3 August 2011
Robert Bagnell Pathology & Lab Med UNC-CH
Bagnell@med.unc.edu
Biosensor_FRET.ijm
INTRODUCTION –
This ImageJ macro processes time-lapse image stacks made from live
cells transfected with the single chain CFP-YFP FRET biosensor from
Klaus Hahn for detection of RAC and RHO activity. The image processing
steps are those outlined in the paper: Imaging Spatiotemporal Dynamics of
Rac Activation in Vivo with FALIR by Hahn et al in Methods in Enzymology,
Vol 325.
While the Hahn biosensor was the impetus for creating this macro, the
macro can be utilized for any single chain biosensor exhibiting FRET,
where it is not necessary to make bleed-through corrections.
WHAT IS REQUIRED ImageJ 1.4 or later is required.
http://rsb.info.nih.gov/ij/
In addition to the standard set of macros and plugins that come with
ImageJ, this macro uses the following macros and plugins that are
available for download from the ImageJ web page or were specially made
for this purpose and are available from the MSL web page
http://www.med.unc.edu/microscopy
Biosensor_FRET macro available from MSL.
Biosensor_FRET_Names is a plugin that ask the user to assign the
names of open stacks to names that Biosensor_FRET expects – available
form MSL.
BiosenProcess1-9 MSL Robert Bagnell March 2011
StackAverage_16 is a16-bit version of the standard ImageJ plugin
StackAverage - available from MSL.
Infinity_to_zero is a plugin that sets any pixel in a 16-bit stack that is
65535 to 0 - available from MSL.
Shading_Corrector plugin form the ImageJ plugins collection at
http://rsb.info.nih.gov/ij/plugins/index.html
SubtractMeasuredBackground from the ImageJ macros collection at
http://rsbweb.nih.gov/ij/macros/SubractMeasuredBackground.txt
TurboReg plugin from
http://bigwww.epfl.ch/thevenaz/turboreg/
DoRT_Curve_Fit_Bob is a plugin that utilizes the built-in ImageJ curve
fitting routines to do a double exponential curve fit in the Photobleach
correction section - available from MSL.
For imaging systems that save an alternating sequence of CFP / FRET
images as a single file, such as Velocity, the plugin SubstackMakerPlus,
from MSL, can be used to easily create separate CFP and FRET image
stacks. This plugin is an adaptation of the ImageJ plugin SubstackMaker.
These plugins should be installed in the ImageJ Plugins folder and the
Subtract Measured Background macro in the ImageJ Macros folder.
Placing the Biosensor_FRET macro in the Plugins folder will make it
available in the ImageJ Plugins menu.
Image stacks for the plugin are made as follows:
1. The CFP stack is a series of images over time in the CFP setting
(CFP excitation, CFP emission).
2. The FRET stack is a companion time series of images in the FRET
setting (CFP excitation, YFP emission).
3. The CFPdark and FRETdark series are taken in the CFPsetting and
the FRETsetting with the light path to the camera blocked (about 20
images each is enough; exposure times should be manually set to
about the same as for CFP and FRET). These are for correcting the
camera’s dark current.
BiosenProcess1-9 MSL Robert Bagnell March 2011
4. The CFPshade and FRETshade sets are taken in their respective
settings from an area of the sample that has no cells, with the
microscope slightly defocused, and with a small amount of very dilute
fluorescein well mixed into the culture medium to produce an overall
fluorescence (about 20 images each is enough). The brightness of
these images should be only about 1/5 the brightness of the CFP and
FRET images. These are for correcting the uneven illumination of the
imaging system.
Before the Biosensor_FRET macro is started, six stacks of images must be
open. The macro will ask you to assign your open stacks to the following
names, so your names can be different from these.
CFP
CFPshade
CFPdark
FRET
FRETshade
FRETdark
HOW TO USE IT -
1.
2.
3.
4.
5.
6.
7.
Brief protocol for using the macro:
Drop the 6 image stacks on ImageJ.
Select the Biosensor_FRET macro in the Plugins menu.
Assign your stack names to the macro’s names.
Use the check box to select/unselect dark correction.
Use the check box to select/unselect sub-pixel registration.
When asked in a dialog box to draw an area of interest (AOI) in a
background region, use the rectangle tool to choose a background
region. Look through the entire stack to be sure no bright artifact drifts
into the region you choose. Click OK in the dialog box. Repeat for the
second stack of images - the box you drew in the first stack is
duplicated in the second stack so you only have to click OK.
When asked to set a threshold, if the Adjust Threshold window is not
open, open it (Image/Adjust/Threshold). Select the Dark Background
BiosenProcess1-9 MSL Robert Bagnell March 2011
check box (ImageJ 1.4 required). Adjust the red threshold to cover
the entire cell without extending the selection beyond the cell border.
