Bujny, Miriam

Kim, Doory

Zhuang, Xiaowei

Elsevier - Publisher Connector 

606a Tuesday, February 18, 2014Currently, we have successfully made small quantum dots with different emis-
sion wavelengths (527 nm, 615 nm, 620 nm and 655 nm), and functionalized
them with streptavidin. Furthermore, we tested the small quantum dots on
cultured neurons and found that all of them bind to AMPA receptors with
high specificity. We found that the diffusion behavior of AMPA receptors
labeled with our small quantum dots is different from that with commercial
quantum dots, indicating that bigger quantum dots may have hindered the
AMPA receptors’ accessibility to the synaptic cleft. In addition, we identified
micro-domains of approximately 100 nm x 100 nm where the AMPA receptors
tend to bind and diffuse within these domains.
3059-Pos Board B751
Thermodynamically Driven Blinking for Super-Resolution Microscopy
Susan Gayda, Richard Haack, Joseph P. Skinner, Qiaoqiao Ruan,
Richard J. Himmelsbach, Sergey Y. Tetin.
Abbott Diagnositics, Abbott Park, IL, USA.
Super-resolution microscopy enables imaging of structures smaller than the
diffraction limit, defined by Abbe’s law. Over the last years, a continuously
increasing toolset of physical methods, fluorescent probes and labeling strate-
gies has emerged, making this imaging approach accessible to a broad range of
applications. Single-molecule based stochastic methods, as STORM, PALM
and their derivates require the least complex instrumentation, which gained
these methods fast growing popularity among scientist of very diverse back-
grounds. However, the relative simplicity of the instrumentation comes with
a trade-off in probe requirements. These methods rely on the partial suppres-
sion of the detectable on-state, which can be achieved by photo-activation of
an initially dark state or reversible switching between a bright (on) and a
dark (off) state. Most synthetic dyes allow for the latter activation scheme,
but require special treatment in order to do so. Chemical additives, as reducing
reagents and oxygen scavenger, complex illumination schemes, molecular
scaffolds, high irradiation intensities or a combination of the above is usually
required to optimize the ratio between on- and off-state that enables a maximal
densely labeling of the structure of interest. Here, we report a rhodamine deri-
vate, which converts between a bright and a dark state in response to pH
changes. At pH7, an average of >1% is fluorescent and tumbles between the
two states on the molecular level. The resulting blinking occurs on timescales
of up to several seconds and can therefore be exploited for e.g. STORM
without further sample treatment or complex illumination schemes. Its ease-
of-use and its outstanding photo-stability and brightness render this dye an
excellent tool for super-resolution imaging techniques based on stochastic
read-out.
3060-Pos Board B752
Multiplexed Imaging of Osteocytes in Bone
LeAnn M. Tiede-Lewis, Yixia Xie, Sarah E. Dallas.
Oral and Craniofacial Sciences, University of Missouri Kansas City, Kansas
City, MO, USA.
Osteocytes are embedded in bone and account for 90% of cells within mature
bone. Although previously viewed as quiescent cells, recent research has re-
vealed the importance of osteocytes in regulation of bone remodeling, both
as a mechanosensor and a source of signaling molecules for regulating oste-
oclasts and osteoblasts. Many osteocyte imaging techniques rely on imaging
the lacunocanalicular space around the cell rather than directly imaging oste-
ocytes themselves. To better characterize differences in osteocyte morphology
and structure in aging and disease, we have developed a multiplexed imaging
technique with the goal of examining osteocytes in 3D within their bone envi-
ronment. 50-100 micron-thick decalcified bone sections were prepared and a
variety of imaging dyes were used in combination with confocal microscopy
to image bone matrix, lacunocanalicular space, osteocyte cell membrane,
nuclei, and cytoskeleton in various combinations. We have simultaneously
imaged collagen matrix (using a transgenic mouse line expressing GFP-
tagged-collagen), lacunocanalicular space (using a fixable Texas Red-
labeled dextran), osteocyte cytoskeleton (using alexa-647-phalloidin) and
nuclei (using DAPI). We have also employed DiO labeling of the cell mem-
brane in addition to dextran labeling of the lacunocanalicular system and
DAPI imaging of cell nuclei. DiI labeling of the membrane in combination
phalloidin labeling of the cytoskeleton was problematic in bone slices with
either incomplete penetration of DiI in the sample or loss of phalloidin spec-
ificity. Mounting of bone slices in 2-2’-thiodiethanol increased imaging depth
and resolution but resulted in loss of GFP and phalloidin signal over time,
therefore requiring imaging immediately after mounting. From multiplexed
