Chapter 3 – Making Light Work in Biology 89
under investigation inside a living cell, which in most applications consists of a modified
DNA repair protein called “O6-alkylguanine-DNA alkyltransferase” (AGT). The cell is then
incubated with a secondary probe, which is labeled with a bright organic dye that will bind
with very high specificity to the primary probe. SNAP/CLIP-tags have the same advantage of
FPs in specificity of labeling since the primary probe is generated at the level of the encoding
DNA. The primary AGT probe has a molecular weight of 18 kDa, which is smaller than FPs
and results in marginally less steric hindrance. A key advantage over FPs, however, is that
the secondary probe is labeled with a bright organic dye, which has significantly superior
photophysical properties.
3.5.10 OVERCOMING CELLULAR AUTOFLUORESCENCE
A strong additional source of background noise is autofluorescence. This is the natural
fluorescence that occurs from a wide range of biomolecules in cells, especially molecules
such as flavins, used in the electron transport chain, and those containing pyrimidines, for
example, one of the two chemical categories for nucleotides in nucleic acids but also a com
ponent of NAD+ and NADH also in the electron transport chain (see Chapter 2). Typically,
autofluorescence is more prevalent at lower wavelengths of excitation, in the blue or long UV
(~300–500 nm).
Many autofluorescent molecules have low photostability as fluorophores and so will
photobleach irreversibly quickly. This trick can be used in fluorescence imaging to prebleach
a sample with a rapid pulse of excitation light prior to data acquisition. Some autofluorescent
components, however, have a longer photoactive lifetime. Certain metabolic tricks can be
applied to minimize the cellular concentration of these components, for example, to cul
ture tissues in nutrient medium that is designed to reduce the expression of flavins and
pyrimidines, but this runs a risk of adjusting the physiology of the tissue unnaturally. A better
approach is to avoid using fluorescent dyes that are excited by blue wavelengths in preference
for longer excitation wavelength, or red-shifted, dyes.
The contrast of fluorescence microscopy images obtained on cellular samples can be
enhanced using a technique called “optical lock-in detection” (OLID) that facilitates discrim
ination between noise components in living cells and the desired specific fluorescence signal
from fluorophores inside the cell by utilizing detection of the correct time signature of an
imposed periodic laser excitation function (Marriott et al., 2008). This can be particularly
useful in the case of FP imaging in living cells that are often expressed in small copy numbers
per cell but still need to be detected over often large levels of noise from both camera readout
and native autofluorescence from the cell.
OLID implements the detection of modulated fluorescence excitation on a class of specific
OLID dyes that are optimized for optical switching. The relative population of the excited and
inactive fluorescence states in a dye is controlled by periodic cycles of laser activation and
deactivation, which is dependent on wavelength. Neither camera noise nor autofluorescence
are so sensitively dependent on wavelength, and therefore only specific biomolecules that are
labeled with an OLID dye molecule will register a fluorescence emission signal of the same
characteristic modulation driving frequency as the laser excitation light. Software can then
lock-in onto true signal data over noise by applying cross-correlation analysis to the individual
pixel data to a time series of acquired images from a sensitive camera detector pixel array,
referenced against the driving excitation waveform of the laser. This allows a precise correl
ation coefficient to be assigned for each pixel in each image. Thus, a pixel-by-pixel 2D map of
correlation coefficients is generated, which provides a high-contrast image of the localization
of the specific switchable OLID dye molecules, largely uncontaminated by background noise.
The first OLID fluorophores developed were organic dyes (e.g., probes based on a dye
called “nitrospirobenzopyran”). The main issues with their use were challenges associated
with how to deliver them into living cells and also nonspecificity of dye binding. Recent cel
lular studies exploit an FP called “Dronpa” and have generated high-contrast images of cel
lular structures in live, cultured nerve cells, as well as in small in vivo samples including
mammalian and fish embryos.