Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. We then used total internal reflection fluorescence microscopy (TIRF) with alternating excitation to measure the single molecule FRET (smFRET) efficiency between these two sites in pol II-TFIIF. We found they exhibited a proximity consistent with that observed in the transcription pre-initiation complex by cryo-electron microscopy (cryo-EM). We further compared our non-covalent labeling approach with an enzyme-enabled covalent labeling method. The virtually indistinguishable results validate our smFRET approach and show that the observed proximity between the two sites represents a hallmark of the pol II-TFIIF complex. Taken together, we present a simple and versatile bio-orthogonal method derived from TAP to enable selective labeling of a protein complex. This method is suitable for analyzing dynamic relationships among proteins involved in transcription and it can be readily extended to many other biological processes.

Improved axial point spread function in a two-frequency laser scanning confocal fluorescence microscope.

A two-frequency laser scanning confocal fluorescence microscope (TF-LSCFM) based on intensity modulated fluorescence signal detection was proposed. The specimen-induced spherical aberration and scattering effect were suppressed intrinsically, and high image contrast was presented due to heterodyne interference. An improved axial point spread function in a TF-LSCFM compared with a conventional laser scanning confocal fluorescence microscope was demonstrated and discussed.

Measurement of the surface effect of a small scattering object in a highly scattering medium by use of diffuse photon-pairs density wave.

The surface effect close to the boundary of a small light-scattering object in a highly scattering medium is experimentally demonstrated. This is the first attempt to measure the surface effect of a small spherical scattering object in 1% intralipid solution by use of developed diffuse photon-pairs density wave (DPPDW) in terms of the amplitude and phase detection. Theoretically, the surface effect of a small scattering object in turbid media is localized close to the boundary according to the perturbation theory, concerning an inhomogeneous distribution of the diffusion coefficient in the frequency-domain diffusion equation. Hence, an improvement of the spatial resolution of the image via an inverse algorithm, which relates to detection sensitivity of localization to the boundary of the image object in a multiple scattering medium, is anticipated. In this study, we demonstrate that DPPDW is able to sense the surface effect of a 2-mm spherical scattering object in 1% intralipid solution, with high sensitivity. Subsequently, an improvement of spatial resolution of imaging in turbid media by using DPPDW in comparison with conventional diffuse photon density wave (DPDW) using inverse algorithm is discussed.

Zeeman laser scanning confocal microscope and its ability on reduction of specimen-induced spherical aberration.

The spherical aberration induced by refractive-index mismatch results in the degradation on the quality of sectioning images in conventional confocal laser scanning microscope (CLSM). In this research, we have derived the theory of image formation in a Zeeman laser scanning confocal microscope (ZLSCM) and conducted experiments in order to verify the ability of reducing spherical aberration in ZLSCM. A Zeeman laser is used as the light source and produces the linearly polarized photon-pairs (LPPP) laser beam. With the features of common-path propagation of LPPP and optical heterodyne detection, ZLSCM shows the ability of reducing the specimen-induced spherical aberration and improving the axial resolution simultaneously.

Dual-frequency heterodyne ellipsometer for characterizing generalized elliptically birefringent media.

This research proposed a dual-frequency heterodyne ellipsometer (DHE) in which a dual-frequency collinearly polarized laser beam with equal amplitude and zero phase difference between p- and s-polarizations is setup. It is based on the polarizer-sample-analyzer, PSA configuration of the conventional ellipsometer. DHE enables to characterize a generalized elliptical phase retarder by treating it as the combination of a linear phase retarder and a polarization rotator. The method for measuring elliptical birefringence of an elliptical phase retarder based on the equivalence theorem of an unitary optical system was derived and the experimental verification by use of DHE was demonstrated too. The experimental results show the capability of DHE on characterization of a generalized phase retardation plate accurately.

Differential phase decoder in a polarized optical heterodyne interferometer.

A differential-phase decoder (DPD) together with a polarization common-path optical heterodyne interferometer is set up. Based on this interferometric configuration and a novel balanced-detector scheme, the performance of the quantum-noise-limited differential-phase decoder is demonstrated and analyzed. The minimum-detectable differential phase is on the order of 10(-7) rad/sqrt Hz when a 2.5 mW He-Ne laser is used. Verified experimentally, the DPD is immune to the common-phase noise induced by an electro-optic phase modulator or by thermal disturbance within the interferometer. This signifies that the minimum-detectable differential phase can become 10(-8) rad/sqrt Hz if a 300 mW continuous wave laser is employed instead.