Click OK in the Action Required dialog box. Repeat for the second
stack.
8. A photobleach curve is generated based on the threshold settings in
step 7. These settings have a large effect on the photobleach
calculation so you will be given a chance to change the settings in
case the result is not satisfactory. When a “Simplex Fitting Options”
dialog box appears click its’ OK. Examine the generated curve in the
“Curve Fit” window and the R^2 value in the “Message” window
(close to 0.95 is good for R^2). Click OK in the Message window. If
you are happy with the curve fit, uncheck the retry checkbox and click
OK. If you are not satisfied with the fit, choose to repeat the curve fit
with a different threshold selection by clicking OK in the Retry box.
When asked, set a threshold on the brightest part of the cell and click
OK in the dialog box. Repeat for the second stack. You can repeat
this process as many times as you like.
9. Select a lookup table (Image/Lookup Tables) and click the play button
on the resulting image sequence. To see the values of the lookup
table select Image/Color/Show LUT.
The Log window shows the values of the curve fit for each image. The
Results window shows the photobleach correction value by which each
image was multiplied.
WHAT IT DOES The macro is extensively documented within the micro file. In general, it
performs the following steps in order:
1. Check to see if 6 stacks are open and exit if not.
2. Assign user file names to macro file names.
3. Ask if the user wants to do dark correction.
4. Ask if the user wants to do sub-pixel registration. In some cases subpixel registration is not necessary and it saves time to omit it.
5. Open a text window to show progress of the macro. The text window
is in the upper left corner of the screen.
6. Create an averaged image from each of the dark noise stacks.
7. Stacks and images that are no longer needed are closed.
BiosenProcess1-9 MSL Robert Bagnell March 2011
8. The averaged dark current images are subtracted from their
respective stacks (CFP, FRET, CFPshade and FRETshade) then
averaged shade images are made from each of the shade stacks.
9. Shade correction is done by dividing the CFP and FRET stacks by
their averaged shade images using the Shading Corrector plug-in.
10.
Background subtraction is done by asking the user to choose
an appropriate background region in the CFP stack. The same region
is also used in the FRET stack. The average intensity of that region is
calculated and subtracted from every pixel of every image in the
stack. NOTE: It is a good idea to look through the stack to make sure
no bright artifact drifts into the background region that you choose.
11.
Sub-pixel registration, if selected, is done using the TurboReg
plugin selecting rigidbody (translation and rotation) registration in
automatic and accurate mode. The image pair being registered are
copied from their stacks, registered and the FRET image returned to
its stack in place of the unregistered image. The FRET images are
registered to the CFP images. Registration is done prior to masking
because TurboReg does some image processing and uses every
pixel in the image during registration so a previously masked image
cannot be used. TurboReg produces a “Refined Landmarks” table
that indicates the amount of adjustment that was made for each of
the three landmarks used in rigid body alignment. Comparing the
source to target values in that table indicates the amount of
adjustment in x and y that was made. Unfortunately only the last
value is left on the table.
12.
Images are masked such that the background intensity is 0 and
the cells are grayscale. This requires that the user set a threshold on
the cells using the Image/Adjust/Threshold dialog box. The user
must select “dark background” in the dialog box before setting
the threshold. A message box prompts the user to do this.
13.
Photobleach correction is started by using the threshold that
was set in the masking step. The user can change the threshold if the
result is not satisfactory. Photobleach correction is based on the ratio
of the change in mean brightness of the thresholded parts of the cells
over time. FRET and CFP mean values are normalized to the first
value in each set and a ratio is calculated of the normalized values.
Thus ((FRETmean#/FRETmean1) / (CFPmean#/CFPmean1)) is
calculated for every time point. This data is the Y-axis of a graph and
image number (i.e. time) is the X-axis. A double exponential curve is
BiosenProcess1-9 MSL Robert Bagnell March 2011
fit to this data. A “Simplex Fitting Options” window is displayed with
default values. The user can change these values, but they should be
OK. A curve fit graph is generated as well as a table giving the R^2
value for the fit (0.95 or better is excellent). Since setting the
threshold can be tricky, the user is allowed to repeat this step, setting
the thresholds themselves, if they are not satisfied with the curve fit or
the R^2 value. After the FRET / CFP raw ratio stack is calculated,
each image is multiplied by the inverse of the curve value at its
respective time point to correct for the amount of photobleach.
14.
The final FRET / CFP ratio is calculated and the photobleach
correction is applied. The result is in 32-bit floating point. Before
converting this to 16-bit integer, a histogram stretch is done to insure
that all values are kept within range. The user should apply a look-up
table of their choice (Image/Lookup Tables and Image/Color/Show
LUT) to the resulting stack to get the best idea of pixel intensities.
BiosenProcess1-9 MSL Robert Bagnell March 2011