Page 18 - ASME_NEMB_2016_Program

Technical Program TRACK 1

strated. Our 3D tracking microscope is built upon spatiotemporally multiplexed Cancer is complex and heterogeneous. Successful diagnostic techniques
two-photon excitation and uses time-gated analysis via a photon counting will require molecular characterization of cancer to interrogate the diverse
histogram to discern the molecular 3D position. Feedback control then steers cell types and functional states within tumors. Next generation sequencing
the excitation to lock-on to the single molecule as it travels at a high speed. The and mass spectrometry offer multiplexed molecular analysis. However, ge-
molecular trajectories are reconstructed from the recorded actuator positions nomic methods are destructive, and transcript levels do not always reflect
from the feedback control loop operating at 1-5 ms. Dynamics down to 50 µs functional levels. These strategies also require interrogation of a popula-
can be inferred from analysis of the photon counting histogram. In our method, tion of cells in bulk, sacrificing spatial resolution. Advancements enabling
the first PMT channel is used for particle tracking while the second and the third single-cell transcriptional analysis still lack the ability to study protein in its
PMT channels can be used for two-photon scanning microscopy, colocalization cellular context, and complex regulatory processes and signaling pathways
analysis, and energy transfer studies. We have coined this technique TSUNAMI cannot be elucidated from the genome alone. In this study, we have devel-
(Tracking Single particles Using Nonlinear And Multiplexed Illumination). oped a nanoparticle targeting platform using fluorescence lifetime imaging
(FLIM) capable of direct detection of functional molecules and extreme multi-
Reference: E. Perillo et al., “Deep and high-resolution three-dimensional plexing while maintaining spatial resolution.
tracking of single particles using nonlinear and multiplexed illumination,” Na-
ture Communications, 2015. We performed FLIM on nanoprobes in solution and analyzed lifetime using
the phasor approach. The phasor approach to FLIM, pioneered by Dr. Enrico
4:50pm Eddy current microscopy for biomedical applications Gratton and the Laboratory of Fluorescence Dynamics, greatly simplifies
lifetime analysis. Instead of fitting complicated exponential functions, the
Technical Presentation. NEMB2016-6004 phasor approach transforms histograms of time delays at each image pixel,
replacing intricate decay curves to a simple graphical representation. We
confirmed distinct phasor locations for 4 nanomaterial probes: a PDA lipo-
Josh Javor, Vish Subramaniam, Joseph West, Travis Jones, The some, organic fluorophore rhodamine, fluorophore-loaded nanoparticle,
Ohio State University, Columbus, OH, United States and cadmium selenide/cadmium sulfide quantum dot. Next, we conjugated
these probes to a monoclonal antibody targeting HER2. Binding assays
The non-invasive, real-time detection and imaging of microscopic mor- of immunoconjugates revealed optimal labeling concentrations between
phological artifacts in biological tissue is imperative to the diagnosis and 1-30 nM. FLIM of HER2-targeted nanoprobes labeled onto SK-BR-3 cells
subsequent treatment of various diseases. Optical microscopy is the stan- demonstrated that phasor locations for each probe closely matched the
dard in biological and biomedical applications and is invaluable in medical solution measurements. Simultaneous targeting of EpCAM and HER2 with
diagnosis. By its nature, it is capable of discriminating between different pairs of nanoprobes resulted in combination phasor results that correlated
morphological structures, tissue types, and cells only after fixing (e.g. with with expression levels. Targeting performed on a panel of cell lines with
formaldehyde or formalin) and staining (e.g. for hematoxylin and eosin) varying biomarker expression levels of HER2, EpCAM, transferrin receptor,
procedures are applied. There is a need therefore to explore non-invasive and folate receptor, also resulted in combined phasor results that correlated
and real-time approaches that might complement optical microscopy, but well with expression levels measured by flow cytometry. To our knowledge,
yet yield different information regarding electromagnetic properties. For this is the first demonstration of resolving 4 probes based on fluorescence
example, when cancer cells or small solid tumors are present in otherwise lifetime. To fully maximize the potential of this technology, a diverse set
normal tissue, there are changes in electromagnetic characteristics in ad- of nanomaterial probes with sufficient phasor resolution is needed. Thus,
dition to morphological changes in the surrounding normal tissue that can future work will focus on creating a library of probes with distinct lifetimes
be detected without the fixing and staining processes required for optical and extending detection into other spectral windows. We can achieve this
microscopy. Eddy-current detection is a technique that has been used to by encapsulating fluorescent nanoprobes within a silica shell, thereby mod-
detect microscopic features, such as carbon nanotubes, beneath the surface ulating lifetime through increased non-radiative decay. The extensive level
in conductive specimens and can be extended to detect physical features in of molecular information offered by our technology would make it possible
heterogeneous biological samples with varying electrical conductivities and to richly characterize heterogeneous tumor specimens to improve detection
morphological structures. The purpose of this study is to develop a scalable and enable host-cell subtyping, and rare cell detection.
