Phasor FLIM analysis in cultured HeLa cells.

(A) The phasor histogram of images shown in panel B. The grey dotted line indicates the axis used for color-coding the FLIM images in (B) and (C). (B) FLIM images 24 h after transfection of the stable control RNA, construct 1, construct 2 and the stable control RNA without TMR in cultured HeLa cells. The scale bar is 30 μm. (C) FLIM images for all measured constructs and time points. These measurements are the same as those shown in Fig 4. The scale bar is 200 μm.</p

Evaluation of the degradation of the dual-labeled RNAs in cell extract by different techniques.

The constructs were incubated in HeLa cell extract for 3 h and the degradation was monitored with a confocal microscope. The degradation was analyzed using FCS (A), FCCS (B) and FRET via intensity (C) and fluorescence lifetime (D). The curves were normalized to 1 for the initial data-point.</p

Quantification of the fluorescence lifetime measurements.

<p>(A-D) Distribution of the pixels along the line connecting the mono-exponential decays at 4.1 ns and at 1.25 ns in the phasor plot for the four modification patterns. (E) Summary of the average fluorescence lifetimes of the cell populations shown in panels A-D using a Gaussian fit to the distribution. The error bars represent the standard deviations of three independent measurements.</p

Design of the dual-labeled RNA oligonucleotide.

<p>(A) 23 nucleotide RNA oligonucleotide conjugated to tetramethylrhodamine (TMR) at its 5’ end <i>via</i> a thioether bond and to Atto488 at its 3’ end <i>via</i> an amide bond. Upon exposure to the cellular environment, the oligonucleotide can be degraded by various RNases. (B) Modification patterns selected to monitor intracellular localization and integrity of the oligonucleotide. RNA backbone modifications to modulate stability towards nucleolytic degradation: 2’-F, 2’-O-Me and phosphorothioate.</p

Fluorescence intensities of HeLa cells in culture after transfection with oligomer <i>278</i>.

<p>(A) A U-shaped, sequence defined cationizable lipo-oligomer <b><i>278</i></b> for complexation of the dual-labeled RNAs (C: cysteine, K: lysine, Stp: succinoyl-tetraethylene pentamine, linA: linoleic acid). (B) Fluorescence intensity images of the HeLa cells, 15 min, 1 h, 6 h and 24 h after transfection of the four different modifications patterns. The contrast level is equal for all images. The scale bar represents 200 μm. (C) Average fluorescence count rate of the cells at the different conditions shown in (B). The error bars represent the standard deviation of three independent measurements.</p

Monitoring oligonucleotide degradation using FCS, FCCS and FRET.

<p>The stability of various RNAs was measured as a function of incubation time in cell extracts. The main changes and parameters corresponding to RNA degradation are shown exemplary for construct 2, representing: (A) the diffusion time from the autocorrelation function (FCS), (B) the amplitude of the cross-correlation function (FCCS), (C) an apparent FRET efficiency determined from the fluorescence intensity and (D) the donor fluorescence lifetime based FRET using a phasor analysis. The colored crosses represent the center of mass in the phasor plot of measurements after 1 min (blue), 60 min (green), 120 min (orange) and 180 min (magenta). The grey arrows indicate the direction of the main changes.</p

Three-Dimensional Tracking of Carbon Nanotubes within Living Cells

Three-dimensional tracking of single-walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map corral volumes (0.27–1.32 μm³), determine an active transport velocity (455 nm/s), and calculate local viscosities (54–179 cP) within the cell. With respect to the future use of SWNTs as sensors in living cells, we conclude that the sensor must change the fluorescence signal by at least 4–13% to allow separation of the sensor signal from fluctuations due to rotation of the SWNT when measuring with a time resolution of 32 ms

A New Photostable Terrylene Diimide Dye for Applications in Single Molecule Studies and Membrane Labeling

A new terrylene diimide-based dye (WS-TDI) that is soluble in water has been synthesized, and its photophysical properties are characterized. WS-TDI forms nonfluorescing H-aggregates in water that show absorption bands being blue-shifted with respect to those of the fluorescing monomeric form. The ratio of monomeric WS-TDI to aggregated WS-TDI was determined to be 1 in 14 400 from fluorescence correlation spectroscopy (FCS) measurements, suggesting the presence of a large amount of soluble, nonfluorescent aggregates in water. The presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption of the aggregates due to the formation of monomers in micelles. This is accompanied by a strong increase in fluorescence. A single molecule study of WS-TDI in polymeric films of PVA and PMMA reveals excellent photostability with respect to photobleaching, far above the photostability of other common water-soluble dyes, such as oxazine-1, sulforhodamine-B, and a water-soluble perylenediimide derivative. Furthermore, labeling of a single protein such as avidin is demonstrated by FCS and single molecule photostability measurements. The high tendency of WS-TDI to form nonfluorescent aggregates in water in connection with its high affinity to lipophilic environments is used for the fluorescence labeling of lipid membranes and membrane containing compartments such as artificial liposomes or endosomes in living HeLa cells. The superior fluorescence imaging quality of WS-TDI in such applications is demonstrated in comparison to other well-known membrane staining dyes such as Alexa647 conjugated with dextran and FM 4-64 lipophilic styryl dye