We thank Stephan Uphoff for discussions. 
R.C. was supported by Linacre College, Oxford University. A.P. was supported by the German Academic Exchange Service (DAAD), the German National Academic Foundation and EPSRC. M.S. was supported by the Wellcome Trust. A.N.K. was supported by a UK BBSRC grant (BB/H01795X/1), and a European Research Council Starter grant (261227).

<ol>
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The authors have nothing to disclose.

Many parameters can be varied during cell electroporation and data acquisition depending on the biological system of interest and the precise nature of the experiment (cell-level or single-molecule analysis). For example, when electroporating DNA into bacteria, 0.25 to 5 pmol of labeled dsDNA fragments leads to a low internalization efficiency, allowing direct single-molecule detection (i.e., without the need for photobleaching beforehand). Above 5 pmol dsDNA, cells tend to be heavily loaded, a regime better suited for single-cell analysis. All labeled DNAs should also be previously gel-purified in order to remove any trace of free dye (non-reacted fluorophore) from the DNA stock solution. Besides, potential issues with DNA degradation, particularly for smFRET experiments, can be addressed by using DNAs with unnatural nucleic acids, or motifs that protect exonuclease-accessible termini such as hairpin loops.
Another adjustable parameter in electroporation is the field strength applied during electroporation. Low field strength (~1 kV/cm) will lead to a low loading efficiency appropriate for single-molecule studies. Higher field strengths (up to 1.8 kV/cm) will increase the loading efficiency; however, there is an inverse correlation between field strength and cell viability after electroporation (see Figure 4). For reference, a normal field strength used for bacteria and yeast electroporation is ~1.5 kV/cm. The time constant, representing the length of this decay, is a convenient parameter to follow, since the time constant drops as soon as any arcing phenomenon occurs in the cuvette. Under normal settings, the time constant should be greater than 4 ms; lower values will lead to low loading efficiency or even non-loaded damaged cells. Most electroporators offer other degrees of freedom (such as &#8220;pulse truncation&#8221; or &#8220;pulse shape&#8221;) which can be modified to tune both cell loading and viability. We applied this method to both bacteria and yeast, however similar procedures should also allow the internalization of labeled biomolecules into mammalian cells using appropriate electroporator settings since their membrane is actually less complex (single lipid bilayer) and since electroporation has already been used with such cells 21.
When internalizing labeled proteins, all free dye needs to be removed from the labeled protein stock solution prior electroporation. Free dye molecules, due to their smaller size, can be internalized preferentially over the proteins of interest, and are difficult to discriminate during data analysis (despite their expected faster diffusion). As a guide, for a sample of organically labeled protein to be suitable for electroporation, the amount of remaining free dye should be below 2% (detected using fluorescent scanning of a SDS-PAGE)22. This process is particularly important, as some molecules might stick to the outer membranes of electroporated bacteria or yeast. In this respect, the negative control sample should display fluorescence intensity per cell clearly lower than electroporated cells, ideally as low as the autofluorescence level of empty cells (cells which have not been incubated with any fluorescently labeled biomolecules nor electroporated, Figure 2).
As with dsDNA, the internalization efficiency of labeled proteins is linked to the amount of biomolecules added to the cells prior to electroporation. However, other parameters, such as size and charge, play a role in internalization. Small proteins exhibit high internalization efficiencies, whereas larger proteins (up to 98 kDa) can be successfully internalized but with lower efficiency (Figure 5)26. The isolelectric point of the protein, potential interactions with the cell membrane and other physicochemical parameters also influence cell loading during electroporation. As a result, users need to optimize experiments for their own system, knowing that a high initial concentration of labeled protein (&#62;50 &#956;M) will give the best chance for successful loading. Electroporation also offers a new tool to perturb and analyze cellular function by introducing proteins and other biomolecules into cells (either labeled or unlabeled). The T7 RNA polymerase experiments (Figure 5C) present such an example of an experiment where we can introduce a biomolecule that can change gene expression in vivo using electroporation.
When performing single-molecule fluorescence experiments, TIR illumination is usually favored over other illumination modes as it offers the best signal-to-noise ratio by exciting only fluorophores within a thin section above the coverslip surface (~100 nm). However imaging labeled biomolecules diffusing inside living microorganisms might require deeper illumination (up to 0.8 &#181;m for E. coli). Deeper illumination is achieved in HILO mode, while preserving a high signal-to-noise ratio. On the other hand, wide field imaging is particularly important for step-wise photobleaching analysis, where the user is estimating the number of internalized molecules by photobleaching an entire loaded cell with high laser power and dividing the initial cell fluorescence intensity by the unitary intensity produced by a single molecule (single photobleaching step, Figure 3). Widefield imaging is also required for long-term molecule tracking in order to localize the diffusing molecules of interest even if their trajectories cover the entire cell volume.
In this protocol, we present how electroporation, a standard technique for biologists and biochemists for delivering nucleic acids in cells, constitutes a simple method for delivering fluorescent biomolecules in various cell types. This novel, high-throughput technique offers a unique tool to observe labeled molecules in their native environment. In addition biomolecules labeled with fluorophores covering a wide range of wavelengths, electroporation can deliver molecules modified with many chemical groups, such as unnatural nucleotides and amino acids, metal chelators, crosslinkers, and caging groups. If the biological system of interest is not essential to the cell development, the gene encoding for the target protein can also be deleted (or knocked-down), ensuring that the proteins observed after internalization represent all (or most) of the intracellular protein pool. In essence, electroporation can &#8220;transplant&#8221; the flexibility of in vitro bioconjugates into living cells and therefore benefit efforts in synthetic biology, systems biology, and in vivo single-molecule detection.

