Biomedical Engineering Reference
In-Depth Information
co-localization studies below 50 nm. Likely better chemistry will
prevail and at least partially solve the problem of multichannel co-
localization. However, depending on how the labeling is done, ste-
ric issues may still remain.
Scientists interested in temporal evaluation of cellular processes
require live cell techniques. The necessity of taking thousands of
frames for PALM/STORM to produce one full image prevents the
ability to conduct live cell imaging experiments, a distinct disad-
vantage. For such temporal studies today, SIM provides the best
time resolution, ~1-2 images per second. An extensive review of
fl uorescent probes and their application in answering biological
questions is provided in reference [
27
].
Preparing samples for super-resolution microscopy demands preci-
sion and cleanliness. Using very clean, very fl at, high-precision
#1.5 coverslips is central to sample preparation. All reagents used
for preparing SIM or PALM/STORM samples should be fi ltered
to reduce possible background noise. Preparation of fi xed samples
for PALM/STORM should include an extra fi xative step post-
secondary antibody incubation. Mounting media is a growing area
of development as soft mount media are recommended for super-
resolution techniques to avoid sample compression and image dis-
tortion. Refractive mismatch should be considered when choosing
mounting media [
21
]. Some commercial mounting media may be
useful for both SIM and PALM/STORM [
21
].
5.2 Sample
Preparation
5.3 Imaging System
Considerations
All super-resolution microscopes require a highly sensitive EMCCD
or CMOS detector, laser modules with optimized fi lter and dichroic
mirror sets, and especially stage stability and vibration reduction
modifi cations. Vibration system tables and very stable microscope
stands are recommended for all systems, and as always, high numer-
ical aperture objectives are recommended for best resolution. Most
systems can also be optimized for wide-fi eld microscopy for general
focusing. The most important microscope requirement for SIM is
an illumination module with different grating frequencies to match
the light illumination pattern to the laser wavelength and objective
lens [
20
]. Pattern rotation is also required. PALM/STORM sys-
tems usually include a TIRF system for minimization of background
fl uorescence, allowing detection of faint emission from single mol-
ecules. Hence, a TIRF numerical aperture objective is used. A laser
system with 50-200 mW is required to give appropriate energy for
single-molecule switching and imaging [
8
,
9
].
STED microscopes require two laser wavelengths. Resolution
depends on the intensity of the STED beam laser, and resolution
directly increases with increase in STED laser intensity [
10
]. As
such, these systems often have lasers that operate at approximately
700 mW of power [
10
]. Additional components required for mul-
ticolor and 3D imaging are considered below.