Degrees of polarization and coherence of paired linear polarized laser beam by scattering glass plates measured using optical coherent ellipsometer.

An accurate optical coherent ellipsometer (OCE) is proposed and setup in which a two-frequency paired linear polarized laser beam is integrated with a common-path heterodyne interferometer. This OCE is able to precisely measure the optical properties of scattering specimen by measuring ellipsometric parameters (Psi, Delta). In the mean time the degree of polarization P, and degree of coherence Chi of incident two-frequency linear polarized laser beam are measured too. In the experiment, both smooth and ground BK7 glass plates were tested in which the optical parameters (Psi, Delta, P, Chi ) were obtained precisely. Comparing with conventional ellipsometers, OCE can characterize scattering specimen precisely and excludes the scattering effect.

Measurement of diffuse photon-pairs density wave in a multiple-scattering medium.

As a continuation of the previously developed theory of a diffuse photon-pairs density wave (DPPDW) [Appl. Opt.44, 1416-1425 (2005)APOPAI0003-693510.1364/AO.44.001416], this research experimentally studies and verifies the DPPDW theory in a heterogeneous multiple-scattering medium. The DPPDW is generated by collecting the scattered linear polarized photon pairs (LPPPs) in the multiple-scattering medium. Theoretically, the common-path propagation of LPPPs not only provides common phase noise rejection mode but also performs coherence technique via heterodyne detection. In addition, the polarization gating and spatial coherence gating of LPPPs would suppress the severe scattered photon in the multiple-scattering medium. In the experiment, the amplitude and phase wavefronts of DPPDWs, which are distorted by a small object embedded in a homogeneous multiple-scattering medium, are measured in one dimension or two dimensions by scanning the source detector pair. The measured distortion of DPPDW wavefronts are detected precisely and are consistent with the theoretical calculation of DPPDW. It implies an improvement on the detection sensitivity of a small object compared with the conventional diffuse photon density wave (DPDW).

Determination of retardation parameters of multiple-order wave plate using a phase-sensitive heterodyne ellipsometer.

To characterize the linear birefringence of a multiple-order wave plate (MWP), an oblique incidence is one of the methods available. Multiple reflections in the MWP are produced, and oscillations in the phase retardation measurement versus the oblique incident angle are then measured. Therefore, an antireflection coated MWP is required to avoid oscillation of the phase retardation measurement. In this study, we set up a phase-sensitive heterodyne ellipsometer to measure the phase retardations of an uncoated MWP versus the oblique incident angle, which was scanned in the x-z plane and y-z plane independently. Thus, the effect on multiple reflections by the MWP is reduced by means of subtracting the two measured phase retardations from each other. As a result, a highly sensitive and accurate measurement of retardation parameters (RPs), which includes the refractive indices of the extraordinary ray n(e) and ordinary ray n(o), is obtained by this method. On measurement, a sensitivity (n(e),n(o)) of 10(-6) was achieved by this experiment setup. At the same time, the spatial shifting of the P and S waves emerging from the MWP introduced a deviation between experimental results and the theoretical calculation.

Properties of a diffused photon-pair density wave in a multiple-scattering medium.

A novel diffused photon-pair density wave (DPPDW) composed of correlated polarized photon pairs at different temporal frequencies and orthogonal linearly polarized states is proposed. A theory of DPPDWs is developed. A DPPDW selected by coherence gating and polarization gating that satisfies the diffusion equation has been verified experimentally. The sensitivity of amplitude and phase detection of the heterodyne signal has been improved by the properties of synchronized detection and common-path propagation of polarized pair photons in a multiple-scattering medium. Both reduced scattering coefficient micro2s' and absorption coefficient micro2alpha of the scattering medium in terms of the measured phase and amplitude of the heterodyne signal have been obtained. The detection sensitivity of micro2s' and micro2alpha and the properties of a DPPDW in a multiple-scattering medium are discussed and analyzed.