Most fluorescence studies inside living cells depend on protein fusions with fluorescent proteins (FPs), such as GFP1. These fluorescent tags allow studies of the copy number, diffusion pattern or localization of proteins involved in processes such as gene expression or membrane transport2-7. FPs offer high labeling specificity, easy implementation, and are available in a large inventory of variants with various photophysical and chemical properties1. However, organic fluorophores remain the prime choice for in vitro experiments due to their greater photostability (up to 100-fold more stable than FPs)8,9, small size (up to 100-fold smaller volume than FPs) and ease of intramolecular labeling (mainly through the use of cysteine residues). All these factors are particularly important for single-molecule fluorescence and FRET studies10.
Several internalization methods combining the advantages of organic labeling and in vivo detection have been introduced over the past decade; however, such methods either employ relatively large polypeptides tags (e.g., TMP, HALO, or 20 kDa SNAP tags)11-14, require the use of unnatural amino acids15, or are limited to large, single-membrane eukaryotic cells (e.g., scrape loading, syringe loading, microinjection)16-19.
This protocol describes a novel, straightforward and high-throughput internalization method that couples the advantages of organic fluorophores with in vivo observation. To develop this technique, we adapted the electroporation procedure commonly used to transform cells with plasmid DNA20,21 in order to load microorganisms, such as E. coli or S. cerevisi&#230; with organically labeled biomolecules. The protocol consists of 4 simple steps: incubation of cells with labeled biomolecules, electroporation, cell recovery, and cell washing to remove non-internalized biomolecules. Here, we present this electroporation protocol, as well as the cell imaging and data analysis processes to study cell-based and single-molecule fluorescence and FRET signals.
Electroporation relies on discharging a high-voltage electric field across a low ionic strength cell suspension to form transient membrane pores through which biomolecules can enter cells (Figure 1)20,21. Just as with transformation of bacteria or yeast with plasmid DNA, cells have to be prepared prior to electroporation to ensure their electrocompetency. This procedure, consisting of several washing steps with water, increases the membrane permeability and lowers the ionic strength of the cell solution to avoid arcing in the electroporation cuvette. In this protocol, cells can be prepared as described below (See PROTOCOL: 1.1) or bought from commercial providers.
<img alt="Figure 1" src="/files/ftp_upload/52208/52208fig1.jpg" /> 
Figure 1: Schematic representation of the internalization protocol. From left to right: add a few microliters of labeled biomolecules to the aliquot of electrocompetent cells (doubly-labeled DNA fragments and bacteria in this example); incubate 1 to 10 min&#160;on ice and transfer to a pre-chilled electroporation cuvette; electroporate and then add 0.5-1 ml rich medium to the cells immediately after; incubate at 37 &#176;C (or the temperature required by the organism, e.g., 29 &#176;C for yeast) to let the cells recover; perform 5 washing steps to remove any excess non-internalized labeled molecules; resuspend the final pellet in 100-200 &#956;l of PBS buffer and pipette 10 &#956;l on an agarose pad; cover the pad with a cleaned coverslip and image on a fluorescence microscope (in wide field mode or HILO mode).
Electrocompetent cells are incubated with the labeled biomolecules just before electroporation, which can be performed using standard electroporators found in most biochemistry laboratories. Immediately after electroporation, cells are incubated in a rich medium allowing their recovery before washing (Figure 1). The excess of non-internalized labeled biomolecules is first removed by washing in a buffer containing a fairly high concentration of salt and some detergent (See PROTOCOL: 3.3). The presence of salt disrupts non-specific electrostatic interactions formed by non-internalized labeled biomolecules which otherwise may stick on the outer membrane. Similarly, the presence of detergent in the washing buffer disrupts non-specific hydrophobic interactions.
While DNA internalization is straightforward (Figure 2), precautions need to be taken when internalizing labeled proteins using electroporation. First, the stock sample of organically labeled protein might still contain a small percentage of free dye. Free dye molecules are much smaller than proteins and might therefore be internalized preferentially. To ensure that the vast majority of the observed internalized fluorescent molecules correspond to the protein of interest, the initial protein sample should contain less than ~2% free dye (Figure 5)22. The excess of non-internalized labeled proteins can also stick to the outer cell membrane after electroporation; this phenomenon is protein-specific and needs to be checked for each new protein. We propose several options that allow the removal of non-internalized proteins from the loaded cell sample (See PROTOCOL: 3.3.3).
Finally, cells are resuspended in a small volume of phosphate buffer and pipetted onto an agarose pad, allowing their imaging on a fluorescence microscope. Immobilization on agarose pads is a simple and efficient way of imaging cells on a coverslip without harming their integrity. The pad should contain a low-fluorescence culture medium.
Cell imaging can be performed either in widefield, total internal reflection fluorescence (TIRF) or using HILO (Highly Inclined and Laminated Optical Sheet) microscopy. In the HILO configuration, the laser beam penetrates deeper into the specimen than in TIRF, yet does not illuminate the entire sample as for widefield, allowing a greater signal-to-noise ratio23. Depending on the laser power and time resolution used, internalized biomolecules can be counted (using stepwise-photobleaching analysis, Figure 3), localized, or tracked24-28. Internalization of doubly labeled constructs with a FRET pair of fluorophores allows the quantification of FRET both at single-cell or single-molecule levels (Figure 6).
Different parameters can be varied depending on the desired output and the biological system studied. First, the amount of internalized material per cell can be tuned by altering the concentration of labeled biomolecules added to the cells prior electroporation (Figure 2). Electroporation field strength will also influence both the loading efficiency and cell viability; as expected, while the loading efficiency increases with increasing field strength, the viability of electroporated cells decreases (Figure 4A). Both parameters can be quantified by recording the percentage of loaded and dividing cells after electroporation. This viability assay coupled with fluorescence imaging also verifies the observation of internalized biomolecules in living cells and allow continuous observation over several generations (Figure 4B).
In summary, this protocol allows the internalization of fluorescently labeled DNA and protein molecules into E.coli or S. cerevisi&#230;26. Individual molecules labeled with organic fluorophores can be tracked with high spatiotemporal resolution for timescales an order of magnitude longer than FPs. Finally, this method is compatible with widefield, TIRF and confocal detection, as well as pulsed excitation schemes, such as ALEX (alternating laser excitation28,29).

