Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low-comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls-these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, ß-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.

Fluctuation correlation spectroscopy and photon histogram analysis of light scattered by gold nanospheres.

Fluorescence correlation spectroscopy (FCS) is a valuable tool in biological research. In recent years there has been growing interest in using light scattered from metallic colloids in place of organic fluorophores. Metallic colloids display optical cross sections for scattering that are orders of magnitude brighter than fluorophores. We used the FCS method to study the scattering properties of varying sizes of gold colloids 38-100 nm in diameter. The optical cross sections of the gold colloids increase rapidly with size, as can be seen by both the G(0) value of the autocorrelation function and the scattering intensity distributions. In mixtures of different size gold colloids the autocorrelation function is dominated by the larger (brighter) colloids, even when present at a small fractional population. We show that it is possible to detect one 100 nm gold colloid in the presence of 10(3)-10(4)smaller 39 nm diameter colloids. Because the scattering cross sections of colloids will increase with aggregation, we believe that FCS can be used to detect a small number of associated bio-labeled colloids in the presence of a much larger population of non-associated colloids.

Oligonucleotide immobilization on micropatterned streptavidin surfaces.

We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n(-3/4).

Real-time velocity of DNA bands during field-inversion gel electrophoresis.

The velocity v of bands of double-stranded, linear DNAs containing 48.5-5700 kbp was determined with 0.3 s resolution during field-inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pulse period T+, keeping the duration of the backward pulse T- = T+/3. Within 0.6 s or less after the field changed sign from-to +, the velocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to -. For long pulses, the magnitude of this spike increased with M0.36, reaching ten times the steady-state velocity for M = 5.7 kbp. After this spike, the velocity dipped to 55-75% of its value in a steady field, then increased to a small secondary peak before reaching a steady-state plateau. The duration of the velocity trough, and the time of the small peak, increased as M1. For standard FIGE conditions (ratio of forward:reverse pulse duration, T+:T- = 3:1; equal forward and reverse field amplitudes, E+ = E-), the mobility mu = integral of vdt over a complete cycle was a minimum when E+ terminated at the end of the velocity trough. The minimum occurred because the velocity during E+ sampled primarily the trough, and because the backward velocity during E- was exceptionally large; the negative velocity spike was maximized when T+ terminated at the end of the velocity trough. Computer simulations of FIGE by Zimm (J. Chem. Phys. 1991, 94, 2187-2206) and by Duke and Viovy (J. Chem. Phys. 1992, 96, 8552-8563) generate real-time velocities that are in excellent agreement with our experimental data.