eddy current detection tool to image morphological structures on the order
of the dimensions of the immediate environment around individual cells (~ 10 5:30pm Comparison of Continuous-Wave and Frequency-Do-
µm-100 µm). A detection scheme using a dual axis coil is used, in which the main Fluorescence Tomography in a Commercial Hybrid Small
inner coil produces a temporally varying magnetic field resulting in eddy cur- Animal Scanner
rents in the specimen whose magnetic fields are detected by the outer coil.
Lock-in amplification is used to amplify small changes (nV) in the magnitude
of the detector coil waveform arising from eddy currents in the sample. First, Technical Presentation. NEMB2016-6163
a fabrication technique is developed to manufacture multi-turn, multi-layer
(25 turns, 2 layers) coils with the smallest diameter of commercially available Eva Sevick-Muraca, Center for Molecular Imaging, The Brown
magnet wire (25µm) around a 300 µm core. A circuit element model is then Foundation Institute of Molecular Medicine, The University of Texas
developed and its governing equations are solved in MATLAB to predict and Health Science Center, Houston, TX, United States
compare with experimental measurements. Images and measurements are
generated using eddy current microscopy on metal ring phantoms, animal Development of nanoimaging probes for detection of cancer metastases
tissue, plant leaves, and superficial skin samples from mouse models for requires sensitive imaging devices for colocalization of imaging probes
squamous cell carcinoma. The results were analyzed in a contour plot using with metastatic lesions in clinically relevant animal models. With the advent
MATLAB and compared to macroscopic (mm) eddy current detection and ob- of far-red fluorescent protein gene reporters, we and others have shown
servable artifacts in the samples. Improving the resolution of eddy current de- the ability to non-invasively track metastases as a function of time to tu-
tection in biological samples using smaller probes (80µm) is also discussed. mor-draining lymph node basins, but have lacked a tomography approach
sensitive enough for detection. Recently, we designed and installed a
5:10pm Direct, Multiplexed Molecular Profiling Using Fluores- miniaturized near-infrared and far-red sensitive, gain-modulated intensified
cence Lifetime Imaging CCD camera system into the CT gantry of a Siemens Inveon PET/CT system
to compare and demonstrate fluorescence tomography on phantoms and
Technical Presentation. NEMB2016-6031 animals. By gain modulating the intensifier at a radio-frequency of 100MHz,
we were able to conduct time-dependent frequency-domain measurements.
By driving the intensifier with a constant voltage, we were able to conduct
18 Maha Rahim, Rajesh Kota, Enrico Gratton, Jered Haun, Universi- continuous wave measurements. Assessment of quantitative recovery of
ty of California, Irvine, Irvine, CA, United States fluorescence absorption cross section was performed using a fully parallel,
regularization-free, linear reconstruction algorithm with diffusion approxima-