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.
A simple apparatus for time-correlated single photon counting (TCSPC) measurements in the near-infrared (near-IR) region for scanning-type applications has been constructed and examined. The apparatus consisted of five major components including a pulsed diode laser source (lasing wavelength equals 780 nm; repetition rate equals 80 MHz; power equals 5 mW; pulse width equals 150 ps), an integrated microscope, a large photoactive area avalanche photodiode (APD), a TCSPC PC-board including the electronics and a windows-based software package for accumulating the fluorescence decay profiles. The instrument response function of this assembly was found to be 460 ps, which is adequate for measuring lifetimes with (tau) f greater than or equal to 500 ps. Due to the small size of the device, it also allowed implementation into scanning experiments where lifetimes were measured. To demonstrate this capability, a three-well microscope slide containing a near-IR dye was scanned. The decay profile of the near-IR dye, aluminum 2,3-naphthalocyanine, was collected and analyzed to obtain its lifetime, which was found to be 2.73 ns, in close agreement to literature values for this particular dye. In addition, a three dimensional image of aluminum 2,3- naphthalocyanine fluorescence decays was acquired by scanning the microscope head over this three-well glass slide. In the scanning mode, the IRFs as well as the decays of the dyes were found to be very stable. The device demonstrated a concentration detection sensitivity of 2.33 nM, however, the dynamic range was limited due to the APD and its slow time constant (passive quenching). In addition, this microscope head was installed in a LI-COR DNA Sequencer 4000 for collection of scanning images.
A LI-COR Model 4000 DNA Sequencer has been modified by removing the internal scanning infrared fluorescence microscope and combining it with an external, orthogonal scanner. Due to the reduced background fluorescence and light scattering of nylon membranes in the near- infrared (8000 nm) as compared to the visible region of the optical spectrum, sensitivity of labeled DNA fragments is enhanced. Dot blots of dilution series of labeled oligonucleotides reveal a detection limit of 25 attomole (25 X 10-18 mole). DNA fragments blotted onto nylon membranes using direct transfer electrophoresis in multiplex DNA sequencing can also be detected and subsequently analyzed.
Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well- to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm well-to-read distance) produces sequence read lengths of up to 1000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases/sample X 2 ten hour runs/day) to be sequenced with the advantages of infrared technology. Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany) and Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer and template combinations. The inclusion of (alpha) -Thio-dNTP's in the labeling reaction increases signal intensity two- to three-fold.
An infrared fluorescence microscope consisting of a laser diode for exciting infrared fluorophores attached to DNA oligo-nucleotides and a silicon avalanche photodiode for detecting the infrared emission has been designed. The microscope was mounted on a scanning platform which could be optimally focused on an electrophoretic gel (0.1 - 0.4 mm thick) sandwiched between two glass plates. Background fluorescence is minimal in the infrared region of the optical spectrum. In addition, the optics were designed to further minimize this background fluorescence while maximizing the signal output. A 5 pM fluorophore-DNA concentration in unpolymerized gel solution (about 2000 molecules in an irradiated volume of 600 pL) gave a signal-to-noise ratio of 4:1, 3:1, and 2:1 for a glass-gel-glass sandwich made using quartz, borosilicate, and soda-lime glass, respectively.
Conference Committee Involvement (2)
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications
25 January 2010 | San Francisco, California, United States
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications
26 January 2009 | San Jose, California, United States
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