Signal Processing - November 2017 - 20

NORTHWESTERN UNIVERSITY

FIGURE 3. Northwestern University Prof. Hao Zhang says his research team has developed an
improved version of a superresolution fluorescence microscopy technique that's used to study
molecular processes in living cells.

on this assumption, only the centers of
the detected individual molecular emis-
sions are extracted and stored in each
acquisition. This process is then repeated
thousands of times to accumulate all the
"emission centers" into a final image. The
spatial resolution is proportional to the
number of photons in each -emission.
SPLM brings, for the first time, spec-
troscopic analysis to photon localiza-
tion microscopy, allowing researchers to
image multiple molecular labels simul-
taneously. The emission spectra of these
molecular labels do not have to be signifi-
cantly different and, in fact, can be largely
overlapping. SPLM can analyze the full
profile of each emission spectrum to dis-
tinguish molecular labels, numerically
improving spatial resolution by com-
bining photons from different imaging
frames. Additionally, SPLM allows the
imaging of multiple molecules, such
as DNA, using their intrinsic fluores-
cence emissions.
"Our contribution to this technology is
that we add an additional spectroscopic
imaging capability to photon localiza-
tion," says Hao F. Zhang, professor of
biomedical engineering in Northwest-
ern's McCormick School of Engineer-
ing (Figure 3). "The earlier technologies
cannot distinguish wavelength differ-
ences from those emissions."
To solve this deficiency, the research-
ers needed to design molecular labels
with desired, separated emission spectra
and use optical filters to separate photon
20

emissions with different wavelengths.
One technical constraint the team faced
is the limited number of filters that can be
incorporated into a single system, which
restricts the number of molecules that
can be simultaneously imaged. Addition-
ally, spatial resolution cannot be further
improved once a particular molecular
label has been selected. "We added a
specially designed optical grating to the
detecting optical path so that both the
intensity of emitted photons and their
associated optical spectra are detected at
the same time," Zhang says.
Optical grating is an optical disper-
sive component. "When light passes
through or is reflected by an optical grat-
ing, two beams will be generated simul-
taneously due to multiple interference,"
Zhang says. One beam, referred to as the
zeroth-order diffraction beam, discloses
the incident beam's intensity. The second
beam, referred to as the first-order diffraction beam, reveals the optical spec-
trum. "We detect both the zeroth- and
first-order beams using the same highsensitivity array detector to obtain the
molecular location and its emission spec-
trum simultaneously," Zhang explains.
"Because no optical filter is used in the
detection, and the complete profile of
photon emission is detected, the num-
ber of molecular contrasts is, in prin-
ciple, unlimited." Additionally, based
on the individual emission spectrum,
the system can combine imaging frames
to numerically increase the number of
IEEE SIGNAL PROCESSING MAGAZINE

November 2017

photons for localization, which improves
spatial resolution.
Signal processing plays a critical role
in building SPLM's high-quality images.
"For example, during the photon localiza-
tion process, we need to find the best way
to fit the point spread function of each
photon emission," Zhang says. "To make
individual emissions recognizable among
different camera frames, we needed to
design pattern recognition algorithms to
identify optical spectral features among
thousands of frames and determine
whether they are from the same single
molecule or not."
SPLM uses a high-sensitivity array
photon detector to capture two images
at different regions of the array detec-
tion simultaneously. The photo detector
has an internal amplification capabil-
ity. Each single frame acquisition takes
about 10 ms, and several thousand such
single frames may be generated during a
single session. "Once all these frames are
acquired and stored, we identify the cen-
ter of each single-molecular emission and
its associate optical spectrum," Zhang
says. "We applied sophisticated signal
conditioning operations to, for example,
reduce background noise and remove
detector dark current." Gaussian fitting
provides the emission center and associ-
ated optical spectrum.
Zhang says the signal processing
used in SPLM is far more comprehen-
sive than what's currently available in the
field. "The most unique part is that our
method takes advantage of the optical
spectrum of all single-molecular emis-
sions, besides their locations, into con-
sideration," he says. "As a result, we are
not constrained by the limited number of
detection channels and pseudo coloring;
we know the full spectra of all the detect-
ed molecules. There are no alternative
approaches because "the optical spectra
information is undetectable otherwise,"
he adds. Zhang says the team is devel-
oping an open-source image processing
package for SPLM.

Author
John Edwards (jedwards@johnedwards
media.com) is a technolgy writer based in
the Phoenix, Arizona, area.
SP

http://www.media.com

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