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Nov 6, 2013 (3 years and 9 months ago)

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©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

MetaMorph Workshop

Ed Rader

September 12
th
-
14
th
, 2012

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Agenda


Images & Image Display


Acquisition


Acquire


Scan Slide


Calibrate Distance


MDA


Working With Stacks


Image Processing


Image Analysis


Presenting Images


Automation

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Goals


Familiarize you with MetaMorph’s tools and where to
find them



Provide a basic understanding of each tool’s use



Not covering every button and setting in every function



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

How to get help

1.
Phone

1.
1
-
800
-
635
-
5577

1.
Press 3 for Tech Support

2.
Press 2 for Cellular Imaging

3.
Press 2 for MetaMorph


2.
Email

1.
Support.dtn@moldev.com


3.
Web

1.
http://support.meta.moleculardevices.com/

2.
http://www.meta.moleculardevices.com/authorize/



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Tools & Display

Ed Rader

September 12
th
-
14
th
, 2012

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Display


Can you guess what this image
looks like?



Our perception is trained to
interpret light intensity from
the eye to images



Our perception is not adapted
well to convert numbers to
brightness in an image

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Display


MetaMorph uses a mapping of
intensities to produce an image


Contrast



Definition of an image


Two dimensional grid of pixels


Each pixel contains an intensity
value



Other information in images


Annotation and properties


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Info


Edit Menu



Besides the pixel data, other
information is stored with
images when they are saved



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Histogram


Measure Menu



A graph of the distribution of
the number of pixels at each
intensity in the image



Why do we care about this
histogram?



Tells us how effectively we are
utilizing the detector

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

What’s wrong here?


These two images are of the same field of view


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scale Image


Display Menu



Use the High Wedge to set the
White



Use the Low Wedge to set the
Black



Changes display, does
NOT

alter
the data, only how it appears to
our eyes

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scale Image


Pixels equal and above the
High intensity value are White



Pixels equal and below the low
intensity value are Black



Changes display, does
NOT

alter the data, only how it
appears to our eyes

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scale Image


AutoScale



Automatically finds the lowest
and highest intensity in the
image and sets the scaling
there



Wedges can then be changed
to select % of pixels below and
above wedges

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Window Tools


Zoom


makes the image
larger / smaller


You can also use the wheel
on the mouse



Look Up Table



Scale Image



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Color Image


What’s the difference between monochrome and color
images?



How can you tell?



Why should you care?

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Color Image


No Scale Image tool



Pick Color Channel



Three intensities per pixel
instead of one



Easier to get images into other
programs



Create color images with
overlays for presentation

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquiring Images

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Acquire Menu



Acquire snaps an image and
displays it



Autoexpose not good if imaging
beads or low signal



Show live displays a
continuously updating image


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


The duration of the collection
of photons to create the image



How much area of the camera
should be used to capture
images



Shows the camera’s
temperature or some warnings
here

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Autoexpose is good when
there is “strong” signal



Watch your target intensity
(Acquire Tab)

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Binning



Sum adjacent pixel intensities
together to form new pixels



Binning of 2x2, 4x4, 8x8, are
common in detectors




©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Why Bin


Faster Frame times


Shorter Exposure times


Smaller files



Giving up


Spatial details



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Display tab mostly used for
setting the scaling



Acquire tab mostly used for
synchronizing shutters &
Autoexpose settings



Correct tab for background
subtraction and flatfield
correction

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Annotate tab sets what
settings to save with the image



Special tab is for device
specific settings



Live Replay is like a
MetaMorph DVR

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Special Tab

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Special Tab


Common Settings


Speed
-

Digitizer



Gain


Amplify signal and intensities



Average Frames


To minimize noise



Driver Information



Triggering


Controlling or being controlled by
external timing signals

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Live Replay


Live Replay is like a MetaMorph
DVR.



