Biology Reference
In-Depth Information
objective. The 488 nm line of the Ar ion laser was used for excitation of enhanced
green fluorescent protein (EGFP). The laser power was maintained at 1% throughout
the experiment with the emission collected through a 493-556 nm filter.
19.1.2
Cellular imaging and analysis
Investigation of peripheral protein association with cellular membranes requires
fluorescent labeling of the protein of interest and a technique that can scan fast
enough to detect the fluorescent protein in different pixels of an image. RICS is a
good method for meeting these objectives as the scanning movement creates a
space-time matrix of pixels within each image. From the stack of frames collected
for a live cell, a spatial correlation map can be obtained to determine the particle's
diffusion coefficient. Thus, when the spatial correlation map is obtained at different
subcellular locations, the diffusion coefficient can be determined at different cellular
sites. This provides information on how the membranes, organelles, or other cellular
components restrict the movement of fluorescently labeled proteins. On commercial
confocal microscopes, the spatial and temporal sampling time of the laser beam
(pixel dwell time) is known as is the scan time between scan lines and time between
images. Thus, using RICS allows for generation of spatial-temporal maps of
dynamics, occurring across the living cell. RICS data was acquired on a commercial
laser-scanning confocal microscope (Zeiss LSM 710 inverted microscope) using a
Plan-Apochromat 63x 1.4 NA oil objective. The 488 nm line of the Ar ion laser
was used for excitation of EGFP. The laser power was maintained at 1% throughout
the experiment with the emission collected through a 493-556 nm filter. The data
were collected as images of 256
s. RICS
analysis was done with SimFCS software using 100 frames from the series of images
(
Adu-Gyamfi et al., 2012
).
256 pixels with a pixel dwell time of 12.6
m
19.1.3
Imaging and analysis at the plasma membrane
In order to more carefully and selectively image VP40 assembly on the inner leaflet
of the plasma membrane, total internal reflection microscopy (TIRF) imaging was
employed (
Adu-Gyamfi et al., 2012, 2013
). TIRF imaging was performed using a
homebuilt TIRF imaging system (model No. IX81 microscope (Olympus, Melville,
NY) as described previously (
Ross et al., 2011
). Briefly, images were collected using
a Cascade 512B EMCCD camera. Samples were excited with the 488 nm line from
an Ar ion laser (Melles Griot, Albuquerque, NM) through a 60
1.45 NA oil objec-
tive (Olympus). To ensure cell integrity, cells were maintained at 37
C using a
thermostated stage (Tokai Hit, Shizuoka, Japan).
Images were collected at
256
256 pixels with a 50 ms exposure time per frame with 4000 total frames col-
lected. Images were saved as 16 bit unsigned and imported into the SimFCS software
(Laboratory for Fluorescence Dynamics, Irvine, CA). TIRF image series were ana-
lyzed using SimFCS (Laboratory for Fluorescence Dynamics, Irvine, CA). For N&B
analysis, 512 frames were analyzed per image series. HEK293 cells expressing
