Biosensor_FRET Macro Descriptionx

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Nov 6, 2013 (3 years and 5 months ago)

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BiosenProcess1
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9 MSL Robert Bagnell March 2011

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 Enz
ymology,
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 a
re
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.


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9 MSL Robert Bagnell March 2011

StackAverage_16

is a
16
-
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 exp
onential curve fit in the Photo
bleach
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 plug
in 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 fold
er.
Placing the Biosensor_FRET

macro in the Plugins folder will make it
available in t
he 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 enou
gh
; exposure times should be
manually set to
about
the same as for CFP and FRET
)
.
These are for correcting the
camera’s dark current.

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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
fluore
scein
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 bright
ness of the CFP
and
FRET
images.
These are
for
correcting

the uneven illumination of the
imaging system
.


Before the Biosensor_FRET macro is started,

s
ix 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
-


Brief protocol for using the macro:

1.

Drop the 6 image stacks on ImageJ.

2.

Select the Biosensor_FRET

macro in the Plugins menu.

3.

Assign your
stack names to the macro’s names.

4.

Use the check box to select/unselect dark correction.

5.

Use the check box to select/unselect sub
-
pixel registration.

6.

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 f
or the
second stack of images
-

t
he box you drew in the first stack is
duplicated in the seco
nd stack so you only have to click OK.

7.

When asked to set a threshold, if the Adjust Threshold window is not
open, open it (Image/Adjust/Threshold). Select the Dark Background
BiosenProcess1
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9 MSL Robert Bagnell March 2011

check box

(ImageJ 1.4 required)
. Adjust the
red
threshold to cover
the entire cel
l 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 photobl
each
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 bo
x. Repeat for the second stack. You can repeat
this process as many time
s

as you like.

9.

Select a lookup table (Image/Looku
p 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 documen
ted 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 sub
-
pixel registration is not necessary and it saves time to omit it.

5.

Open a text window to show progress of the macro.
The

text wind
ow
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.

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9 MSL Robert Bagnell March 2011

8.

The averaged dark current images are subtracted from their
respective stacks (CFP, FRET,

CFPshade and FRETshade) then

average
d

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
automati
c 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 maski
ng
because TurboReg does some image processing and uses every
pixel in the image during registration so
a previously

masked image
can
not be used
. TurboReg produces a
“Refined Landmarks”

table
that indicates the amount of adjustment that was mad
e for each o
f
the three landmark
s 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 threshol
d.

A message box prompts the user to do this.

13.

Photo
bleach 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.

Photo
bleach correction is based on the ratio
of th
e 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
ca
lculated 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
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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
, sett
ing
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 amo
unt 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.