Co-reporter:Daniel C. Kim and Robert C. Dunn
Analytical Chemistry 2016 Volume 88(Issue 2) pp:1426
Publication Date(Web):December 12, 2015
DOI:10.1021/acs.analchem.5b04187
Whispering gallery mode (WGM) resonators are small, radially symmetric dielectrics that recirculate light through continuous total internal reflection. High-Q resonances are observed that shift in response to changes in surrounding refractive index, leading to many applications in label-free sensing. Surface binding measurements with WGM resonators have demonstrated competitive analytical detection metrics compared to other sensing schemes. Similar figures of merit for detecting bulk refractive index changes, however, have proven more challenging. This has limited their use in applications such as capillary electrophoresis (CE), where their compact footprint and refractive index sensitivity offers advantages in nondestructive, universal detection. Here we couple WGM detection with CE by introducing a modulation scheme to improve detection limits. Phase sensitive WGM (PS-WGM) detection is developed to monitor real-time shifts in the WGM spectrum due to changes in surrounding refractive index. We directly compare phase sensitive detection with spectral measurements normally used to track WGM shifts. We report an improvement in detection limits by almost 300-fold using the PS-WGM method. The integrated CE with PS-WGM approach is demonstrated by detecting the separation of a three-component mixture of cations (Na+, Li+, and K+).
Co-reporter:Sarah M. Wildgen and Robert C. Dunn
ACS Photonics 2015 Volume 2(Issue 6) pp:
Publication Date(Web):May 14, 2015
DOI:10.1021/ph500480x
Scanning resonator microscopy (SRM) is developed to integrate whispering gallery mode (WGM) sensing with atomic force microscopy (AFM). The hybrid technique combines the exquisite refractive index sensing of whispering gallery mode resonators with the topography mapping capabilities of AFM. A 45 μm diameter barium titanate microsphere is attached to the end of a conventional AFM cantilever and acts as both a WGM resonator and stylus for mapping surface topography. Calibration plots, taken in contact-mode feedback, show that the WGM spectrum responds to changes in both solution and substrate refractive index. SRM imaging of a glass substrate reveals changes in surface refractive index that correspond to a small, 36 nm high feature measured simultaneously in the contact-mode topography image. Spectral measurements confirm that the contrast arises from refractive index changes and not coupling with sample topography, thus validating the approach. Additional measurements on thin polymer films and protein-coated surfaces are presented and discussed in terms of possible areas of application for SRM.
Co-reporter:Brittany N. DeWitt and Robert C. Dunn
Langmuir 2015 Volume 31(Issue 3) pp:995-1004
Publication Date(Web):December 22, 2014
DOI:10.1021/la503797w
Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir–Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10–8 mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.
Co-reporter:Heath A. Huckabay, Sarah M. Wildgen, Robert C. Dunn
Biosensors and Bioelectronics 2013 Volume 45() pp:223-229
Publication Date(Web):15 July 2013
DOI:10.1016/j.bios.2013.01.072
Small optical microresonators that support whispering gallery mode (WGM) resonances are emerging as powerful new platforms for biosensing. These resonators respond to changes in refractive index and potentially offer many advantages for label-free sensing. Recently we reported an approach for detecting WGM resonances based on fluorescence imaging and demonstrated its utility by quantifying the ovarian cancer marker CA-125 in buffer. Here we extend those measurements by reporting a simplified approach for launching WGM resonances using excitation light coupled into a Dove prism. The enhanced phase matching enables significant improvements in signal-to-noise, revealing the mode structure present in each resonator. As with all label-free biosensing techniques, non-specific interactions can be limiting. Here we show that standard blocking protocols reduce non-specific interactions sufficiently to enable CA-125 quantification in serum samples. Finally, fluorescence imaging of WGM resonances offers the potential for large scale multiplexed detection which is demonstrated here by simultaneously exciting and imaging over 120 microsphere resonators. For multiplexed applications, analyte identity can be encoded in the resonator size and/or location. By encoding analyte identity into microresonator size, we simultaneously quantify the putative ovarian cancer markers osteopontin (38 μm diameter sphere), CA-125 (53 μm diameter sphere), and prolactin (63 μm diameter sphere) in a single PBS assay. Together, these results show that fluorescence imaging of WGM resonances offers a promising new approach for the highly multiplexed detection of biomarkers in complex biological fluids.Highlights► Fluorescence imaging technique for WGM location and intensity measurements. ► Detected CA-125 in serum suggesting non-specific binding is overcome. ► Simultaneously detect three proteins in buffer using WGM imaging technique. ► Multiplexed potential demonstrated by exciting and imaging over 120 resonators. ► WGM mode structure in each resonator can be measured with improved signal-to-noise.
