingly not
normally compatible with life processes. Also a large number of rather simple organic
molecules have been developed as fl uorescent probes [ 1 - 5 ]. These can be injected
directly into cells or tissues and depending on their nature they will bind to certain
proteins or molecules. One of these is DAPI which specifi cally binds DNA and emits
blue light. Virtually, all imaging modalities are now digital : the images are acquired by
a computer and are made up of individual picture elements, or pixels .