Use is based on visually
identifying an expected event



Specify the number of images
before and after the event to
capture



Memory is allocated to hold
Images

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Live Replay


Start Live to begin capturing
pre
-
images



Press F11 to lock pre
-
images
and begin capturing post
-
images



Result stack displayed, current
plane is the “marked”
timepoint

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Acquire


Under the hood


Send Acquisition parameters to
camera


Open Shutter / Set Illumination
to Current


Expose CCD (Exposure Time)


Transfer Image to MetaMorph


Display Image in Window



How can we optimize this for
faster performance?


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Advanced Acquisition Topics


Streaming


Captures images at the maximum rate provided by the hardware


Device Streaming


Allows sequencing of wavelength or z
-
series while streaming, useful for “real time” thru
-
focal sections


Synchronizing image capture with fast devices


Streaming to Hard Drive


When streaming for long time periods, such as minutes, the data can be accumulated on
a fast hard drive. Virtual Stack memory allows the large dataset to be viewed in
MetaMorph.


Stream Preview


During streaming, a pseudo
-
live image is displayed, allowing for interactive change in
focus or adjustment of sample conditions during the acquisition

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stream Acquisition


Optimized mode of camera
operation



Acquire Images as fast as
possible



Typically, Streaming & Live use
the same mode of camera
operation


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stream Acquisition


Captures images at the
maximum rate provided by the
hardware



Only half the available
memory can be used



Integrated as a tab / window
for MDA




©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Device Streaming


Software Synchronizing of
Acquisition with microscope
peripherals



Patented Process



Multiple High speed devices
coordinated together



Devices not asked for position
information

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Device Streaming


Piezo, Galvanometer,
Monochromator, AOTF, DAQ



Typically devices move within
1
-
2ms



Fixed acquisition times for all
images



Devices move during readout



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Overlapped / non
-
Overlapped Mode


Non
-
overlapped: Expose then
readout



Overlapped: Expose and readout at
the same time (sort of)



Effects sampling frequency (live cell
specimens)



Synchronize with relatively slower
devices (on the order of 10s of
Milliseconds.


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Streaming to Hard Drive


Streaming to RAM is limited to
the amount of RAM



Hard drive space is much more
plentiful



Slower to write images to the
hard drive vs RAM

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stream Preview


How do I know that my Stream
Acquisition is still collecting
valuable data



Every N
th

frame displayed to
user in an Image window.



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Timelapse Acquisition


How do I improve the frame rate
of my timelapse?



MDA Automatically Saves
images



CPU Compatibility Mode
Preference


MetaMorph has
more processor time



Minimize image window



Annotations

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Sample bigger than the
field of view



A set of images collected
at different fields of view



Montage together to
present image of entire
sample (low resolution)



High Resolution detail of
any specific region

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Apps Menu


Need motorized stage


Work from top down


Similar controls for Live and Snap

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Main tab for selecting magnification


Description and file saving


Save all settings to a file for later use

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Select Camera Binning and Number of Wavelengths


One Wavelength tab for each


Shading Correction allows for correction of uneven
illumination

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Select Illumination Setting


Select Exposure duration


Image Autofocusing available in case sample is not
level on stage

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Journals can be run at different times during the
acquisition sequence



Check the event and browse for the journal

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Calibration step required to map the size of the field
of view and correlate the movement of the stage to
the field of view

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Select the bounding corners of the sample location to
image


Typically use the Live image and the stage joystick to
set the position


Press Scan to start imaging

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Scan Slide


Show low resolution montage of images


Draw a region on montage


Press Show Image to see high resolution


Stitched result

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Calibrate Distance


Measure Menu



Set the relationship of pixel size
to a real physical dimension



Each pixel is how many microns



Calibrations typically setup for
each magnification

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Calibrate Distance


List of calibrations available in
table



Edit changes in the table for
existing calibrations



Save calibrations to a file



Define new calibrations and
measure with regions

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Calibrate Distance


Calibrate by region, image of stage
micrometer



Select region type and resize region
across the image to known marker
dimensions



Enter the name (typically the
magnification) and calibrated length



Associate the calibration to
magnification and acquisition hardware

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Calibrate Distance

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Calibrate Distance


Quick estimate of the calibration
in your images?



