Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations
in photostability. This compromises their performance in applications that require a photostable fluorescent reporting
group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important
for observing and tracking individual molecular events over extended period of time. We report in this paper an
extremely photostable and highly fluorescent phthalocyanine dye, IRDyeTM 700DX, as a near-infrared fluorescence
labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly
pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g.
Alexa Fluor"R"680, CyTM 5.5, CyTM 7 and IRDyeTM 800CW dyes; and about 27 times more photostable than
tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent
requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high
ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with
IRDyeTM 700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The
IRDyeTM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in
Western blot and cytoblot assays.
Ultrasound tomography is a bioimaging method that combines the geometry of X-ray computed tomography with the non-ionizing energy of ultrasound. This modality has potential clinical utility in breast cancer screening and diagnosis. In conventional ultrasound tomography, data sets from different interrogation angles are used to reconstruct an estimate of a biomechanical property of the tissue, such as sound velocity, in the form of an image. Here we describe an alternative method of reconstruction using novel algorithms which weight the data based on a "quality" score. The quality score is derived from beamforming characteristics, for example, the weighting of angle-dependent data by its distance from the transmit focal zones. The new approach is that for each data set (taken at a different view angle), the reliability of the data (in the range dimension) is assumed to vary. By fusing (combining) the data based on the quality score, a complete image is formed. In this paper, we describe the construction of a rotational translation stage and tissue-mimicking phantoms that are used in conjunction with a commercial medical ultrasound machine to test our reconstruction algorithms. The new algorithms were found to increase the contrast-to-speckle ratio of simulated cysts by 114% from raw data over a 77% improvement by spatial compounding (averaging), and to decrease wire target width by 54% over a 39% reduction by spatial compounding alone. The new method shows promise as a computationally efficient method of improving contrast and resolution in ultrasound images.
Single molecules of unconjugated Bodipy-Texas Red (BTR), BTR-dimer, and BTR conjugated to cysteine, in aqueous solutions are imaged using total-internal-reflection excitation and through-sample collection of fluorescence onto an intensified CCD camera, or a back-illuminated frame transfer CCD. The sample excitation is provided by the beam from a continuous-wave krypton ion laser, or a synchronously-pumped dye laser, operating at 568 nm. In order to essentially freeze molecular motion due to diffusion and thereby enhance image contrast, the laser beam is first passed through a mechanical shutter, which yields a 3-millisecond laser exposure for each camera frame. The laser beam strikes the fused-silica/sample interface at an angle exceeding the critical angle by about 1 degree. The resultant evanescent wave penetrates into the sample a depth of approximately 0.3 microns. Fluorescence from the thin plane of illumination is then imaged onto the camera by a water immersion apochromat (NA 1.2, WD 0.2mm). A Raman notch filter blocks Rayleigh and specular laser scatter and a band-pass-filter blocks most Raman light scatter that originates from the solvent. Single molecules that have diffused into the evanescent zone at the time of laser exposure yield near-diffraction-limited Airy disk images with diameters of ~5 pixels. While most molecules diffuse out of the evanescent zone before the next laser exposure, stationary or slowly moving molecules persisting over several frames, and blinking of such molecules are occasionally observed.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.