- Phosphate Buffer Solution (PBS) for cell washing procedures.
- Latrunculin B solution for actin depolymerization. Store at
20 C. - pcDNA3 plasmid for eukaryotic cell transfection, encoding for
a GFP-tagged variant of the trans-membrane transferrin recep-
tor (GFP-TfR) or a GFP-tagged variant of glycosylphosphati-
dyl inositol (GPI-GFP).
2.3 Imaging
Equipment
and Software
- DMI6000 microscope equipped with TIRF modulus and iXon
Ultra 897 camera (seeNote 1). - LAS AF image acquisition and processing software.
- MatLab software for data analysis.
3 Methods
3.1 Sample
Preparation
- 48 h before the experiment, wash three times a 100 mm Petri
dish of confluent cells (CHO-K1, in the example reported
here) with PBS, add 1 ml of trypsin, and store the Petri in the
incubator (37C, 5% CO 2 ) for 5 min. Resuspend detached
cells by adding 9 ml of DMEM-F12 medium supplemented
with 10% of FBS and seed 100μl of cell solution to a Petri dish
containing 900μl of the same medium (seeNote 2). - Incubate the cells for 24 h at 37C and 5% CO 2.
- 24 h before the experiment transfect cells accordingly by using
Lipofectamine 2000 (following the manufacturer’s instruc-
tions) using the desired plasmid and incubate the cells for
24 h at 37C and 5% CO 2 before imaging.
3.2 Camera and PSF
Calibration
- Turn on the camera and wait for it to cool down. Set the proper
camera acquisition parameters (i.e., for the experimental sys-
tem proposed here, the exposure time is typically set to 0.5 ms,
the EMgain to 1000, the acquisition mode to “Cropped
Mode” (seeNote 3), the ROI size to 32128 (seeNote 4),
and the total number of repetitions to 10^4 ). More technical
details can be found elsewhere [23] - Start the acquisition of the camera background signal.
- Import acquired frame series to a data processing program (e.g.,
Matlab). Calculate and inspect the average intensity in each pixel
in order to verify that the camera background is approximately
flat in the selected region of the chip. Create a histogram of the
values(alsodefinedDigitalLevels,DLs)inacquiredimagesstack
(e.g., by using the “hist” command in Matlab) and plot the
logarithm of the resulting frequency (e.g., by using the “semil-
ogy” command in Matlab) (Fig.2,seeNote 5).
Fluctuation-Based Diffusion Laws 281