Size of pixels on camera / total
system magnification



Example:


6.45 um per pixel / 60x
objective (no tube lens
magnification) = ~.1um per
pixel

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multi Dimensional Acquisition

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


What can you do with MDA?


Timelapse


Multiple Stage Positions


Multiple Wavelengths


Z Series


Streaming


Run Journals



It is possible to do all of the above
at the same time

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Saving


Describe experiment


Select a directory


Name the experiment


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Timelapse


Number of time points you want


Length of experiment you want


Elapsed time
between

time points
you want


MetaMorph will suggest a
minimum interval that you cannot
go below


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Multiple Stage Positions


Unlimited number of positions can
be acquired


Move the stage with your sample
on it to each position you want
imaged


Push Plus or Right Arrow to add
positions to the list


Use the red “X” to delete positions


Use the up and down arrows to
move positions up and down in the
list


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Multiple Wavelengths



Up to 8 wavelengths can be
acquired



As you add wavelengths, the
number of tabs down the side
of the wizard will increase
accordingly

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Multiple Wavelengths


Choose your wavelength in
the illumination dropdown
menu


The most important element
is the exposure time for each
wavelength

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Z series



Two ways to acquire a Z series

1.
From user
-
defined known
positions of Top and Bottom

2.
Range around current: the Z
series will go, in this example, 15
steps above and below the
current Z.



The step size recommended will
depend on your objective’s
magnification and NA

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Streaming


Select the length of time to stream


Select the location to stream to


Select overlapped or non
-
overlapped camera state



MetaMorph will calculate and let you
know what memory you have and
what’s required



If you don’t have enough memory
the acquisition won’t start

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Journal


At a given time
point


For a given
wavelength


At “special” times
during the
experiment

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


When all parameters are set to your
specifications, click the Acquire
button to start the acquisition



Also, once you have all your
parameters set within the MDA
dialog box, you can save them as a
state to be recalled later for another
experiment

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Snaps a single image



Starts the live

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Don’t have to acquire every
wavelength at every timepoint



Beginning, End, Every N
timepoints



Periodic Transmitted light
reference image

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Image Autofocusing to help
correct for drift, sample depth,
stage level



Find the best contrast



3 Search Methods


Step


Binary


Advanced Binary



Configure Steps sizes and range



Align Wavelengths to correct for
Registration & aberrations

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Acquisition


Multiple Z Axis control



Offset positions for auto focus
stability

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multi Dimensional
Data

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


What can you do with RMD?


Enables you to view, filter,
organize, and analyze
MetaMorph images from a
Multi Dimensional Data Set
(*.nd) and generate new
images based on your
viewing criteria


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


Select Base File


Opens the Multi Dimensional
Data Set Utilities dialog box. Use
this dialog to select the file that
you want to use as the base file
for your multi dimensional data.
Choose the file from the list of
the data sets in the Data Sets
(*.nd) box or click Select
Directory to select a data set
from a different directory.

Note: After you select or change your
base file selection, you must click
View

to implement your selection and
return to the Review Multi
Dimensional Data dialog box.


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


Enable Montage


Activates and deactivates the
Montage controls and creates a
montage image of thumbnails for
each time point in a wavelength.



If multiple wavelengths are selected,
a montage image is created for each
wavelength.


After the montage image is
displayed, click a thumbnail in the
montage image to open a full image.
You must have at least one
wavelength selected to use this
feature.


If you click Enable Montage before
selecting any wavelengths, a
montage for a specific wavelength
will be generated when you select
the wavelength.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


Load Images


Loads selected images into
MetaMorph as new images and
displays the images. The image can
be saved to a file using the Set Up
Sequential File Name dialog box.


Select Best Focus


Selects the best focused image from
the Z
-
planes at each time point.


Clear


Clears all the image selections from
the image grid.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


Full Image


Displays an entire image without regard to active
regions.


Active Region


Displays only the active region of an image.


Color Composite


Combine the defined and selected image sources
into a single image, assigning the selected primary
color to each of the respective images.


Source R:


Assigns the primary color
Red

to the wavelength
that you select as source.


