User Manual 2024 04 06 - User Manual 2024 04 06.pdf

Instructions for Running the DTC M

orphometrics

Macro

Things to know:

•

Macros

ere

tested with

live

anesthetized

worms mounted on standard agar pads.

•

Images were

single channel fluorescence

acquired

0.5 μm

step

with

a Nikon

spinning disk

confocal

microscope with

a 60X N.A. 1.4 lens to produce

0.22 μm pixels

with an Ander EMCCD camera. Files were saved

“.nd2”

to preserve metadata

•

Alternative microscopy setups can be used; see additional information below under “

Plugins >

Macros > Rotate

based on straight line

”

•

Fiji bundles BioFormat plugins with ImageJ which can read proprietary formats of most, if not all, microscopes

•

Image data

must have the correct spatial scaling as the macros report results in the scale and units provided in

the files.

•

Laser scanning confocal images may need to be very low noise to work with the

background subtraction and

gamma settings

(see comments in macro code)

•

If using detectors with offset,

is critical that the offset is not set to make the background uniformly zero as this

excludes low intensity voxels which are necessary to retain.

•

The macros rename images during processing, but it is a b

est

practice

to save the original raw images in a folder

separate from

processed images and

subsequent measurements.

Things you need:

•

Single channel

stacks from

a confocal microscope

•

ImageJ or Fiji

1.54i

open

source image processing program on your computer

o

https://fiji.sc/

o

Schindelin, J.; Arganda

Carreras, I. & Frise, E. et al. (2012), “Fiji: and open

source platform for biological

image analysis”,

Nature methods

9(7)

: 676

682, PMID 22743772, doi: 10.1038/nmeth.2019

Installing ImageJ:

Download ImageJ/Fiji on your computer

(

https://imagej.net/software/fiji/

https://fiji.sc/

)

Unless you have full administrative permissions, do not install in the Programs Files directory or Applications (on

Mac).

Also,

recommend do not install on

to the

desktop, especially if your computer has a home drive on a

network, unless you specifically want to be able to access from

multiple computers

Recommended: install in your documents folder. If you are on a network, make sure this is the local documents

folder (unless you specifically want it on the network).

Installation is just copying a folder containing a bunch of files to your computer. It's this simple. You can even

keep it on a USB key to run it anywhere USB drives work.

Make sure that ImageJ is updated to 1.54i or later. This is important since the macros will not run on previous

versions of ImageJ. This update only needs to be done once.

Open ImageJ/Fiji

; this

tool bar appears

(the buttons at the right may look a little different on your

system)

Go to the Help option on the Menu bar:

lick on “Update ImageJ...”

and use the pull down menu to select “daily build.”

Help > Update ImageJ... > daily build

Every Time You Run Fiji:

Install the

DTC Morphometrics

macro to your

ImageJ/Fiji:

On ImageJ/Fiji

menu bar, click

Plugins > Macros > Install...

Select

“DTCmeasurements_

v21

.ijm”

macro file and click ‘Open’

In the tool

bar,

you will see new buttons along the lower right row.

lick

Plugins > Macros

see

another route to these added functions along with additional

options:

Many of these may also be accessed by right clicking on an open image

which will result in a

pull

down

Now you are ready to collect measurements from your image

NOTE:

To automatically install the package every time ImageJ is launched, replace StartupMacros.fiji.ijm

in the Fiji.app macros folder with DTC_measurements_v

217.

ijm.

DTC

markup and

measurements step

step guide:

Goal: To mark location

length

displacement

of various features

and record the values in a table

that links the values

unambiguously to the image file

Image optimization component

Preparing a DTC image for measurements

The images

/stacks

you collect on the spinning disk may have

a wide dynamic range making it difficult to see dim

fluorescence without blowing out brighter features,

random orientation, and

/or a

curved path of DTC extensions.

Ideally, we want DTC images such that

distal is

to the left and

proximal is

to the right. We also

need

the DTC and its

processes to

linear

, as distance

measurements

are

from the distal end.