<table><tbody><tr><td>ElectroMax DH5-alpha Comptent cells</td><td>Invitrogen</td><td>11319-019</td><td>or any other commercial or lab-mage electrocompetant bacteria or yeast.</td></tr><tr><td>EZ Rich Defined Madia</td><td>Teknova</td><td>M2105</td><td>low fluorescence rich media</td></tr><tr><td>MicroPulser Electroporation Apparatus&amp;nbsp;</td><td>Biorad</td><td>165-2100</td><td>or any classical electroporator for microorganism transformation</td></tr><tr><td>Certified Molecular Biology agarose</td><td>Biorad</td><td>161-3100</td><td>low fluorescence agarose for agarose pad</td></tr><tr><td>Microscope coverslips No 1.5 thickness</td><td>Menzel</td><td>BB024060SC</td><td>remove background particles by heating slides in furnace at 500 &amp;deg;C for 1h</td></tr><tr><td>Single-molecule fluorescence microscope</td><td>Home-built</td><td></td><td>described in REFs</td></tr><tr><td>Localization software</td><td>Custom-written, available online</td><td></td><td>MATLAB and C++ software package that can be adapted for localization analysis.&amp;nbsp;</td></tr><tr><td>Tracking software</td><td>Available online</td><td></td><td>MATLAB implementation by Blair and Dufresne.&amp;nbsp;</td></tr></tbody></table>

