Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy

We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris­(2-carboxyethyl)­phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with UV light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for super-resolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell

Single color 3D imaging of hippocampal neurons by STORM and confocal.

<p><i>(A)</i> Mosaic 3D STORM image of hippocampal neurons. The color indicates z-position information according to the colored scale on the right. This image spans a volume of 147×80×1.4 µm <i>(B)</i> A zoomed-in view showing 2D maximum intensity projection of a neural process in confocal (<i>left</i>), confocal after deconvolution (<i>middle</i>). and STORM <i>(right)</i>. The left graph shows the intensity profile in the deconvoluted confocal image (<i>grey plot</i>) and the STORM image (<i>black plot</i>) across the red line indicated on both images. Similarly, the right graph shows the intensity profile in the deconvoluted confocal image (<i>grey plot</i>) and the STORM image (<i>black plot</i>) across the green line indicated on both images. The diameter of the neural process at the measured locations is on average 63 nm (FWHM) in STORM and 250 nm (FWHM) in confocal. <i>(C)</i> A zoomed-in view showing 2D maximum intensity projection of neural processes imaged by confocal (<i>left</i>), confocal after deconvolution (<i>middle</i>) and STORM (<i>right</i>). Two neural processes in close proximity are resolved in the STORM image but are not as clearly resolved in the confocal image. <i>(D)</i> The graphs show the intensity profile plotted across the red line shown in (<i>C</i>) for the confocal image after deconvolution (<i>grey plot</i>) and the STORM image (<i>black dotted plot</i>). Two peaks are visible in the STORM plot indicating the two distinct neural processes in the STORM image. <i>(E)</i> xy cross-section of a 100 nm thick slice of a small neural process taken from the midpoint image of a confocal (<i>left</i>) and STORM (<i>right</i>) stack. The middle panel shows the confocal slice after deconvolution. The membrane boundaries contain more labels and are clearly evident in the STORM slice. <i>(F)</i> Intensity profile across the cyan line shown in (<i>E</i>) for the confocal image after deconvolution (<i>grey plot</i>) and the STORM (<i>black plot</i>) image. The two membrane boundaries appear as two well-separated peaks in the STORM plot. <i>(G)</i> Vertical cross-section images across the three yellow lines shown in (<i>E</i>) for the confocal image after deconvolution and the STORM image. The STORM cross-sections look hollow in the middle, as expected for membrane labeling.</p

Two-color and multicolor (Brainbow-like) imaging of hippocampal neurons by STORM.

<p><i>(A)</i> A zoomed-in field of view of neural processes with (<i>left</i>) and without (<i>right</i>) the color information. The neurons were separately transfected with YFP and mCherry, mixed and co-cultured. For the STORM imaging, each fluorescent protein was immuno-stained with antibodies conjugated to different dye pairs. Neural processes that are clearly distinct in the two color images (<i>left, arrows</i>) are difficult to distinguish in the absence of color (<i>right, arrows</i>). <i>(B)</i> STORM image of neural processes labeled with a combination of three fluorescent proteins. The neurons were co-transfected with a mixture of the three fluorescent proteins. The co-transfection resulted in co-expression of different amounts of each fluorescent protein inside individual neurons and hence to different color combinations. Each fluorescent protein was immuno-stained with antibodies conjugated to different dye pairs. The arrow and arrowhead point to two neural processes that show different color combinations. <i>(C)</i> The STORM image of the same region of neural processes (<i>upper panels</i>) is shown in the presence (<i>left</i>) and absence (<i>right</i>) of color. The tracing results for these two cases are shown in the bottom panels. <i>(D)</i> Tracing results with (<i>left</i>) and without (<i>right</i>) color information for the image shown in (<i>B</i>).</p

Tracing of hippocampal neurons.

<p><i>(A)</i> Z-stack showing two neural processes that cross each other at different heights in confocal (<i>upper panels</i>), confocal after deconvolution (<i>middle panels</i>), and STORM (<i>lower panels</i>). <i>(B)</i> The xz cross-sections are plotted across the three green lines in (<i>A</i>) for the confocal image after deconvolution (<i>left</i>) and the STORM (<i>right</i>) image. The xz cross-section of the “top” and “bottom” neural processes cannot be easily discerned in the confocal images at the crossing point (<i>left</i>) as they merge together. Thus the two neural processes <i>(red</i> and <i>green circles</i>) appear to merge into one process (<i>yellow circles</i>). On the other hand, the membrane that separates the two neural processes is clear in the STORM cross-sections and a “top” (<i>red oval</i>) and “bottom” (<i>green oval</i>) neural process can be identified at all locations. <i>(C)</i> xy cross-section taken from the midpoint image of a 3D confocal (<i>left</i>) and STORM stack (<i>right</i>). The middle panel shows the confocal slice after deconvolution. <i>(D)</i> The graphs show the intensity profile across the red rectangle shown in (<i>C</i>) for the confocal image after deconvolution (<i>grey plot</i>) and the STORM (<i>black plot</i>) image. Three clearly separable peaks are seen in the STORM plot. The first peak is the membrane edge of the first neuron and the last peak is the membrane edge of the second neuron. The peak in the middle is the membrane boundary that separates the two neurons. <i>(E)</i> The difference in tracing results for this region in confocal (<i>left</i>) and STORM (<i>right</i>). The confocal tracing leads to one parent process splitting into two branches (<i>red</i>) whereas the STORM tracing leads to two neural processes (<i>red and green</i>) in close proximity. <i>(F)</i> Tracing results for an identical region of neurons in confocal (<i>left</i>) and STORM (<i>right</i>). Distinct processes are assigned different colors.</p

