MicroscopyInnovations
they report similar speed-up factors. Even though these meth- ods are only a year old, there is strong reason to suspect that any light microscopy dataset that requires deconvolution, image registration (for example, multiview light-sheet micros- copy), or stitching is likely to enjoy massive acceleration in the rate of these post-processing methods.
Isotope-Resolved Electron Energy-Loss Spectroscopy
Oak Ridge National Laboratory, University of Illinois at Chicago, and Nion Co.
Developers: Juan-Carlos Idrobo, Jordan Hachtel, Robert F. Klie, Jacob R. Jokisaari, and Ondrej Krivanek
Te identification of
isotopes is oſten accom- plished using destructive characterization approaches, such as mass spectrometry. Alternatively, isotopes can be resolved through shiſts in
the vibrational modes using infrared or Raman spectroscopy, as well as neutron scattering. However, these characterization approaches all lack the spatial resolution to identify the loca- tion or dynamics of the isotopes and, moreover, require rela- tively large quantities of sample for accurate measurements. Improvements in spatial sensitivity have been achieved using tip-enhanced Raman spectroscopy (TERS) and scanning near- field optical microscopy (SNOM) to examine the vibrational spectra of biomolecules with spatial resolutions of 10 to 25 nm. Recent electron monochromators on aberration-corrected
STEM instruments have allowed electron energy-loss spec- troscopy (EELS) to access vibrational modes in solids with spatial resolutions down to 0.2 nm. When the energy spread of the electron beam is limited to 10 meV or less, the spectral resolution is sufficient to resolve vibration peaks in molecules. Even different isotopes of the same substance can be resolved by this method (Isotope Resolved-EELS). To examine vibra- tional peaks in a liquid, two layers of hexagonal boron nitride were configured to encapsulate the liquid. Te liquid cell was prepared by growing BN films using chemical vapor deposi- tion. Liquid water was sealed between two BN layers by Van der Waals interactions and the surface tension of the water. Examination of a water-deuterium mixture at 60 keV in such a wet cell allowed the O-D stretch peak (310 meV loss, 2500 cm−1
)
to be clearly distinguished from the O-H stretch peak (421 meV loss, 3400 cm−1
). As expected, the heavier isotope is observed at
a lower energy loss. Te signal from deuterium is weak; thus, the experiment above required the average of 100 spectra and careful subtraction of the background. Other experiments have revealed 13
C labels in L-alanine with atomic-site resolution.
When combined with cryogenic sample prepara- tion or liquid cells consisting of ultra-thin graphene or BN
2020 September •
www.microscopy-today.com
window-layers, isotope-labeled ions or molecules can be tracked in battery cathodes, solid oxide-fuel cell membranes, or biological cells with the spatial resolution of a transmission electron microscope. Tis capability enables direct observation of intracellular molecular chemistry.
Label-Free Super-Resolution Localization Photoacoustic Microscopy
Pohang University of Science and Technology and Opticho Inc.
Developers: Jongbeom Kim, Jin Young Kim, and Chulhong Kim
In photoacoustic micros-
copy (PAM) an image is reconstructed from ultrasonic waves induced by a transient thermal expansion of mol- ecules receiving a short pulse of laser light. Te physical characteristics and arrange-
ment of the laser, mirror scanner, and the acoustic detector determine the attainable signal-to-noise ratio (SNR), spatial resolution, and temporal resolution. Existing systems may have high SNR and spatial resolution but operate rather slowly. Label-free super-resolution localization PAM employing
a galvanometer scanner (L-PAM-GS) delivers a high-quality photoacoustic image at a faster rate than previously available PAM systems. A nanosecond pulsed laser with a pulse repe- tition rate of 600 kHz at a wavelength of 532 nm was used to enable ultra-fast, high-contrast vascular imaging capabilities. Using a ring transducer that allows the laser beam to pass through a hole in its center, the ultrasound or laser beam is aligned to maximize a signal-to-noise ratio (SNR). Te galva- nometer scanner is installed vertically, submerging only the mirror part of the scanner in the water. Tis submerged mirror part steers both the laser beams and the photoacoustic waves underwater in a coaxial and confocal formation, leading to a fast imaging speed, a high SNR, and a wide scanning range. Tis configuration allows application of a localization process to in vivo PAM images without a contrast agent. Tis method locally highlights the PA signals generated from red blood cells (RBC), momentarily captured during the PA imaging experi- ments, leading to high resolution with a low optical NA. Improvements with the L-PAM-GS allow noninvasive
observations of microvasculature in small animals and humans in vivo. Further, functional hemodynamics, the blood flow rate in microvasculature, was successfully monitored and quanti- fied in vivo. By localizing photoacoustic signals from RBCs without contrast agent, unresolved microvessels were clearly distinguished, and the spatial resolution in vivo was improved by a factor of 2.5. Te L-PAM-GS device can potentially be useful
for noninvasively delineating stroke lesions in vivo. 23
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