While

DTCs vary with respect to gonad

curvature

straightening the image generates linear measurements

that are

obustly comparable across samples

This

also allows for more uniform qualitative comparisons and ease of presentation.

tep

step

and purpose

each command

in the image optimization component

“Max project and change contrast”

does two things:

Given

a stack, it automatically creates a maximum projection

. It

can also be run on a

previously obtained maximum projection file

ii.

applies a non

linear

gamma

contrast transformation to visualize very low signal features and

very bright features in a very similar intensity range.

NOTE:

Images collected with a different type of microscope may require different contrast adjustment; see

notes in macro code.

NOTE:

Worms

in images below

bear the

qIs57

[

lag

::GFP] transgene (Seigfried et al., 2004)

“Rotate based on straight line”

rotates the image so that the cell body of the DTC

(distal)

is on the left

hand side

and the extensions

(reaching proximally)

are on the right

hand side

NOTE

his

method is used when the DTC is straight; that is, when a straight line can be drawn that defines the

distal

proximal position of the entire cell. See below

“Rotate and straighten based on curved line”

for an alternative

method if the DTC is not straight.

BEFORE

AFTER

On the ImageJ/Fiji toolbar, right click on the ‘line tool’ icon, and select the ‘Straight Line’

option:

ii.

lick on the distal end of the

gonad

(approximately in the center) and drag a straight line on

the axis

about which

you want your image to be rotated.

NOTE:

To obtain a final properly

rotated image (with distal to the left, a position from which subsequent

measurements are made), it

is important

draw the line starting from the distal end of the

gonad

and

drag it

proximally (

towards the direction of the extending processes

in the adult)

NOTE

eave

gap beyond

(distal to)

the DTC cell body before clicking and dragging the line segment

. If

you start the line on the DTC itself, the DTC will be cropped

. Also, drag the line segment far enough

capture the full length of the desired

axis.

iii.

Plugins

Macros > Rotate based on straight line

The image rotates to standard orientation and a box sized 1000 x 250 pixels appears,

which

can be dragged and placed to frame the DTC in the center of the image. This step is done to

standardize each DTC image

The resulting image

shown below.

NOTE:

e rectangular box

size i

based on

the size of the largest cell we would measure as

imaged with

our microscope

. Users may need to specify

alternative box

size

for different

pixel and cell sizes

This may be changed in the macro code

(see comments in code)

The box

size would be set larger than the maximum possible biological feature you would ever

measure.

The macros will work with other single channel 16 bits Z series from a variety of

confocal

systems

Listed below are the

three tested

microscopes

(

and

different detectors) and seven

lenses. Those lenses with N.A. ranging from 1.2 to 1.49 gave outstanding images; N.A. less

than 1.0 did not provide sufficient detail.

LSM800

40X N.A. 1.3 oil lens

(requires larger box size in macro; can be calculated from pixel size)

GaAsP detector

LSM700

40X N.A. 1.2 water lens

PMT detector

Crest X3 on Nikon Ti2

60X N.A. 1.4 oil Ph3

100X N.A. 1.35 silicone (too big for straighten command)

100X TIRF N.A. 1.49 oil (too big for straighten command)

40X N.A. 1.25 silicone

20X N.A. 0.95 water

Kinetix sCMOS camera

NOTE:

he box

should be placed

such that the DTC cell body is aligned on the

center

left with a gap from the

margin; this ensures

inclusion of all

DTC fragments and processes that extend on the right

hand side. You may

also click and drag on one of the small white square boxes

(red arrows)

on the perimeter of the rectangle to

adjust the size

if necessary.

NOTE:

Do not resize the box while moving it.

iv.

Once you have the rectangular box properly placed on the image,

Image > Crop

Your Image will now be aligned

, cropped

and sized properly as shown above.

“Rotate and straighten based on curved line”

NOTE

: this

method is used when the DTC is not straight as it allows the user to place multiple anchors along the

DTC to define its path

for subsequent straightening

example of such a curved DTC morphology

shown below:

Click on the “Segmented Line”

button on the ImageJ/Fiji toolbar:

vi.

eft click beyond the distal end of the DTC (you don’t have to drag) and keep clicking over the

curvature of the DTC

(see

picture below

)

These

clicks

should be made

throughout the extent

of the DTC

to prevent

distortion.

NOTE:

Make sure that you leave at least gap beyond (distal to) the DTC cell body before clicking

creating

the line segments

. If you start on the DTC itself, the DTC will be cropped. Also,

make the line

long

enough to capture the full length of the desired x

axis.

vii.