Comparison between cytoplasmic and membrane labeling for neuron imaging.

<p><i>(A)</i> STORM images of microtubules demonstrating the effect of label density. In the first panel the localizations from only the first few hundred frames of a STORM movie are included in the reconstructed image to simulate the effect that would be observed in the case of low label density. In the last panel localizations coming from the entire STORM acquisition are included to simulate the effect that would be observed in the case of high label density. The panels in between include progressively increasing number of localizations in the final reconstructed image. It is not possible to reconstruct the actual microtubule structure from the first image due to the low number of localizations, whereas the ability to reconstruct the microtubule structure increases with increasing number of localizations. <i>(B)</i> 2D STORM image of a neural process expressing YFP in the cytoplasm. The YFP was immuno-labeled with antibodies conjugated to photoswitchable A405-A647 pair for STORM imaging. The zoomed-in view shows a region with small neural processes. The small volume of these processes results in a low localization density in STORM images. <i>(C)</i> 2D STORM image of a neural process expressing mCherry attached to the membrane through a palmitoylation sequence. The mCherry was similarly immuno-labeled with antibodies conjugated to photoswitchable A405-A647 pair. The zoomed-in view shows a region of small neural processes. The membrane targeting resulted in a 3.6-fold improvement in label density.</p

CD81 is not required for virus binding, internalization or delivery into early endosomes.

<p>A) CD81-knockdown does not affect virus binding, as measured by flow cytometry. The magenta, blue and orange curves correspond to the intensity profiles measured for cells without adding viruses, control cells after influenza virus binding, and CD81-knockdown cells after influenza virus binding, respectively. B) The number of virus particles internalized is not affected by CD81 knockdown. The number of internalized virus particles was shown in a dot plot, with the middle line representing the mean value, and top/bottom line representing standard deviation. At least 40 randomly chosen cells were analyzed for each condition. C) The percent of virus particles colocalizing with early endosome is not affected by CD81 knockdown. Early endosomes were immunostained with anti-EEA1 antibody. Data was plotted similarly as in (B). At least 40 randomly chosen cells were analyzed for each condition. D) CD81 depletion does not affect RSV or pseudo-typed MLV infection. siRNA-treated A549 cells were infected with different doses of RSV and pseudo-typed MLV virus for 24 hours. For RSV virus infection, RSV fusion protein expression was quantified by flow cytometry, while for pseudo-typed MLV virus, the GFP signal was analyzed. A two-tailed student <i>t-test</i> was performed for all of the numerical data, and the p value of the data is shown.</p

CD81 is recruited to the virus budding sites.

<p>A) CD81 is recruited to the virus budding zone in X-31 infected cells. A549 cells were infected with X-31 for 16 hours. Cells were stained with anti-CD81 antibody (green) and anti-PB1 antibody (red). Images are confocal XY cross-sections. Scale bar: 10 µm. B) CD81 is incorporated into budding filamentous virions of Udorn-infected cells. A549 cells were infected with Udorn virus for 16 hours, and stained with anti-CD81 antibody and anti-PB1 antibody. Scale bar: 10 µm. C) Remaining CD81 in CD81-knockdown cells is incorporated into budding filamentous viruses of Udorn infected cells. Similar to (B) except that CD81-knockdown cells were used. The CD81 expression level in Udorn-infected cells was calculated based on confocal images of more than 100 cells, and was found to be decreased by ∼88% upon CD81 depletion as compared to control cells. The amount of CD81 per viral filament was reduced by 63% compared to that in untreated cells. Scale bar: 10 µm.</p

CD81 is enriched at specific sub-viral sites of budding virions and CD81 knockdown impairs virus scission.