Source G:


Assigns the primary color
Green

to the
wavelength that you select as source.


Source B:


Assigns the primary color
Blue

to the wavelength
that you select as source.


After you assign a wavelength to these sources,
you can turn the image selection off and on in the
Image Window.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


Run Journal Loop



Post acquisition Processing /
Analysis






©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Review Multidimensional Data


Run Journal Loop



Post acquisition Processing /
Analysis






©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multidimensional Data Utilities


Experiment Set Maintenance



Thumbnails are for montage


Preference to turn off
Thumbnails by default




©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Working with Stacks

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stacks


What are stacks?



A set of images organized in a
series, displayed one image at a
time in an image window



Time Series


Z Series


Wavelength Series



Many people think of stacks as
movies

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stacks


Usually saved in one file


2.0


7.1 an .stk file


7.5 or greater a multi
-
image
tiff



Stack Player controls



“Thumb” for picking a single
plane



Play Preferences on Stack Menu

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

4D Viewer


File Menu



2D and 3D Display



Different wavelengths overlaid
simultaneously



Original and volume displays
available



Similar controls as regular image
window

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

4D Viewer


Use the mouse to rotate and
spin the 3D projection



Make movies of rotations, time
series or both in 3D


Select a Codec
-

Cinepak



Surface projection to identify
discrete volumes and make
measurements


Volume size and intensity


Distances


Colocalization

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

4D Viewer


Scaling



Switch between 2D and 3D
views



Select wavelengths



Select rendering option and
level of detail

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

4D Viewer


Set the X & Y calibration


Or set the Z calibration from
Edit menu



“High performance” display card
required to see finest detail



Large data sets are problematic



As much physical RAM as possible
for best performance


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

4D Viewer


IsoSurface enabled



Select individual objects with
the mouse, double
-
click



Object volume measurements
available


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

4D Viewer


Distance measurements



Colocalization available when
multiple wavelengths are in
the same data set


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Keep Planes


Stack Menu



Used to copy a portion or crop
a stack



Can sub
-
sample a data set
with the Step Size setting


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Keep Planes


Specify a range of planes to
select



Press Select Planes in range



Scroll list to see check marks



Pick the action



Click Apply


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Montage


Stack Menu



Arrange planes side by side to
create a single image of the
stack



Rows and columns can be
configured



Result can be zoomed or
stitched

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Montage


Select the fill order



Select the rows and columns if you
don’t like the defaults



Zoom the image if necessary



Stitch the result if source is an X,Y
Stage Scan



Press OK

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Processing

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.


Filtering helps prepare images for segmentation and/or analysis



Filtering accomplishes:


Noise removal


Correction of uneven background


Elimination of image details other than the objects of interest



No image filter will work perfectly on all images

Image Filtering

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Basic Filters


Process Menu



Weighted filtering and rank filtering



Examining each pixel and its
neighborhood of adjacent pixels



The size of the neighborhood is
called the “kernel”

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Basic Filters


The center of the kernel defines the
pixel location being processed



Larger size kernels enhance the
effect of any specific filter



Kernels are placed at each pixel in
the image and the filter operation is
performed



Watch for edge effects

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Basic Filters


Low Pass



suppresses high frequencies by
replacing each pixel intensity with
the local average intensity


blurs edges



Sharpen


enhances edges by applying a high
pass filter


multiplication of each kernel’s
value with pixels and summing
results

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Basic Filters


Median


removes noise by replacing each
pixel intensity with the local
median intensity


good at noise removal while
preserving edges



Rank


removes noise by replacing each
pixel intensity with a local rank
statistic intensity such as
minimum, maximum, or median


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Basic Filters


Low Pass
: suppresses high frequencies by
replacing each pixel intensity with the local
average intensity


Median
: removes noise by replacing each
pixel intensity with the local median intensity


Rank
: removes noise by replacing each pixel
intensity with a local rank statistic intensity
such as minimum, maximum or median


