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Description

TIRF time-lapse imaging of EB1-GFP (green) and mCherry-tubulin (red). EB1-GFP is seen on microtubule tips that grow to the cortex but does not accumulate on stable microtubule bundles at the equator, in contrast to kinesin-6 . Corresponds to video 8 from J Cell Bio 186:727-738, 2009. Total internal reflection microscopy was performed using a microscope (Perfect-focus TE2000; Nikon) with a 100×, 1.45 NA objective (Nikon) and illumination from either a 488-nm argon laser (100 mW) or a 491-nm solid-state laser (100 mW) and a 561-nm solid-state laser (50 mW). For dual-color TIRF microscopy, we used a triplepass dichroic filter (z491/561/633rpc) and changed the emission filter (ET525/50 or ET595/50; both from Chroma Technology Corp.) with a filter wheel placed before the camera. In some cases, an excitation notch filter was used in the filter cube (NF01-405/488/561/635; Semrock, Inc.). Images were typically captured every 2-3 s with a 50-200 ms exposure with an EM charge-coupled device camera (iXon; Andor Technology). The microscope was controlled and images were acquired using open source MicroManager software (http://www.micro-manager.org).

Biological Sources

NCBI Organism Classification
Drosophila melanogaster
(Common fruit fly)
Cell Type
epithelial cell
Cell Line
S2
Cellular Component
EB1
microtubule

Biological Context

Biological Process
microtubule polymerization

Attribution

Names
Ronald D. Vale
James A. Spudich
Eric R. Griffis
Published
J Cell Bio 186:727-738, 2009
Pubmed
19720876

Grouping

This image is part of a group.

Imaging

Image Type
recorded image
Imaging Mode
TIRF
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of a specific protein
Visualization Methods
GO:0046785
mCherryFP
Processing History
unprocessed raw data
color combine
Data Qualifiers
raw, unprocessed data

Sample Preparation

Methods
living tissue
Relation To Intact Cell
dispersed cells in vitro

Dimensions

Spatial Axis Image Size Pixel Size
X 360px ——
Y 322px ——
Channel Wavelength
1 488, 561 nm
Time 8 sec
*CIL – Cell Image Library accession number. Please use this to reference an image.