116 4.2 Super-Resolution Microscopy
Irreversible photobleaching, primarily due to free radical formation, can be suppressed by
chemical means using quenchers. In essence, these are chemicals that mop up free radicals
and prevent them from binding to fluorophores and inactivating their ability to fluorescence.
The most widely used is based on a combination of the sugar glucose (G) and two enzymes
called “glucose oxidase” (GOD) and “catalase” (CAT). The G/GOD/CAT freeradical quencher
works through a reaction with molecular oxygen (O.):
(4.5)
G
O
GA+H O
H O
H O
O
GOD
CAT
+
+
→
→
•
2
2
2
2
2
2
2
2
where glucose is a substrate for GOD, which transfers electrons in glucose to form a product
glucuronic acid (GA), and hydrogen peroxide (H2O2). In a second reaction, CAT transfers
electrons from two molecules of hydrogen peroxide to form water and oxygen gas. The
downside is that as GA accumulates, the pH of the solution potentially drops, and so strong
pH buffers are required to prevent this, though there are some newer quencher systems avail
able that have less effect on pH.
As a rough guide, at video-rate imaging of a typical FP such as GFP, a high-magnification
fluorescence microscope optimized for single-molecule localization microscopy can achieve
a localization precision in the lateral xy focal plane of a few tens of nanometers, with irrevers
ible photobleaching occurring after 5–10 image frames per GFP, or ~200–400 ms at video-
rate sampling. If faster sampling time is required, for example, to overcome motion blurring
of the fluorophore in cytoplasmic environments, then detection may need to be more in the
range of 1–5 ms per image frame, and so the total duration that a typical FP can be imaged
is in the range ~5–50 ms. However, as discussed in the previous section, strobing can be
implemented to space out this limited photon emission budget to access longer time scales
where appropriate for the biological process under investigation.
4.2.4 ADVANCED APPLICATIONS OF LOCALIZATION MICROSCOPY
Localization microscopy super-resolution approaches have been successively applied to multi
color fluorescence imaging in cells, especially dual-color imaging, also known as colocalization
microscopy, where one biomolecule of interest is labeled with one-color fluorophore, while
a different protein in the same cell is labeled with a different color fluorophore, and the two
emission signals from each are split optically on the basis of wavelength to be detected in
two separate channels (see Chapter 8 for robust computational methods to determine if two
fluorophores are colocalized or not). This has led to a surplus of acronyms for techniques that
essentially have the same core physical basis. These include single-molecule high-resolution
colocalization that can estimate separations of different colored fluorophores larger than
~10 nm (Warshaw et al., 2005, for the technique’s invention; Churchman et al., 2005, for
invention of the acronym). Also, techniques called “single-molecule high-resolution imaging
with photobleaching” (Gordon et al., 2004) and “nanometer-localized multiple single-
molecule fluorescence microscopy” (Qu et al., 2004) both use photobleaching to localize two
nearby fluorophores to a precision of a few nanometers up to a few tens of nanometers.
Single-particle tracking localization microscopy (TALM) uses localization microscopy of
specifically mobile-tagged proteins (Appelhans et al., 2012).
4.2.5 LIMITING CONCENTRATIONS FOR LOCALIZATION MICROSCOPY
Localization microscopy super-resolution techniques are effective if the mean nearest-
neighbor separation of fluorophores in the sample is greater than the optical resolution limit,
permitting the PSF associated with a single fluorophore to be discriminated from others in
solution. Therefore, there is a limiting concentration of fluorescently tagged molecules in a