Efficiency of light projection is dependent on the LEDs etendue.

<p>A lens (blue) is used to collimate the light from an LED with multiple emitters (green squares). While light from the central emitter (green rays) is projected onto the objective aperture, light from off-center LEDs does not reach the objective aperture (grey rays). In addition, outer beam angles produce a wider beam, which also cannot be directed into the objective aperture (black rays).</p

Filterset choice determines LED choice.

<p>The excitation (blue, thin line), emission (red, thin line) and dichroic filter properties (green, thin line) for the Chroma ET-EGFP/mCherry set (Chroma 59022) provided by Chroma [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref013" target="_blank">13</a>] are plotted and overlaid with the spectra of the Oslon SSL 80 470nm LED (Oslon 470, blue, thick line), the Epitex SMBB490-1100-02 490 nm LED (Epitex 490, cyan, thick line) and the Luxeon PC amber 595 nm (Luxeon 595 amber, thick line) provided by Thorlabs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143547#pone.0143547.ref014" target="_blank">14</a>]. The 470 nm LED spectrum fits well with the first excitation of the filter set, while the 490 nm LED light, although more ideal for the excitation of EGFP is largely blocked by the excitation filter. The phosphor-converted 595 LED spectrum is broad and only parts of the emitted spectrum can be used.</p

Open LED Illuminator: A Simple and Inexpensive LED Illuminator for Fast Multicolor Particle Tracking in Neurons

<div><p>Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution.</p></div

Characterization of PRV recombinants.

<p>(A) Cells infected with parental viruses (PRV Becker, 180) or gM-pHluorin expressing viruses (PRV 483, 486) were harvested at 12 hpi, and lysates were analyzed by Western blot. Parallel blots were probed with polyclonal antiserum against gM (αgM), or a monoclonal antibody that recognizes pHluorin (αGFP). (B) Single-Step Virus Replication. Parallel cell cultures were infected in triplicate with the indicated parental viruses (PRV Becker, 180), gM-pHluorin expressing viruses (PRV 483, 486), or a gM-null virus (PRV 130). Cells and supernatants were harvested at indicated times, and infectious virus titer was measured by plaque assay. Error bars represent range. (C) Membrane Topology. Particles produced by the indicated viruses were imaged to detect gM-pHluorin or gM-EGFP, mRFP capsid, and immunofluorescence targeting the pHluorin or EGFP epitopes (αGFP IF). Immunofluorescence labeling was performed without membrane permeabilization. The schematic represents the predicted topology of gM-pHluorin or gM-EGFP. Images depict single representative virus particles (each image is 2.5 µm by 2.5 µm). Bar graph represents classification and quantification of particles based on fluorescence (n≥237 particles per condition). (D) pH Sensitivity. Particles produced by the indicated viruses were imaged to detect gM-pHluorin or gM-EGFP, and mRFP capsid after addition of buffers at pH 6 or 7. Images depict single representative virus particles (each image is 2.5 µm by 2.5 µm). Graph represents relative particle fluorescence after each indicated buffer change (n≥154 particles per condition).</p

Verification of the cell-based complementation assay.

<p><b>(A)</b> Flpe-NIH were nucleofected with the indicated BACs and plasmids and mixed with NIH/3T3. Samples were fixed at 6 dpt, stained with an antibody specific for the IE1 protein and IE1-positive plaques quantified. Depicted are average and SD of triplicate samples of two independent experiments. EV, empty vector. <b>(B)</b> Flpe-NIH were treated as in (A), stained with antibodies specific for the IE1 (pp89) and the major capsid protein (MCP) and images taken using a fluorescence microscope. <b>(C)</b> Flpe-NIH were nucleofected with the ΔM53 BAC and a rescue plasmid expressing the indicated M53 mutants and treated as in (A). Depicted are mean and SD of duplicates of two experiments. Mutants were generated by transposon-based random mutagenesis and described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094918#pone.0094918-Lotzerich1" target="_blank">[33]</a>. Their capacity to complement the loss of the wt pM53 protein is indicated below the diagram. EV, empty vector; i, insertion of five amino acids; s, stop mutation at this position.</p

High time-resolution tracking and MSD analysis of particle movement.

