ImageJ

Load image into ImageJ

    Rotate so the the axon
is coming up from the bottom

a.
    “image” ---- “transform”
---- rotate, or flip to produce mirror image

    “Process” ---- “smooth”
makes the neurons a little smoother looking

4.     Select a section mostly including the neuron in
interest

    “Image” ---- “duplicate”
to duplicate the selected region (this step is important to keep your original)

    “Image” ---- “type” ----
“8-bit” (turns the image black and grey)

    “Edit” ---- “invert”
(image now becomes white and grey)

    “Image” ---- “adjust”
---- “brightness/contrast” to make neurons stand out more

    “Save” Note: save as
.jpeg in order to see changes without opening in ImageJ

Class 1 neuron in segment A1 (Gal4^221, UAS-MCD8-GFP). Same image
from previous page, transformed with ImageJ.

**Isn’t it beautiful????**

Arvind’s Pruning Analysis Protocol

Sample preparation

     Clear
all pupae to start time course

     Check
vial every hr to look for pupationàmark time when pupated and remove pupae from
vial and place on glass slide with double sided tape in following orientation:

3.      [insert image here]

a.
     90degree
turns and ~75degree turns seem to place the class I ddaD/E cell body cluster
near the center of the pupae—over turning can lead to dendritic
projections/cell body to be cut of because they are “turning” around the edge
and as a result difficult to image

b.      [insert diagram for what
happens when overturning pupae]

c.
     Look
for distinct ddaD morphology—primary dendrite will be pointing towards center
of body(towards trachea) and secondary branches will point posteriorly
innervating the denticles (often accompanied by ddaE with secondary dendrites
pointing anteriorly but sometimes these two cells can be separated if pupae is
moved around too much after mounting coverslip)

     Once
correct neuron is located set end and start points for the z stack (end point
should be the cell bodyàas you move deeper in to the body you should see the
dendritic projections and set the start point once you pass all dendritic
projections)

     Start
assembling z stack

Analysis

     Open
z stack in imagejàclick ok to bioformats pop up

     Scroll
through the z stack to see if the cell body of the neuron is included in the z
stack and visible—

a.
     If
not then note this on data analysis—do not include in quant

b.
     If
cell body is visible proceed with analysis

     Click
imagesàStacksàz project

a.
     If
a lot of noise/background gfp from pupal casing, you can remove some slices of
the z stack at beginning or end of stack with this pop up window as long as you
are not cutting off dendrite or cell body

     Convert
z projection to 8bit(imageàtypeà8bit)

     Click
editàinvert so that neuron is now black and background is white

     Transform
image so that dendrites pointing up and axons pt down(this should be a 90deg
left turn unless pupae was mounted in a way that deviated from standard
mounting above—note this in data analysis)

     Look
at black/white max projection image to see if there are any discontinuities in
primary or secondary branches

a.
     if
it appears like there is, return to the z stack and scroll through the stack to
verify that this is actually the case. Occasionally this discontinuity is just
be an artifact of the black/white conversion—look to see if the same region of
the dendrites is severed when scrolling through the z stack (transform this z
stack in the same orientation as the binary image if this helps locate the
region of interest)

b.
     in
the data analysis spreadsheet label this neuron as “primary
yes/no(distal/proximal); secondary yes/no(distal/ proximal)” under the
appropriate time pt

c.
     also
note whether dendritic debris is cleared (ie is there severed branch

es
lingering)—“yes cleared/no not cleared”

     save
binary image as tiff file in folder labelled “[insert date here] pruning”

\*things to note:

Images from same neuron at different time points should be
analyzed in a separate folder(the first image can be used with the other quant
data but to avoid counting severing from the same neuron from the same
biological replicate “twice” and skewing data, analyze the subsequent images in
a separate time course folder