|
With the sensitivity turned up, low intensity
excitation-light can be used; a key to successful live cell
imaging.
The software
controlled filter block carousel in the Axiovert 200M microscope
allows automated switching between filter block combinations
ensuring optimal fluorophore separation in multi-wavelength
experiments. |
|
 HeLa cells growing at 37°C in
a sealed chamber with normal experimental buffer supplemented with
10 µM staurosporine to induce apoptosis; 2 µl/ml Annexin
V-AlexaFluor 488 (green) to indicate phoshotydylserine flipping; and
2 µM propidium iodide (red) to reveal loss of membrane integrity.
Images were acquired with a conventional widefield, fluorescence
imaging system equipped with time-lapse functionality (Perkin Elmer) using a
×20 phase objective.
Images
were acquired every 6 minutes. (T. Collins, Babraham Institute, UK).
|
|
A Sutter
Lambda 10-3 filter wheel system allows more rapid switching
between excitation wavelengths for dual excitation ratio-imaging
(e.g. the Fura divalent cation sensitive dyes; the SNAFL pH
sensitive dyes).
The system also has a Uniblitz brightfield shutter, allowing
acquisition of transmitted phase-contrast or DIC images alongside
fluorescence images.
The ASI
M2000 motorised x, y-stage allows acquisition of multiple fields of
view during long-term time lapse work. This can be from multiple
wells in a tissue culture plate or multiple regions of a single
coverslip. It also allows acquisition of large image montages of
fluorescent samples. |
SHSY5Y cells
loaded with Fura2 and treated with 20 µM carbachol. Click
here for higher resolution AVI. |
|
This hardware is controlled by IPLab software allowing custom
acquisition routines to be written for fast or long-term
acquisition.
The facility has ordered
(due Dec 2005) a
Bioptech's microperfusion pump for perfusion of acute tissue
and a Neue LiveCell 37°C/5%CO2 microincubator for long term
imaging of cultures cells and tissue. Objective heaters and stage
heaters are also available.
A stage and objective heater are also
available as well as a Sutter MP-285 micromanipulator and Eppendorf micromanipulator and transjector.
|