confocal image stacks it is possible to simultaneously study several aspects
of osteocyte structure in demineralized bone matrix with sufficient resolution
to render detailed 3-D reconstructions of imaged volumes. These methods can
be applied to studying osteocyte structure/morphology in normal and diseased
bone tissues.3061-Pos Board B753
Structural Studies by Correlative Stochastic Optical Reconstruction
Microscopy and Electron Microscopy
Doory Kim, Miriam Bujny, Xiaowei Zhuang.
Harvard Univ., Cambridge, MA, USA.
In cell biology, visual techniques such as light and electron microscopy are
essential tools for analysis of cellular structures and function. The development
of a super-resolution fluorescence microscopy method, stochastic optical
reconstruction microscopy (STORM), has allowed multicolor and three-
dimensional imaging with nanometer-scale resolution and enabled the study
of cellular processes at the molecular scale. Electron microscopy (EM) can
reveal not labeled structures, like organelles, membranes, macromolecules in
higher resolution. By combining STORMwith EM, we were able to understand
cellular complexity and localization of molecules of interest in relation to other
structures in high resolution. Here, we demonstrate that this method can be used
to elucidate the ultrastructural details of cellular events by bridging the gap
between light and electron microscopy in cell biology applications.
3062-Pos Board B754
4D Multiplexed Functional Imaging in Deep Tissue
Ming Zhao1, Xiaoyang Wan2, Weibin Zhou2, Leilei Peng1.
1College of Optical Sciences, The University of Arizona, Tucson, AZ, USA,
2Department of Pediatrics and Communicable Diseases, University of
Michigan, Ann Arbor, MI, USA.
We present a deep tissue multiplexed functional imaging method that probes
multiple cellular conditions in live model organisms. The method uses
FRET-based biosensors to sense cellular conditions such as calcium and
cAMP concentration levels, and uses fluorescence lifetime quantification to
interpret the cellular conditions reported by the FRET biosensors. The method
is based on Fourier lifetime excitation-emission matrix (FLEEM) spectroscopy
[1] that simultaneously measures fluorescence lifetimes at multiple excitation
and emission wavelengths within 23 microseconds, allowing quantifications
on multiple FRET biosensors simultaneously in live model organisms. Samples
are imaged in 3D by combining FLEEM spectroscopy with scanning laser
optical projection tomography (SLOT) [2]. We demonstrate the method in
zebrafish embryos transiently expressing cAMP FRET biosensor, which
showed an increase in cAMP concentration upon physiological stimulus with
forskolin and IMBX. Calcium and cAMP concentration levels during zebrafish
embryonic development were moni-
tored with time lapse 3D functional
imaging of the embryo from 12 hours
to 22 hours post fertilization. The
method opens the door to multiplexed
functional imaging of cellular biochem-
istries in whole live organisms.
[1] M. Zhao and L. Peng, Optics Letters. 35,2910. (2010).
[2] R. A. Lorbeer, et al., Optics Express. 19,5419. (2011).
3063-Pos Board B755
Design and Implementation of 3D Focus Stabilization for Fluorescence
Microscopy
Karl Bellve, Clive Standley, Lawrence Lifshitz, Kevin Fogarty.
Program in Molecular Medicine, University of Massachusetts Medical
School, Worcester, MA, USA.
Focus stabilization is critical formany imagingmodalities likeTIRF, PALMand
STORM. The focus stabilization device presented here, named pgFocus, is an
open source and open hardware solution that can be integrated into microscopes
with an existing objective positioner. pgFocus is a programmable and inexpen-
sive circuit board consisting of a micro-controller, linear sensor array, DAC and
an ADC. While pgFocus can stabilize on a single focal plane within53nM at
30Hz, it can also follow and correct 3D focus changes when imaging multiple
Z positions. pgFocus works by monitoring the reflection of an 808nm laser
beam that is internally reflected at a glass/water interface. The translation of
the reflected laser beam is converted into D distance change between the objec-
tive and the glass/water interface. The
relationship between movement of the
objective and the translation of the return
laser beam is determined through an
calibration procedure. This D distance
change measurement is used to modify
and adjust a pass-through voltage signal
that is directed to a piezo objective
positioner. The pass-through voltage is
continually adjusted tomove the reflected
laser beam back to the original focus
position.


Structural Studies by Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy 

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Structural Studies by Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

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