<p>A) CD81 knockdown does not change the number of budding virions attached to infected cells. siRNA-treated cells were infected with WSN virus with the acid-bypass treatment for 15 hours. Cells were directly fixed for transmission electron microscopy and the number of budding virus particles per cell cross-section is quantified for over 250 sections, and presented in the dot plot. A two-tailed student <i>t-test</i> was performed and the p value is provided. B) CD81 knockdown causes a substantial reduction in the number of released virus particles. siRNA-treated cells were infected with WSN virus with the acid-bypass treatment for 17 hours. The amount of viral M1 protein in the supernatant was probed with ELISA. The number of M1 positive and HA positive virus particles in the supernatant was counted using immunofluorescence imaging. The error bar is standard deviation from three independent measurements. C) CD81 localizes at the tip of growing X-31 viruses during the early budding stages. Cells were infected with X-31 for 12 hours and CD81 was immunogold labeled for electron microscopy. An enlarged image of the area in the white box is shown in the upper right corner. Scale bar: 100 nm. D) CD81 mainly localizes at the tip and budding neck of the X-31 viruses during late budding stages. Similar to (C) except the infection time was 16 hours. Scale bar: 200 nm. E) Distribution of gold particles in budding X-31 viruses at 16 hour post infection. To align the virus particles, the length of each virus is normalized to 1, with its middle point assigned with coordinate value of 0. For individual gold particles on the budding virus, their coordinate values were calculated based on their relative distance to the middle point. Coordinates with negative values correspond to positions close to the plasma membrane. A total of 105 budding viruses were analyzed. F) Budding WSN viruses exhibit a spherical morphology with fully enclosed membrane envelope in control siRNA-treated cells. A549 cells were infected with virus with the acid-bypass treatment for 13 hours. The region in the white box is magnified and shown in the upper right corner. Scale bar: 200 nm. G) Budding WSN viruses are more elongated in CD81 siRNA treated A549 cells. A substantial fraction of budding viruses have an open membrane neck connected to the plasma membrane (indicated by arrowheads). The region in the white box is magnified and shown in the upper right corner. Scale bar: 200 nm. H) Budding WSN viruses are elongated upon CD81 depletion, as shown by the distribution of budding virus length in control or CD81-knockdown cells.</p

Scattered distribution of CD81 along budding filamentous Udorn virus.

<p>A) CD81 localizes along the filament of budding Udorn viruses. A549 cells were infected with Udorn virus for 18 hours and CD81 was immunogold labeled for electron microscopy. Shown here is a bundle of virus filament budding from the cell (the cell is not shown in order to magnify the virus filaments). Scale bar: 200 nm. B) CD81 and viral PB1 proteins appear to take an alternating distribution along filamentous Udorn virus. A549 cells were infected with Udorn virus for 16 hours and immunostained with anti-CD81 (green) and anti-PB1 (red) antibodies. CD81 was further probed with Alexa Fluor 405/Alexa Fluor 647-conjugated secondary antibody while PB1 was labeled with Atto 488 conjugated antibody for two-color 3D STORM imaging. Two example filamentous viruses were shown in B-1) and B-2). Left: xy projection images. Middle) xz projection images. Right) Localization distribution of CD81 and PB1 along the filament long axis. Scale bar: 500 nm.</p

A major fraction of viruses are trafficked to and fuse in CD81-positive endosomes.

<p>A) CD81 substantially colocalizes with Rab5. A549 cells were electroporated with CD81-mEmerald and RFP-Rab5. At 24 hours, the cells were fixed and imaged. An enlarged image of the boxed region is shown on the right. Scale bar: 10 µm. B) Influenza virus particles traffick into CD81+ endosomes. A549 cells were cold bound with Alexa Fluor 647-labeled X-31 virus (red) on ice for 30 minutes and then chased for 15 minutes at 37°C. The samples were fixed and immunostained against CD81 (green). An enlarged image of the boxed region is shown on the right. All of the images are confocal XY cross sections. Scale bar: 10 µm. C) An influenza virus particle enters and fuses within a CD81-positive endosome after entry. Live-cell confocal imaging of DiD-labeled X-31 added <i>in situ</i> to CD81-mEmearld expressing A549 cells maintained at 37°C. The images were collected with a 0.5 s interval. C-1) Several snapshots taken at different time points with the virus indicated by the white circles. C-2) The fluorescence signal of the indicated DiD-labeled virus as a function of time. Note that there is a sudden increase of DiD signal at 515 s, which indicates a viral fusion event. D) Influenza virus can also fuse in a CD81-negative endosome. D-1) Several snapshots taken at different time points with the virus indicated by the white circles. D-2) The fluorescence signal of the indicated DiD labeled virus as a function of time. The virus particle fused at 422 s. E) Among 61 virus particles tracked from binding to fusion, 52±8% enter and fuse within CD81+ endosomes whereas the remaining 48±8% fuse in CD81- endosomes. The results are taken for four independent experiments, and the ±error indicates the standard deviation derived from these experiments. F) Virus fusion is impaired upon CD81 depletion. DiD-labeled X-31 was allowed to bind with A549 cells on ice for 30 minutes, and then chased for the indicated times at 37°C. Cells were trypsinized and fixed immediately, and analyzed by flow cytometry. The increase in the DiD intensity versus the initial DiD intensity is plotted. The error bars are standard deviation derived from duplicate experiments.</p