I S Methods IV : Image processing

IS A Calculation of ErbBl-GFP diffusion coeBicient

Each frame from an individual FRAP time lapse sequence (as recorded on the confocal microscope) was saved as a 12-bit TIFF file. The data was analysed by importing the raw data into a program which was written in the software package Mathematica (Wolfram research). The program was designed to calculate the diffusion coefficient, D, from an individual FRAP time lapse sequence. Firstly, the fluorescence intensity in the bleached ROI was corrected for any bleaching of the sample that occurred during image acquisition. This was achieved by calculating the average intensity of the fluorescence in a region outside the bleached ROI, and dividing the signal from the bleached ROI by this value, for every image in the time-lapse sequence. D was then calculated by fitting the normalised data to the following function:

f i t ) = exp ( - 2 t d / 1) [/„ ( 2 t d / O + / ; ( 2 t d / / )]

where, and / , are modified Bessel functions and T^ = w ‘‘ 14D , where w = is the radius of the bleached ROI (Soumpasis, 1983). The value of D was then estimated by solving the equation: / 4D for D (i.e. D = w ^ I 4Td). The process was repeated for 10 time lapse sequences allowing the mean value of D, and the standard deviation over the

10 measurements, to be calculated.

2.5.2 Analysis of FLIM data

FLIM data series were saved as IPlab image files consisting of a stack of 17 images (16 phase-dependent images and 1 background image). Image processing was performed using a PC running the Linux operating system, and proceeded via a series of sequential steps. 7. Correction fo r backgound. The average intensity of the background image was subtracted from all phase images in the FLIM series. This background- corrected series was used for all further image processing. 2. Calculate DC image. An

___________________________________________________________________ C hapter! image representing the mean average fluorescence intensity at each pixel across the series (‘direct current’ image or DC image) was calculated by summing all the images across the series. 2. Intensity thresholding. Using the DC image, an image ‘mask’ was generated where pixels of interest = 1 and unwanted pixels = 0. Unwanted pixels included any pixels below a certain threshold intensity value (as defined by the operator) and any pixels where the fluorescence signal was saturated. Note that the mask is a separate image from the data. 3. Correction fo r photobleaching. Each of the pairs of equivalent phase images of the forward and reverse cycles were summed to first order correct for photobleaching of the donor. 4. Application o f the analysis to determine fluorescence lifetimes. Each background- and photobleaching-corrected FLIM data series was analysed using only pixels where the corresponding pixels in the mask = 1. Two types of analysis were performed on the data to resolve the lifetimes. Firstly, each FLIM data series was analysed individually using a standard Fourier transform to calculate frequency-weighted lifetime estimates according to the phase shift and the demodulation at each pixel of the image, producing two separate images showing the phase lifetime and modulation lifetime at each pixel, respectively (see Squire & Bastiaens, 1999; Harpur & Bastiaens, 2000; Verveer et al., 2001). Secondly, the pixels from many FLIM data sets were linked in a global analysis which assumed the presence of two populations of receptors associated with one of two spatially invariant lifetimes. The details of the algorithm employed are described elsewhere (Verveer et al., 2000a). The global analysis generated the following outputs: 1. the spatially invariant lifetimes of each ErbBl-G FP species: ErbBl-G FP in the presence of FRET (Tj) and ErbB l-G FP in the absence of FRET (Ti), and 2. a fractional population map, for each FLIM data series, showing the molar fraction of ErbBl-G FP molecules with lifetime Tj at each pixel (a map). Application of an appropriate pseudocolor table to the population maps allowed the level of phosphorylation at every pixel of the images to be examined. For statistical purposes, the a map raw data was used to calculate the percentage of ErbBl-G FP molecules with lifetime Tj in each image i.e. the percentage of phosphorylated ErbBl-G FP molecules present was calculated for each FLIM series.

Fig. 2.1

Figure 2.1 Frequency-domain FLIM set-up

See main text for details.

Chapter 3

Chapter 3

Quantitative Imaging of ErbBl Lateral