Remove haze
: subtracts out of focus blur
estimated from neighboring planes


Sharpen
: enhances detail by applying a high
pass filter


Unsharp mask
: subtracts out of focus blur
estimated by applying a low pass filter

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stack Arithmetic


Stack Arithmetic



Compress the data from an entire
stack into one image



Can be used:



to improve signal to noise


compress stack of images into
single plane


show the path of motion in a
single plane

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Morphology Filters


Process Menu



Spatial and intensity
-
based
filtering



Settings for basic shape, size,
and area



Most intuitive results on binary
images, but can operate on
grayscale images

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Morphology Filters


Dilate


grow bright things



Erode


shrink bright things



Close


dilate then erode



Open


erode then dilate



Open and Close are equivalent
to where does the shape fit



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Morphology Filters


Close to fill small black
holes in white



Open filters out small
white in a black
background

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Morphology Filters


Top Hat


Residue of Open Filter


Keeps the things that the open filter
would have removed


Top Hat to find small features



Bottom Hat


Residue of Close



H
-
Dome


Local intensity peaks



H
-
Basin


Local intensity troughs



Top Hat to find small features



H
-
Dome to flatten an uneven
background

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Morphology Filters


Gradient


Difference
between Dilate and
Erode



Watershed Lines


Helps with splitting
touching features


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Segmentation

Ed Rader

September 12
th
-
14
th
, 2012

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Segmentation


Differentiate between what to
and what not to measure



Regions of Interest


Thresholding


Morphology


Morphometry


Application Modules




©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Regions of Interest


Regions Menu



Rectangle


Ellipse


Polygon


Line


Multi
-
Line


Multi
-
Line Trace


Region Properties

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Regions of Interest


Active region



Region tooltips for position
and size (in calibrated units)



Some filtering tools operate in
active region


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Regions of Interest


Double
-
click on line regions to
edit positions of points



Use Rectangle region and Ctrl
-
D to crop an image or Ctrl
-
Shift
-
D to crop a stack



Tooltips are displayed when
regions are being created

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Regions of Interest


Right
-
click menu: copy, paste,
delete regions



Save and load regions to a file



Transfer regions to another
image



Create Region function

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Thresholding


Measure Menu and on image
window toolbar



Select pixels in image based on a
range of intensities



Three states


Off


Inclusive Threshold


Exclusive Threshold



Subjective to user

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Thresholding


Colored overlay visible on image



Even illumination/background
necessary for good thresholding



Pixels are either thresholded or
not so binary images can be
created



A prerequisite step for some
analysis functions

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Thresholding


Blue Wedges on image
windows can be used to adjust
threshold



Autothreshold to find either
bright or dark objects in image



Preference to select between
two different algorithms

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

ROI + Thresholding


You can combine using
Regions of Interest and
Thresholding



Threshold Image



From the Regions menu select
Create Regions around Objects



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

ROI + Thresholding


Each discrete object will be
traced with a region



Must be at least two adjacent
pixels to create a region


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Combining Tools


Threshold Image


Create Binary Image


Open Filter


Erode Filter


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Combining Tools


Threshold Image


Create Binary Image


Open Filter


Erode Filter



Watershed Lines Filter
(using Binary and Erode
images)


Invert Watershed


Logical AND between
Binary and Invert Result

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Combining Tools


Notice some of the new lines
separating the nuclei



Use the Arithmetic function to
create a final image using the
original image and this filtered
image to get the original image
intensities combined with the
Watershed Segmentation
applied


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Analysis

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Analysis


What questions can MetaMorph
answer about images?


