ECFP was excited with a 405 nm diode laser, EYFP was excited with the 515 nm line of an argon ion laser and AF647 and Cy5 were excited by a 635 nm diode laser. For ECFP a 480–495 nm band pass filter was used to filter emission, for EYFP a 535– 565 nm band pass filter, and for AF647 and Cy5 a 655–755 nm band pass filter was used. For all images, the pinhole was set to obtain a 1 mm thick optical slice. Single particle tracking fluorescence microscopy For live cell imaging an inverted microscope (Olympus IX71) in an epi-fluorescent configuration with a 1.45 N.A., 60x, oil immersion objective (Olympus) was used to collect images. Excitation was supplied by two lasers: a tunable argon ion laser operating at 457 nm (35-LAP-431-208, Melles Griot, Carlsbad, CA) and a green diode (Green 532, Crystalaser, Reno, NV). Excitation beams were overlapped and focused on the back focal plane of the microscope objective. Cells were illuminated by both laser lines using a dichroic mirror (Z458/532/633RPC, Chroma). Excitation light was filtered from the emission by the appropriate filters: ECFP-Brightline 483/32 (Semrock, Rochester, NY) and EYFP-HQ580/50 (Chroma). Emission filters were mounted in a rotating filter wheel (FW103, Thorlabs, Newton, New Jersey). Images were recorded sequentially at a rate of 0.5 frames per second with a 200–300 ms exposure. The relatively fast switching time of the filter wheel (400 ms) in comparison to the motion of the vesicles (,1 mm/s) means that sequential imaging is equivalent to simultaneous imaging. Shutters (Uniblitz, Rochester, NY) limited exposure of the cells to the lasers. A full description can be found in Szymanski, et al [16]. Images were detected on an EMCCD camera (DU-888, Andor, South Windsor, CT). A heated stage plate and an objective heater were used to maintain the cells at 37uC during the course of experiments. Data analysis ImageJ (http://rsb.info.nih.gov/ij/) was used for data analysis. Colocalization was scored manually using ‘‘Image5D’’ (http://rsb. info.nih.gov/ij/plugins/image5d.html). Vesicles for analysis were chosen randomly by overlaying each image with a 10 mm610 mm grid. The vesicle closest to the center of the grid was used for analysis. Tracking was performed using the ImageJ measurement tool in combination with a frame-forwarding macro. Intensity levels for image analysis were determined by imaging cells labeled with single fluorophores and measuring the cross-talk between channels. Images for publication were background subtracted and intensities were adjusted equally within each data set. Significance testing was performed using a two-tailed Welch’s t-test to obtain a p-value. Supporting Information Figure S1 Rab7 and LAMP1 are highly colocalized in BS-C-1 cells and in HeLa cells. (A) A representative confocal microscopy image shows the overlay of ECFP-Rab7 (blue) from BS-C-1 cells stably expressing ECFP-Rab7 and LAMP1 (red), labeled with a primary antibody against LAMP1 (1:100, ab25630, Abcam, Cambridge, MA) and a Cy5-labeled secondary antibody (1:500, AP160S, Chemicon, Temecula, CA). Smaller images show the individual color components. We measured 8567% coloca- lization of Rab7-vesicles with LAMP1-vesicles and 9066% colocalization of LAMP1-vesicles with Rab7-vesicles. Colocaliza- tion values were calculated for 9–2 vesicles per cell for 10 cells in 2 distinct experiments. (B) A representative confocal microscopy image shows the overlay of endogenous Rab7 (red) and LAMP1 (blue). Smaller images show the individual color components. Endo-Lysosomal Vesicles Rab7 was labeled with a primary antibody against Rab7 (1:100, 9367, Cell Signaling, Danvers, MA) and a Cy5-labeled secondary antibody (1:500, ab97077, Abcam). LAMP1 was labeled with the same primary antibody as described above and a FITC-labeled secondary antibody (1:500, ab7064, Abcam). We measured 8568% colocalization of Rab7-vesicles with LAMP1-vesicles and 82611% colocalization of LAMP1-vesicles with Rab7- vesicles. Colocalization values were calculated for 10–15 vesicles per cell for 10 cells in 2 distinct experiments. (C) A representative confocal microscopy image shows overlaid ECFP-Rab7 (blue) and LAMP1-EYFP (green) images resulting from transient expression in HeLa cells. Smaller images show the individual color components. We measured 9166% colocalization of Rab7- vesicles with LAMP1-vesicles and 9164% colocalization of LAMP1-vesicles with Rab7-vesicles. Colocalization values were calculated for 12–14 vesicles per cell for 5 cells in 2 distinct experiments. (TIF) Figure S2 Similar levels of Rab7- and LAMP1-vesicle colocalization were observed with an alternate labeling scheme. A confocal microscopy image of BS-C-1 cells in which Rab7 is labeled with EYFP and LAMP1 is labeled with a primary antibody against LAMP1 (ab25630, Abcam) and a Cy5-labeled secondary antibody (AP160S, Chemicon). The inset, split into its individual color components and enlarged, shows a Rab7-vesicle (green, circled). The colocalization of Rab7-vesicles with LAMP1- vesicles (8964%) and the reverse (8966%) were similar to those obtained with the ECFP-Rab7/LAMP1-EYFP labeling scheme. Colocalization values were calculated for 25 vesicles per cell for 9 cells in 3 distinct experiments. (TIF) Figure S3 The colocalization of LAMP1- and Rab7/ LAMP1-vesicles with M6PR is not due to non-specific binding. (A) Confocal microscopy image showing the Cy5 emission from a BS-C-1 cell labeled with a primary antibody for M6PR and a Cy5-labeled secondary antibody. The corresponding three color image is shown in Figure 3A. (B) The Cy5 emission of a BS-C-1 cell with the same fixation, permeabilization, blocking, and imaging conditions in the absence of the primary M6PR antibody. (TIF) Figure S4 Colocalization of M6PR with Rab7-, LAMP1-, and Rab7/LAMP1-vesicles in BS-C-1 cells stably ex- pressing ECFP-Rab7 and in HeLa cells. (A) Confocal microscopy image of ECFP-Rab7 (blue) from a BS-C-1 cell line stably expressing ECFP-Rab7, the transient expression of LAMP1- EYFP (green), and an antibody against M6PR (MA1-066, Fisher Scientific) labeled with a Cy5 secondary antibody (red, AP160S, Chemicon). (B) A significant fraction of Rab7-, LAMP1-, and Rab7/LAMP1-vesicles are positive for M6PR; 4864%, 2767%, and 52612%, respectively. Error bars show standard deviations. The graph shows the analysis of 10 of each type of vesicle per cell in 9 cells. Similar results were obtained for BS-C-1 cells transiently expressing ECFP-Rab7, Figure 3. (C) Confocal microscopy image of ECFP-Rab7 (blue), LAMP1-EYFP (green), and an antibody against M6PR labeled with a Cy5 secondary antibody (red) in HeLa cells. (D) As with the BS-C-1 cells, a significant fraction of Rab7-, LAMP1-, and Rab7/LAMP1-vesicles are positive for M6PR; 39613%, 3364%, and 6466%, respectively. Error bars show standard deviations. The graph shows the analysis of 10–15 of each type of vesicle per cell in 5 cells. P-values,0.001 are indicated by ***,,0.01 by **. N.S. indicates p-values .0.05. (TIF) PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26626