Co-reporter:Kevin P. Armendariz and Robert C. Dunn
The Journal of Physical Chemistry B 2013 Volume 117(Issue 26) pp:7959-7966
Publication Date(Web):June 7, 2013
DOI:10.1021/jp405312a
Single molecule fluorescence measurements are used to probe the effects of GM1 in DPPC monolayers. Langmuir–Blodgett films of GM1 and DPPC were doped with ∼10–8 mol % of the fluorescent lipid probe, BODIPY-PC, and transferred onto glass substrates at 23 mN/m. As shown previously, the individual orientation of each BODIPY-PC probe in the membrane can be measured using defocused polarized total internal reflection fluorescence microscopy, revealing changes in film properties at the molecular level. Here, BODIPY-PC tilt angle histograms are used to characterize the effects of GM1 in DPPC films from 0.05 to 100 mol % GM1. At high GM1 levels (>5 mol % GM1), trends in the single molecule measurements agree with previous bulk measurements showing the turnover from condensing to expanding influence of GM1 at 15–20 mol %, thus validating the single molecule approach. At biologically relevant, low concentrations of GM1 (<5 mol % GM1), where bulk fluorescence measurements are less informative, the single molecule measurements reveal a marked influence of GM1 on film properties. The addition of trace amounts of GM1 to DPPC films leads to an expansion of the film which continues to 0.10 mol % GM1, above which the trend reverses and the condensing effect previously noted is observed.
Co-reporter:Philip W. Livanec, Heath A. Huckabay, Robert C. Dunn
Thin Solid Films 2012 Volume 520(Issue 19) pp:6233-6237
Publication Date(Web):31 July 2012
DOI:10.1016/j.tsf.2012.05.028
An approach for reducing photobleaching of dyes doped into lipid films formed using the Langmuir–Blodgett technique is discussed. Fluorescent lipid analogs are often doped into lipid membranes to characterize microscopic domains present in the films. Dye photobleaching can limit the information from these studies which can be especially problematic for single molecule fluorescence measurements. Here we show that a protective polymer coating grown on the film using a fuming process can reduce dye photobleaching without perturbing the underlying lipid structure. Following the transfer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers to a glass substrate using the Langmuir–Blodgett technique, the films are exposed to vapors of ethyl 2-cyanoacrylate. Polymer formation produces a thin protective barrier on the lipid film that reduces photobleaching of the underlying fluorescent lipid analogs. Single molecule orientation measurements reveal that the molecular structure of the underlying monolayer is unaltered by polymer formation, suggesting that this method is a general, nonperturbative technique for the reduction of photobleaching in fluorescence studies of Langmuir–Blodgett films.Highlights► The photobleaching of fluorescent probes doped into lipid monolayers is studied. ► Fuming lipid monolayers leads to reduced photobleaching of the probe dye. ► Single molecule fluorescence measurements confirm that film structure is not altered.
Co-reporter:Nicholas E. Dickenson;Kevin P. Armendariz
Analytical and Bioanalytical Chemistry 2010 Volume 396( Issue 1) pp:31-43
Publication Date(Web):2010 January
DOI:10.1007/s00216-009-3040-1
Near-field scanning optical microscopy (NSOM) is an emerging optical technique that enables simultaneous high-resolution fluorescence and topography measurements. Here we discuss selected applications of NSOM to biological systems that help illustrate the utility of its high spatial resolution and simultaneous collection of both fluorescence and topography. For the biological sciences, these attributes seem particularly well suited for addressing ongoing issues in membrane organization, such as those regarding lipid rafts, and protein–protein interactions. Here we highlight a few NSOM measurements on model membranes, isolated biological membranes, and cultured cells that help illustrate some of these capabilities. We finish by highlighting nontraditional applications of NSOM that take advantage of the small probe to create nanometric sensors or new modes of imaging.
Co-reporter:Elizabeth S. Erickson, Philip W. Livanec, Jessica F. Frisz and Robert C. Dunn
Langmuir 2009 Volume 25(Issue 9) pp:5098-5102
Publication Date(Web):March 3, 2009
DOI:10.1021/la804104k
Lipid monolayers of l-α-dipalmitoylphosphatidylcholine (DPPC) are used to pattern substrates using the Langmuir−Blodgett (LB) technique. Lipid monolayers are deposited onto freshly cleaved mica surfaces or glass capillaries under conditions that lead to distinct patterns in the film. Exposure of the supported monolayer to ethyl 2-cyanoacrylate fumes leads to preferential polymerization in the more hydrated regions of the patterned monolayer. This method enables surfaces to be micropatterned where the lateral features are controlled by the structure present in the underlying LB film, and the vertical feature size is controlled by the length of the fuming process. Atomic force microscopy (AFM) measurements confirm that the original structure in the LB film is preserved following fuming and that the lateral and vertical feature sizes can be controlled from nanometers to micrometers. This method, therefore, provides a rapid and versatile approach for micropatterning both flat and curved surfaces on a variety of substrates.