internalization and observation of fluorescent biomolecules in living microorganisms via electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Sample preparation
The different steps of the protocol are presented as schematics in Figure 1. As an example, we represented the loading of bacteria with doubly labeled (donor and acceptor dyes) DNA fragments. Representative results for DNA internalization are shown in Figure 2. For each electroporated sample, data for empty cells and non-electroporated cells were also recorded (Figure 2). &#8220;Empty cells&#8221; correspond to electrocompetent cells neither incubated with fluorescent biomolecules nor electroporated; their intensity in the fluorescent channel reflects the autofluorescence level under identical experimental conditions (laser power, time resolution, temperature, etc.). &#8220;Non-electroporated cells&#8221; (also called &#8211;EP, i.e., minus EP) correspond to a negative control in which electrocompetent cells have been incubated with the fluorescent biomolecules but not electroporated. These non-electroporated cells should exhibit a fluorescence level similar to the autofluorescence of the empty cells and significantly lower than the fluorescence intensity displayed by loaded, electroporated cells. This confirms the removal of any non-internalized labeled biomolecules that could have adhered to the outer cell membrane.
<img alt="Figure 2" src="/files/ftp_upload/52208/52208fig2.jpg" /> 
Figure 2: Representative results for the internalization of dsDNA labeled with different fluorophores at different concentrations in bacteria (A-E) and yeast (F). Left to right: white-light, fluorescence and overlay images. -/+ EP denotes incubation without/with electroporation. Scale bars: 3 &#181;m. A. Cy3B dsDNA, 10 pmol, E. coli. B. ATTO647N dsDNA, 10 pmol, E. coli. C. Alexa647 dsDNA, 5 pmol, E. coli. D. Cy3B dsDNA, 100 pmol, E. coli. E. ATTO647N dsDNA, 100 pmol, E. coli. F. ATTO647N dsDNA, 30 pmol, Yeast. This figure has been modified from reference 26. <a href="https://www.jove.com/files/ftp_upload/52208/52208fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Counting the number of internalized biomolecules per cell
The procedure to estimate the number of internalized labeled biomolecules per cell using photobleaching analysis is presented in Figure 3 and Supplementary Movie 2, together with representative results obtained with different concentrations of labeled DNA. Cell loading efficiency increases with the initial amount of incubated labeled DNA, allowing the user to tune the number of labeled molecules per cell from a &#8220;single-molecule&#8221; level (&#60;10, Supplementary Movie 2B) to an &#8220;ensemble&#8221; level (&#62;10, Supplementary Movie 2A). A robust way of estimating the percentage of loaded cells is to count the number of electroporated cells displaying a cell intensity above the average cell intensity of non-electroporated cells plus 3 times their standard deviation (i.e., Av(I&#8211;EP)+3*StdDev(I&#8211;EP) where Av = average, I = intensity per pixel, Std. Dev. = standard deviation and &#8211;EP = non-electroporated) as shown in Figure 3.
<img alt="Figure 3" src="/files/ftp_upload/52208/52208fig3.jpg" /> 
Figure 3: Counting the number of internalized molecules using photobleaching analysis. (A) Single-cell photobleaching analysis. Examples of fluorescence intensity timetraces (blue: raw data;&#160;red: fit; insets:&#160;WL&#160;and&#160;fluorescence images&#160;of&#160;E.&#160;coli&#160;loaded with ATTO647N-labeled dsDNA before and after bleaching). Top: single-step bleaching event. Middle: cell containing &#177;3 molecules showing bleaching and blinking. Bottom: cell containing &#62;10 steps corresponding to at least 10 molecules. (B) Histogram of single-step height intensities from an automated step-fitting algorithm from 57 cells containing less than 6 distinguishable steps. Single-Gaussian fit is centered at 11 &#177; 3 a.u., corresponding to a unitary fluorophore intensity of 8100 photons per second. The asterisk marks the bin gathering all the step heights above or equal 50 a.u. (C) Histogram of internalized molecules per cell electroporated with different amounts of ATTO647N dsDNA, calculated after dividing the initial fluorescence intensity by the unitary fluorophore intensity. Top to bottom: empty cells (i.e., not incubated with fluorescent molecules and not electroporated), non-electroporated (but incubated with fluorescent molecules, named &#8211;EP), and electroporated cells incubated with 10 and 100 pmol dsDNA (named +EP). Empty and non-electroporated cells correspond to autofluorescence, whereas electroporated cells show a broad distribution of internalized molecules, with a higher proportion of highly loaded cells at 100 pmol (&#8805; 4 molecules, see asterisk-marked bin). Internalization efficiency (fraction of cells with Int. &#62; mean + 3x Std. Dev. of non &#8211;EP sample) for the 10 and 100 pmol samples was 94% and 90%, respectively. Mean number of internalized molecules per cell: 121 &#177; 106 molecules for 10 pmol dsDNA, and 176 &#177; 187 molecules for 100 pmol dsDNA. Settings: 100 ms exposure, widefield illumination. Scale bars: 1 &#956;m. This figure has been modified from reference 26. <a href="https://www.jove.com/files/ftp_upload/52208/52208fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cell loading and viability
In addition to changing the amount of labeled biomolecules added to the cells prior to electroporation, the user can tune the amount of internalized molecules by choosing different field strengths during electroporation (Figure 4, Supplementary Movie 1). Higher field strengths lead to greater internalization efficiencies, but lead to a slight decrease of cell viability. For protein internalization, the use of a filtration step may help remove non-internalized labeled proteins (see 3.3.3.1.1). In such cases, cell filtration ensures that observed fluorescent proteins are indeed internalized inside the bacterial cytoplasm; we note, however, that filtration also has a negative impact on cell viability (for more details, see REF 22).
<img alt="Figure 4" src="/files/ftp_upload/52208/52208fig4.jpg" /> 
Figure 4: Influence of electroporation voltage upon cell loading and viability. (A) Bar chart representing the effect of electroporation field strength on loading efficiency (red bars) and viability of the cells (green bars). 84 &#177; 8 % of the non-electroporated cell (0 kV/cm) divide after 1 h on an agarose pad at 37 &#176;C. Under the same conditions, 78 &#177; 3 % and 49 &#177; 3 % of the cells electroporated at 0.9 kV/cm and 1.8 kV/cm respectively divide after 1 hr. For loading efficiency, 73 &#177; 8 % of the cells are loaded at 0.9 kV/cm, while 92 &#177; 6 % of the cells are loaded at 1.8 kV/cm. The error bars represent the standard deviation calculated from three independent measurements; more than 200 cells were analyzed for each sample and each repeat. Overall loading efficiency increases with electroporation voltage to the slight detriment of cell viability. (B) Cell-based fluorescence measurements over several generations show that the overall fluorescence intensity is shared equally between both daughter cells. Cell 1 and 2 refers to the cell number in the white light image (left) and fluorescence image at t = 0. Scale bar: 1 &#181;m). This figure has been modified from reference 26.
Protein internalization
Representative results for protein internalization are in Figures 5A &#38; B. It is particularly important to remove as much of the remaining free (unreacted) dye from the protein sample as possible prior to electroporation. In the examples in Figures 5A &#38; B, the Cy3b-labeled Klenow Fragment sample (Cy3b-KF, where KF is the Klenow fragment of the E. coli DNA Polymerase I, 66 kDa) only contains 1% free dye; such dye contribution to the overall cell loading is negligible. Comparisons of the electroporated sample of interest with both the non-electroporated cells (incubated with the same amount of labeled protein) as well as cells electroporated with the equivalent amount of free dye constitute two required controls to ensure that the observed fluorescent molecules are indeed internalized labeled proteins.
<img alt="Figure 5" src="/files/ftp_upload/52208/52208fig5.jpg" /> 
Figure 5: Protein internalization in live bacteria. (A) Representative fluorescence overlay field of views. Cells electroporated at 1.4 kV voltage with 50 pmol RNAP &#969; subunit from a protein stock solution that contained only 1% free Cy3b dye. Non-electroporated (non &#8211;EP) and empty cells are defined as previously. Free dye was internalized at the same concentration as in the RNAP &#969; electroporated sample. Imaging in wide field mode, 532-nm excitation at 1 mW, 50 ms exposure. (B) Distribution of uncorrected cell-averaged intensities for samples in (A), given in proportion of the total cell count. More than 400 cells per sample were segmented. This figure has been modified from reference 22. (C) Internalization of unlabeled T7 RNA polymerase (T7 RNAP, 98 kDa) into electrocompetent DH5&#945; carrying the pRSET-EmGFP plasmid encoding emerald GFP (EmGFP) under control of a T7 promoter. Left: Schematic of assay. Middle: fluorescence overlay. Right: histograms of cell-based fluorescence intensities for the non-electroporated sample (top) and cells incubated and electroporated with T7 RNAP (bottom); approximately 11% of the electroporated cells show high fluorescence intensity (fraction of cells with Int. &#62; mean + 3x Std. Dev. of non &#8211;EP sample) indicating expression of EmGFP. The asterisks indicate bins gathering all the intensities above or equal 1,100 a.u. Scale bar: 3 &#956;m. This figure has been modified from reference 26.