How Many


How Bright


How Big


How Long


How Far


How much Overlap



Quantify Results


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Image Analysis


All Analysis function can send
data to a text file or Microsoft
Excel; also known as “Logging”



Simplest measurement is a
pixel intensity with the mouse

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Line Scan


Measure Menu



Intensity profile along a line
region



Results shown in graph



Good for checking the flatness
of the background



One line at a time

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Show Region Statistics


Measure Menu



Measurements of a single region



Measurements on a single
image



Intensity and spatial
measurements

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Show Region Statistics


Pick the active region or the
entire image



Check Use Threshold to
measure on thresholded pixels
in the region



Updates even if measuring on
the Live Image

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Region Measurements


Measure Menu



Measure many regions all at
once



Measure single plane or all
planes in a stack



Data in table or graph



Summary data available

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Region Measurements


Graph measurements for all
the planes in a stack



Pick the X axis as Plane
Number, Elapsed Time, Z, or
Wavelength



Y axis is the available list of
measurements enabled

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Colocalization

red & green probes colocalized


What is colocalization?
Assume we are overlaying
two separate images


Provides quantitative
data regarding the
amount of overlap of two
fluorescent probes


Aggregate measurements

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Measure Colocalization


Apps Menu



Threshold images



Measures the amount of overlap in
wavelengths





©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Measure Colocalization

A

B

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Measure Colocalization

A

B

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Measure Menu



Measures attributes of objects like
intensity, size, shape, and count



Presents data in different tables and
graphs



Filter objects that you don’t want to
measure

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


To measure with the IMA the
image must be thresholded



After being measured, a green
object overlay is displayed on the
image



Toggle the overlay on/off



The object overlay can become a
new binary image

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


The list of measurement
parameters to display and or filter



Check the box to display, filter or
both



To filter, enter the range of values
to include in the measurement


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Pick the measurements that you
want to either view or filter on
the image



Expand the + to see the available
measurements in each group



Check the measurements to
make


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Summary statistics for each
measurement parameter



Count = how many objects were
measured



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Histogram



Bar graph of distribution of
measurements of the objects



Can filter parameter by calipers
on graph



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Scatter Plot graphs the relationship
between two measurements



Correlation coefficient is calculated



Pick which measurements to graph



Can filter objects here also with
calipers on graph

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Preferences



Mouse clicks on image


find measurements


exclude/include object


Define filter range for acceptable
objects



Object overlay drawing



Measure all regions

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


The data display tables and graphs
can be:


Docked


In a separate window


On a tab in the IMA


Not displayed



If you close docked window, it
displays on a tab



Configure data display on View tab

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Integrated Morphometry Analysis


Reset the filters to their defaults



Save/Load IMA configuration
to/from a file



Reset measurements



Create object mask

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Presentation Images

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Duplicate as Displayed


Want to show regions and
other overlays on image



Create a “snapshot” of the
image and all the items on it



The new image can be saved
and opened in other
applications


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Duplicate as Displayed


Edit


Duplicate


Duplicate as
Displayed



Dropin



Representative of the original
data, but not identical



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Color Combine

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Color Combine


Display Menu



Combine one to three images together
to make a color result



Only does red, green, and blue



Does not handle transmitted light
images (brightfield, DIC, phase
contrast)


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Color Separate


Display Menu



Separates color images into
their component amounts

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Overlay Images


Display Menu



Create a color image
combining/overlaying up to 7
different images together



Select 5 predefined colors or use
the hue selection



Pick the images corresponding
to each hue

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Overlay Images


Automatic balancing



Adjusting scaling in images also
effects result



Preview result



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Make Movie


Stack Menu



Make an AVI or Quick Time move



Duration of each frame; e.g., 30
-
30ths = 1 second in movie



Pick a file name and location



Select a compression Codec

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

3D Deconvolution

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Why Deconvolution?


Imaging a point source of light
doesn’t give you a point in your
image


you get an Airy Disk



Tries to take the information in
the 3D image dataset and
restore the original specimen



Improved Contrast & Resolution
laterally and more so Axially

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Point Spread Function


Point Spread Function



PSF is the 3D representation of
the diffraction pattern of light in
the microscope



Use either a mathematical or
measured PSF as information on
how the optical system is
distorting the object into the
image


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Deconvolution


Process Menu



MetaMorph Licenses
algorithms from Autoquant



Uncheck “Faster Processing…”



Number of Iterations to
converge to original specimen



Expert Settings are good default

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Deconvolution


Enter information on the optical
system



Fully automated and calibrated
systems get information from
Images


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Deconvolution


Select either the Theoretical
(Blind) or Measured PSF
algorithm



Full frame stacks takes on the
order of 10’s of minutes to
process

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Deconvolution


Optical Sectioning Spacing &
Range?



