Localized measurements of scattering in biological tissue provide sensitivity to microstructural morphology but have limited utility to wide-field applications, such as surgical guidance. This study introduces sub-diffusive spatial frequency domain imaging (sd-SFDI), which uses high spatial frequency illumination to achieve wide-field sampling of localized reflectances. Model-based inversion recovers macroscopic variations in the reduced scattering coefficient (μs′) and the phase function backscatter parameter (γ). Measurements in optical phantoms show quantitative imaging of user-tuned phase-function-based contrast with accurate decoupling of parameters that define both the density and the size-scale distribution of scatterers. Measurements of fresh ex vivo breast tissue samples revealed, for the first time, unique clustering of sub-diffusive scattering properties for different tissue types. The results support that sd-SFDI provides maps of microscopic structural biomarkers that cannot be obtained with diffuse wide-field imaging and characterizes spatial variations not resolved by point-based optical sampling.

Cellular membranes contain a variety of shapes that likely act as motifs for sorting lipids and proteins. To understand the sorting that takes place within cells, a continuous, fluid bilayer with regions of membrane curvature was designed and characterized using confocal fluorescence and total internal reflection fluorescence microscopy techniques. A supported lipid bilayer was formed over fluorescently labelled nanoparticles deposited on a glass surface. The lipid composition and membrane shape are separately controlled and the nanoparticle dimensions (d = 40-200 nm) determine the extent of curvature. The bulk membrane is fluid as demonstrated by fluorescence recovery after photobleaching (FRAP) using dye labelled lipids. In bilayers that contain fluorescently labelled, single-tailed lipids, accumulation is observed at regions of curvature, yet the molecules retain fluidity. Using single particle imaging methods, lipids are observed to visit regions of curvature and exchange with the surrounding flat membrane. The nanoparticle patterned substrate described here allows for quantitative measurement of the transient interactions between fluorescently labelled biomolecules and regions of membrane curvature.

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding kcat and KM, values of 1.02 ± 0.06 s−1 and 15 ± 3 μM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in >2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.

Integrins, particularly the alpha(v)beta(3) heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the alpha(v)beta(3) integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308) bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and a lipophilic near-infrared (NIR) fluorescent dye cypate. The alpha(v)beta(3) integrin binding affinity and the internalization properties of LS308 mediated by the alpha(v)beta(3) integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of (111)In-labeled LS308 in a 4t1luc tumor-bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT). The results show that LS308 has high affinity for alpha(v)beta(3) integrin and internalized preferentially via the alpha(v)beta(3) integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in alpha(v)beta(3)-positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of alpha(v)beta(3) integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.