Figure 5C presents another application of protein electroporation. Here, the electroporated protein is unlabeled, but its internalization triggers an observable fluorescent response. This experiment verifies the presence and functionality of electroporated proteins in the cell cytoplasm. Unlabeled T7 RNA polymerase (98 kDa) was internalized into E. coli strain DH5&#945; containing a plasmid encoding for fluorescent protein EmGFP under the control of a T7 promoter26. As the gene for T7 RNA polymerase is absent in DH5&#945;, EmGFP expression in our experiments requires that functional T7 RNA polymerase is introduced into the cells via electroporation (Figure 5C). Following electroporation with 1 pmol T7 RNAP, &#62;11% of the cells (blue bar, Figure 5C) exhibited fluorescence higher than the negative control (incubated with the same amount of T7 RNAP, but not electroporated). This result establishes that a proportion of the T7 RNAP molecules internalized by electroporation retain their integrity in vivo and can perform their intended functions in the cell cytoplasm.
In vivo FRET at the single-molecule and single-cell levels
Finally, the internalization and analysis of doubly labeled species in living bacteria is presented in Figure 6 and Supplementary Movie 3. As fluorescent protein fusions are not ideal for in vivo smFRET studies, the ability to deliver doubly labeled biomolecules into living cells using electroporation is one of the great assets of this method. Figure 6A presents single-cell FRET analysis of bacteria loaded with different FRET DNA standards (using Cy3B and Atto647N fluorophores as donor-acceptor FRET pair). Cells are electroporated with 20 pmol of three short doubly-labeled dsDNA FRET standards with apparent FRET efficiencies (E*) of 0.17, 0.48, and 0.86 in vitro (previously determined26). All DNAs enter cells efficiently (Figure 6A, left) and the main peak of each single-cell E* distribution agrees well with the in vitro results (Figure 6A, right). In the intermediate- and high-FRET samples, cell populations with lower E* than expected are observed, presumably due to a combination of acceptor bleaching and photophysical inactivity, variable cell loading (thus, variable signal-to-noise ratio) and DNA degradation.
<img alt="Figure 6" src="/files/ftp_upload/52208/52208fig6.jpg" /> 
Figure 6: Representative results for single-cell and single-molecule FRET observation in living bacteria. Ensemble and smFRET studies in single bacteria. (A) Analysis of cells loaded with 20 pmol each of three DNA FRET standards exhibiting low (~0.17), intermediate (~0.48), and high (~0.86) FRET (as measured using in vitro single-molecule measurements; see REF 26). Left: white light and green/red (FRET) fluorescence overlay images (Scale bar: 3 &#956;m). Examples of FRET values from different cells are indicated (white). Right (top to bottom): uncorrected cell-based FRET (E*) histograms for donor only (dark green), low (light green), intermediate (yellow), and high (red) FRET DNA standards. (B&#8211;D) In vivo smFRET. Cells are loaded with 0.25 pmol intermediate-FRET DNA (panel B), 0.25 pmol high-FRET DNA (panel C), and 5 pmol doubly-labeled KF (panel D). Left column: green/red fluorescence overlay of single frame before and after acceptor photobleaching. Middle column: time traces corresponding to the molecule in the yellow circle. FRET efficiencies, donor emission intensities, and acceptor emission intensities are displayed in blue, green, and red, respectively. Right column: FRET histograms of donor only molecules (green) and donor-acceptor molecules (yellow, red, and gray) from 20 time traces for each sample. Scale bars: 3 &#956;m for A, 1 &#956;m for B&#8211;D. This figure has been modified from reference 26. <a href="https://www.jove.com/files/ftp_upload/52208/52208fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To observe smFRET in vivo for DNA or protein samples, low