Co-reporter:Philip W. Livanec and Robert C. Dunn
Langmuir 2008 Volume 24(Issue 24) pp:14066-14073
Publication Date(Web):November 16, 2008
DOI:10.1021/la802886c
Biological membranes are highly heterogeneous structures that are thought to use this heterogeneity to organize and modify the function of membrane constituents. Probing membrane organization, structure, and changes therein are crucial for linking structural metrics with function in biological membranes. Here we report the use of single-molecule fluorescence studies to measure membrane structure at the molecular level. Several groups have shown that polarized total internal reflection fluorescence microscopy using p-polarized excitation can reveal single-molecule orientations when spherical aberrations are introduced into the optics train. We use this approach to measure the orientation of fluorescent lipid analogs doped into Langmuir−Blodgett films of DPPC and arachidic acid. We compare two commonly used fluorescent lipid analogs, BODIPY-PC and DiIC18, which have their fluorophores located in the tailgroup and headgroup, respectively. We find the tilt orientation of BODIPY-PC is very sensitive to the surface pressure at which DPPC films are transferred onto the substrate. At low surface pressures, the tailgroups are largely lying in the plane of the film and evolve to an orientation normal to the surface as pressure is increased. For DiIC18, however, no evolution in orientation with surface pressure is observed, which is consistent with the headgroup located fluorophore being less sensitive to changes in membrane packing. Single-molecule orientation measurements of DiIC18 in multilayer films of arachidic acid are also measured and compared with previous bulk measurements. Finally, single-molecule measurements are utilized to reveal the ordering induced in DPPC monolayers following the addition of cholesterol.
Co-reporter:Nicholas E. Dickenson;Kathy A. Suprenant;David Moore
Photochemistry and Photobiology 2007 Volume 83(Issue 3) pp:686-691
Publication Date(Web):14 MAR 2007
DOI:10.1111/j.1751-1097.2007.00050.x
Nuclear pore complexes (NPCs) are macromolecular pores that span the nuclear envelope and undergo conformational changes in response to changes in cisternal calcium levels. Depletion of cisternal calcium leads to the appearance of a mass within the pore. The identity and role of this central mass remain unknown, although some studies suggest they are vault complexes. Vault complexes are 13 MDa ribonucleoproteins found in the cytoplasm and recently in the nuclei of some species, suggesting that they associate with NPCs to cross the nuclear envelope. Using Förster resonance energy transfer (FRET) measurements between labeled vaults and NPCs, we find significant energy transfer suggesting that vaults and NPCs are closely associated at the nuclear envelope. This is supported by high-resolution electron microscopy measurements revealing significant spatial correlations between gold-labeled vaults and NPCs. As the location of the central mass in the NPC is dependent on cisternal calcium levels, FRET signals under conditions of varying cisternal calcium were also measured and shown to undergo significant changes. Together, these findings suggest that the central mass observed in NPCs may be, at least in part, due to the presence of vaults in the pore. Possible roles in cyto-nuclear trafficking are discussed.
Co-reporter:Rachel Sibug Aga
Photochemistry and Photobiology 2004 Volume 80(Issue 3) pp:471-476
Publication Date(Web):30 APR 2007
DOI:10.1111/j.1751-1097.2004.tb00116.x
Survanta is a replacement lung surfactant (LS) used in the treatment of respiratory distress syndrome (RDS), the fourth leading cause of infant mortality in the United States. It consists of purified LS from bovine sources and retains the surfactant proteins (SP) SP-B and SP-C, both thought to be important in proper respiratory function. As such, it provides a useful and biologically relevant model system to probe the structure and function of natural LS. Here, we report results from high-resolution studies on model monolayers formed from Survanta to probe the mechanism of collapse at high surface pressure. Our results show the formation of two different collapse structures. At 62 mN/m, slightly below the collapse pressure, monolayer collapse occurs through buckling. Confocal fluorescence measurements on supported films reveal regions of overlapping phase structure in the films that mark the transition from monolayer to multilayer. Simultaneous near-field scanning optical microscopy fluorescence and force measurements show that the transition seen in the fluorescence measurements accompanies corresponding ∼4–5 nm changes in membrane topography. This change in height is consistent with bilayer formation on monolayer collapse. Analysis of the phase structure near the transitions also suggests that the buckling occurs from a continuous film. However, when the film is compressed to its collapse pressure of 65 mN/m, buckling is no longer evident in the collapsed region. In addition, multilayers and lipid-protein aggregates that are up to 40 nm higher than the monolayer are observed in the collapsed film at this pressure.
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