<p>(A) PRV 483 infected cells were imaged at a rate of 25–50 frames/second, and particles were tracked before and after exocytosis. Graph shows one representative particle track, color-coded to indicate relative gM-pHluorin fluorescence. The location of gM-pHluorin dequenching is indicated (arrow). Bracketed regions correspond to pattern of movement indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat-1004535-g002" target="_blank">Figure 2E and F</a>: (2) Terminal pause. (3) Sharp jerk. (4) Mostly immobile. (B) Average MSD curves of particle tracks before and after exocytosis. Based on slope and MSD values, particles are confined an area approximately 400 nm in diameter before and after exocytosis (n = 43 exocytosis events, in 9 cells, in 3 independent experiments). Dotted MSD curve represents particles immobilized on glass (n = 249 particles). Shaded areas represent standard error of the mean.</p

Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

<div><p>Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.</p></div

Viral exocytosis occurs most frequently near patches of LL5β.

<p>(A–B) Cells were transduced to express mRFP-LL5β, infected with PRV 486 expressing gM-pHluorin, and imaged at 4.5–5 hr after PRV infection. Data represent 150 exocytosis events in 9 independent experiments. Scale bars represent 2 µm in all images. (A) Image is a maximum difference projection depicting exocytosis events over a 10 min time course. Particle exocytosis events are classified according to their proximity to mRFP-LL5β patches (yellow circles), or lack thereof (green squares). (B) Still images of a single exocytosis event, corresponding to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s006" target="_blank">Movie S6</a>. (C) Schematic of molecular and cellular mechanisms that coordinate viral transport and exocytosis. Please refer to the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#s3" target="_blank">discussion</a> section for references supporting the depicted molecular links.</p

Live-cell fluorescence microscopy of particle transport and egress.

<p>(A) Schematic of virus particle transport and exocytosis assay. gM-pHluorin incorporated into virus particles or secretory vesicles is quenched in the acidic lumen of secretory vesicles (black circles), but the mRFP capsid tag is not (red hexagon). Upon exocytosis, pHluorin is exposed to neutral extracellular medium, and becomes fluorescent (green circles). (B) Virus particle exocytosis. PRV 483 infected cells were imaged at 4.5–5 hpi. Image is a maximum difference projection corresponding to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s001" target="_blank">Movie S1</a>, depicting viral exocytosis events over a 13 min time course. Exocytosis of gM-pHluorin particles that do not contain capsids (green squares) and particles containing both gM-pHluorin and mRFP capsids (yellow circles) are indicated. Scale bar represents 2 µm. (C) Still images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s001" target="_blank">Movie S1</a>, depicting a single viral exocytosis event. Images correspond to the boxed area in panel B. Scale bar represents 1 µm. (D) Transcytosis of virus inoculum is almost never observed. Cells were infected with PRV 495 and imaged as in panel B. Image is a maximum difference projection corresponding to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s002" target="_blank">Movie S2</a>, depicting exocytosis events over a 13 min time course. Exocytosis of gM-pHluorin particles that do not contain capsids are indicated by green squares. Particles containing mRFP capsids are almost never observed (not shown). Scale bar represents 2 µm. (E) Kymograph of a virus particle exocytosis event from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s001" target="_blank">Movie S1</a>, depicting mRFP capsid (red) and gM-pHluorin (green) fluorescence over time. (F) Ensemble average of relative gM-pHluorin fluorescence (top, green line), mRFP capsid (top, red line), and instantaneous velocity (bottom, blue line) over 32 exocytosis events, in 10 cells, in 4 independent experiments. Shaded area represents standard deviation. (D and E) Virus particles exhibit stereotyped pattern of movement. (1) Fast directed transport. (2) Terminal pause. (3) Sharp jerk. (4) Mostly immobile.</p