amounts (0.25 pmol) of intermediate- and high-FRET DNA standards (Figure 6B, C) or 5 pmol of double-labeled KF (Alexa647/Cy3B fluorophores as FRET pair, Figure 6D) are electroporated into E. coli. Such concentrations led to many cells loaded with few (n = 1&#8211;10) labeled molecules, allowing direct localization, tracking, and FRET monitoring for single molecules. Some molecules diffuse freely, whereas others appear immobile or diffuse slowly (Supplementary Movie 3). Timetraces of immobilized doubly-labeled biomolecules (Figure 6, middle) last for 1 to 30 s and show the hallmarks of smFRET: anticorrelated changes in the donor and acceptor fluorescence upon acceptor bleaching (for example, t~16 sec; Figure 6B, middle), followed by single-step donor bleaching (for example, t~;19 sec; Figure 6B). FRET distributions generated from such timetraces (Figure 6, right) result in a mean value that is in excellent agreement with published in vitro studies26,31,32. These results establish the capability for quantitative smFRET studies on internalized DNAs and proteins, and suggest that proteins maintain their integrity and structure upon electroporation and internalization (as supported by the T7 RNAP internalization experiments).
Supplementary Movie 1: Cell viability. Left: White light images. Right: fluorescence images. Animated GIF animated showing the division after electroporation (1.8 kV/cm) of bacteria loaded with 10 pmol Atto647 labeled DNA. The overall apparent decrease of fluorescence is due to the dilution of labeled DNA upon cell division and also partially to the photobleaching which occurs during each measurement.
Supplementary Movie 2: Cell-based photobleaching studies. A. Representative example of a heavily loaded cell (containing &#62;100 Atto647N-labeled DNA molecules). Top left, white light image of the cell of interest (red rectangle). Top right, movie of the loaded cells showing their fluorescence decay over several minutes. Bottom, time trace of the overall fluorescence intensity decay of the cell of interest. Organic fluorophores can exhibit photobleaching lifetimes 2 orders of magnitude higher than FPs (here, ~41 sec for Atto647N). B. Representative example of a cell loaded with less than 10 labeled molecules (3 in this case). Top, same as panel A. Bottom, time trace of the overall fluorescence intensity of the cell of interest showing single step bleaching and/or blinking corresponding to single organic fluorophores. The average height of these steps corresponds to the single-molecule unitary intensity (here ~12 a.u.) used to estimate the initial number of internalized molecules per cell. Movies under continuous red laser excitation at 300 &#181;W power and 100 msec per frame.
Supplementary Movie 3: In vivo single-molecule FRET. Top: Cells loaded with 0.25 pmol high-FRET DNA (as in Figure 6C) continuously monitored at 50 ms per frame under nTIRF illumination using 1 mW green (532 nm) laser. Each frame is a green/red (FRET) fluorescence overlay of each channel. Diffusing and immobile red (intact) and green (single active label) DNA molecules can be observed. Bottom: Time trace corresponding to the molecule in the yellow circle. FRET efficiencies, donor emission intensities, and acceptor emission intensities are displayed in blue, green, and red, respectively. Anti-correlated acceptor bleaching event (red-to-green transitions) corresponds to the signature of single-molecule FRET.

Watch this Scientific Journal Video about Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation at JoVE.com

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

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Biology

16.8K Views.  University of Oxford. Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Video: Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Immunology and Infection

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Source: Sontag, R. L. et al., <a href="https://app.jove.com/v/52296">Electroporation of Functional Bacterial Effectors into Mammalian Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated antibodies.

Wprowadzenie bakteryjnych białek efektorowych do komórek ssaków za pomocą elektroporacji

Source: Sontag, R. L. et al., Electroporation of Functional Bacterial Effectors into Mammalian Cells. J. Vis. Exp. (2015)
In this video, we demonstrate ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

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Electroporation of Functional Bacterial Effectors into Mammalian Cells

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