All measurements of bead FWHM, except the measurement of the 2P excitation PSF, were calculated using the ImageJ plugin “Plot FWHM” that fits a Gaussian function to vertical and horizontal cuts centered on the brightest point in the image (http://www.umanitoba.ca/faculties/science/astronomy/jwest/plugins.html courtesy of Jennifer West, University of Manitoba). The emission PSF was measured by exciting the bead sample with filtered light (Semrock, FF02-482/18-25) from a halogen lamp (i.e., using the transillumination pillar of our microscope to provide wide-field illumination), and collecting emission through a 525 bandpass (Semrock, FF01-525/50-25) filter. The 2P excitation PSF lateral FWHM was manually estimated from a plot of the average intensity of a 6 × 6 pixel region centered on a bead, as the excitation light was sequentially stepped across the field of view (note that the emission galvo was held stationary for this measurement; only the excitation galvos were scanned). Images were cropped prior to analysis.
Ff01 525 50 25
Manufactured by IDEX Corporation
The FF01-525/50-25 is a narrow bandpass optical filter manufactured by IDEX Corporation. It is designed to selectively transmit light within a specific wavelength range while blocking unwanted wavelengths. The filter has a center wavelength of 525 nm and a full width at half maximum (FWHM) of 50 nm. It is housed in a 25 mm diameter enclosure.
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Lab products found in correlation
2 protocols using ff01 525 50 25
1
Characterizing Fluorescent Bead Imaging PSF
2
Rab13 Biosensor Imaging and FRET Analysis
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Constitutively active (Q67L) and dominant-negative (S22N) Rab13 biosensors were transiently transfected into HEK293T cells using the JetPRIME reagent following the manufacturer’s protocols. Cells were plated onto coverslips and imaged with an inverted microscope (IX81-ZDC; Olympus) equipped with an 40× oil immersion DIC objective lens (NA 1.3) and a charge-coupled device camera (CoolSNAP ESII; Roper Scientific), using the fluorescence excitation and emission optical configuration as used for imaging of the FRET biosensor MCF10A and MEF cell lines. The mCer3 fluorescence emission intensities were recorded from 10 random fields of view followed by additional 10 random fields of view but postirradiated through a filter (FF01-525/50-25; Semrock) for 2 min to photobleach the mcp229Venus, using the 100-W Hg Arc lamp as the light source. The flat-field–corrected and background-subtracted mCer3 images before and after photobleaching were analyzed for the mean per pixel gray levels, and the FRET efficiency limits were calculated using the following equation:
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