Once you

reach the

desired

end

point

make

last click

with a

right click (marked with a red

arrow in the picture below) to release the segmented line. The outcome should look like this

–

viii.

Plugins

> Macros > Rotate and straighten based on curved line

ii.

The

result

will

be a straightened DTC

iii.

Once the

curved

line has been drawn, click on the ‘Plugins’ option on the menu bar, and then

Macros > ‘Rotate

and straighten

based on

curved

line’.

The image rotates to standard

orientation and a box sized 1000 x 250 pixels appears, that can be dragged and placed to

frame the DTC in the center of the image. This step is done to standardize each DTC image

(see section IB above

Rotate based on straight line

regarding the box)

NOTE:

Images should be saved as Tiff... to preserve metadata and bit depth.

File > Save as... >

Tiff...

***

To use this image for DTC

manual markup

, proceed to

section II

***

To use this image for Sholl analysis, proceed to

section

III

II.

DTC

manual markup:

Setup for DTC manual markup

•

mages

from the section above will be

in TIFF format

contrast adjusted, straightened

and cropped to 1000 x

pixels

•

To open an image, d

rag

and drop

on the ImageJ/Fiji toolbar

desktop icon

or use

File > Open...

he ‘ROI Manager’ window pops up

(shown below)

. T

his window will keep

rack of

measurements

on the left

hand panel (all the markups).

NOTE:

For all the manual markup features below, t

he ‘Delete’ option is very useful

. If

you mark

some

thing

unintentionally/incorrectly

click on the incorrect entry

the left

column of the ROI Manager

and then click the ‘Delete’ button on the right

side to

remove it.

Features

The following

features

can be

measured (counted/positioned

)

relative to the distal end

center point.

User

placed marks can

be made to quantify the following (see details below):

•

Distal end: The d

istal end center point is specified using a circle of 20 μm drawn with the left side

, placed

such that the

intersection of the distal end of the gonad with the inner arc of

the

circle

This point is the left

most

point

the circle and is the 0 point for subsequent distance/position measur

ements.

•

Nucleus

smaller circle is centered and placed over the

DTC

nucleus

, recording the center of which as a

distance along the x axis

from the distal end center point

•

End point

arks record

the length of

DTC

processes

(as defined by the user)

from the distal end

•

Branch point

: M

arks record

the distance of each branchpoint from the distal end.

•

Puncta

arks record

the distance of the isolated dot

like fragments from the distal end.

•

Line segment

: Marks record the start and end points of isolated DTC fragments, as well as the total distance

across a segmented line or across a straight line from start to end.

“Mark Distal End”

. T

his will be first thing to mark since all the measurements are from this reference

. There can

be only one distal end

;

you will receive an error message if you try to mark more than one DTC distal end (same

with marking the nucleus).

Plugins

> Macros > Mark Dista

l End

(or right click in pull down menu)

circle appears on the image

along with a popup saying ‘Position circle and click ok’.

ii.

Hover

over the circle on

the image,

and the

pointer

will appear

as an arrow

. C

lick and

drag the

circle

such that the

distal end of the gonad

aligns

with the inner arc of

the

circle

(distal end of

gonad; see

nd

note below)

on the left side.

NOTE:

Only drag the

pointer

when

you see the arrow

(you may see a ‘+’ crosshair or a hand

), o

therwise you

may

inadvertently change

the size of the specified circle.

ust hover over the center of the circle to get the arrow

pointer and

then

drag it.

NOTE:

or cases where the distal end of the gonad is not clearly coincident with the distal end of the DTC, or

where the distal end of the gonad is

not clearly outlined by the DTC cell body, we recommend collecting

DIC

images

in addition

to determine how to properly designate the distal end of the gonad

iii.

The position of the distal end

registers an entry on the ROI Manager window

iv.

Alternatively, you may right click on the image

, select ‘Mark Distal’

, and the circle

that

pop

can be

drag

ged into p

osition as described above.

NOTE

The

perpendicular tangent drawn on the

left side of this circle (

indicated by the

red line

in the figure

below

, but which will not appear in the image you see

) will be the reference from which

feature

distances

(

e.g.,

end points

a,b,c

in figure below

)

will be measured

. The distance is measured relative to the x axis along the

bottom of the image as indicated by intersection with the

green lines

in the figure below

“Mark Nucleus”

The nucleus is

marked

with a small

circle

to measure

any

nuclear displacement relative to the

distal end.

NOTE:

The nucleus

will

marked with an oval that is

smaller than the

distal end

circle

;

you should aim to place

the center of

the smaller

circle at the center of the nucleus.

Plugins > Macros > Mark Nucleus

(or right click in pull down menu)

circle of a specific size appears on the image

along with a popup saying ‘Place oval centered

over the nucleus and click ok’.

ii.

Drag and drop the circle centered over the nucleus

iii.

Once the

nucleus

circle is positioned, click

‘

’

on the popup.

iv.

Alternatively, right click on the image, select ‘Mark Nucleus’, and the smaller circle

will

pop up,

which you can drag and position as described above.

he position of the nucleus relative to the distal end

will appear as an

entry on the ROI

Manager window.

NOTE

The ROI Manager window stays open throughout the markup

this is where all the markups

are

recorded (red arrow

on figure below

“ENDPTS”

(endpoints)

. T

here are several processes emanating from the

adult hermaphrodite

DTC cell body.

The goal of this markup is to mark the end points of

processes.

For purposes

here

we are marking only

the ends of apparently

contiguous processes

(CPs)

Click on the ‘ENDPTS’ button on your toolbar.

ii.

Click the end points of

each process

on the image.

NOTE:

Alternatively, function keys

for this and other markups below

can be used while hovering over the

position for the markup.

iii.

The

‘ENDPTS’

markup

will appear as a tiny hollow circle

on the image and the position will

be recorded on the ROI Manager each time you click to mark an end point.

iv.

If you incorrectly position an end point, just click on the ‘Delete’ button on the toolbar

(as

noted previously)

–

this will delete the most recent marked point on the image.

Alternatively,

you may select an erroneous endpoint on the ROI Manager window by clicking on it and then

clicking ‘Delete’ option on the right

hand. You will see the corresponding tiny circle marking

the end point disappear on the image.

Once you are done marking the end points, press the

“ALL OFF” button on the toolbar before

performing any other functions.

“

BRANCH

”

(branch points):

Branch points can be marked in a manner similar to End Points, above, but

using the “Mark

branch point” button on the tool bar.

“

PUNCTA

”

(puncta):

Puncta can be marked in a manner similar to End Points, above, but using the “Puncta” button

on the tool bar.

“Line

egment”

disconnected

segments of DTC processes can also be measured.

Segments can be marked by

click

ing

the ‘line tool’ (red circle) on the Fiji dashboard

ii.

tarting from the distal side,

click

on the path of this

segment

iii.

Go to Plugins > Macros > Mark Line Segment [F8]

iv.

The segment will be registered

into the ROI manager and will be measured once the

desired markups on the image are complete and the “Measure all markups” command

is used

(see below)

The results window will look like below, with the parameters pertaining to this “fallen

off DTC process” on the extreme right columns

vi.

The column titles are described below

•

“lines_start” shows the linear distance from where the line segment begins

relative to the

distal

end.

•

“length”

the total length of the entire line segment that you manually dr

over the segment

This

the

sum

of all the lengths between each anchor dot that you mark along the path of the

segment

•

“netlength”

the linear length measurement between the first and the last anchor point of the

segment. Note that

this parameter does not consider how curved or convoluted the

segement

is.

NOTE:

For all features,

it is important to

specify scoring criteria among users for each feature. Different transgenes

will also accentuate different DTCs features.

“Save Markups”

Once all

desired markups are made,

you

must

save all these markups.

Plugins > Macros > Save Markups

ii.

You will be prompted to enter your initials

/name

and click OK

–

NOTE:

This feature is particularly useful to allow for independ

ent

users to mark up similar images and

compare the

ir choices

User

initials

are appended to the file name

iii.

Once you click OK, the following popup appears displaying the file path of the saved markups

–

the markups

should be

saved in a zip file in the same folder that contains the

corresponding

image. Click OK.

These files are paired so that markups may be edited later and measurements may be

performed later.

“Measure all markups”

Plugins > Macros > Measure all markups

(or right click pull down menu)