Implication on Live Cells



No Recipe for the best
Deconvolution


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Deconvolution Results


Typical Contrast Results



Similar Analysis for contrast and
resolution can be done axially

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Tracking

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stack Arithmetic


Stack Arithmetic is used to find
object tracks or a background for
the image series.


For fluorescence or luminescence
use stack maximum for object
paths and stack minimum for a
background.


For transmitted light methods use
stack average or maximum for the
background.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stack Arithmetic

Original Image

Result Image after subtracting
background from every plane.

Background Image after Stack
Arithmetic, Average

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Stack Arithmetic


Using Stack Arithmetic,
Minimum, the path of the
object can be identified.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Difference Image


Advantages


Simple to do in a journal.


Very graphical, shows extension
as well as retraction. ) Areas
moving into appear black, areas
moving out of appear white and
areas that stay the same appear
gray)


Easy to quantitate how much
(

m2/s) has moved.



Disadvantages



Little positional information.


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Difference Image


Do Tracking Exercises 1
-
4


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Kymograph

Original usage: A device which graphically records
motion or pressure.


The Kymograph Image shows a linescan verses
time. Stationary objects appear as vertical lines.
Objects in motion appear as diagonal lines.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Kymograph

Advantages


Provides a pictorial representation of
motion verses time.


Changes in velocity and duration of
movements are readily apparent.


Multiple objects can be followed.
Objects can cross paths, or be
identical.


Can analyze some objects that are
not easily discernible.

Disadvantages


Only analyzes linear motion.
Movement perpendicular to the axis
of analysis is not taken into account.


Calibrations are not automatically
taken into account



©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

The Track Points command allows you
to track one or more selected points
through each frame in a series of
images and to derive measurements of
the paths, positions, and velocities of
the points.


Advantages


Fairly fast


Work from OMDR, stack or live / updating image


The best (most flexible) at not losing the target


Disadvantages


Requires constant manual interaction


Accuracy limited to a few pixels


Only one object can be tracked at a time


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

Click on Add Track


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

Data Table displays
selected Data Type

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

You can Print or
Log the data
table to a
spreadsheet

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

Configures the settings associated with the
time intervals between planes in a stack.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

Opens the Track Points Overlay Options
dialog box. Use this dialog box to
change the default settings of the Track
Points marker and path displays.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

The default origin of the coordinate system used
in image windows is in upper left corner of the
window (or lower left, if you chose Y
-
Coordinate
Increases up the Screen in the Set Graphics
dialog box). Set Origin allows you to obtain point
coordinates that are defined in terms of a
particular location in the image that you select
as a different point of origin.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

Creates a blank image with a copy of the track
overlay, as currently displayed on the tracking
image.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Points

Displays object data over time or plane number in
graphical format. You can select the Data type to
display

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects

Tracks one or more selected objects
through each image in an image stack or a
sequential series of single images. You can
derive measurements of the paths,
positions, and velocities of the points.


Advantages


Can require little user intervention


Multiple targets can be tracked with
subpixel

accuracy


Very reproducible


Many display/analysis tools available


Disadvantages


Requires certain targetable features


Does not work if targets bump


Can be slow


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects

Different Source Image options

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Configuration

Determines the Algorithm
MetaMorph uses to find the
objects as it moves

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Configuration

Configures the Time interval, Display options and where the origin will be.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Placing Regions to track Objects

Pressing Track button opens Select Objects dialog

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Displaying and Editing Data

Displays the statistical data for each object's track.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Displaying and Editing Data

Displays the point
-
by
-
point measurement data for individual objects.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Displaying and Editing Data

Displays object data over time or plane number in graphical format.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Displaying and Editing Data

Allows you to edit specific points in the data set.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects
-

Displaying and Editing Data

Creates a blank image with a copy of the track overlay, as currently
displayed on the tracking image.


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Multi Dimensional Motion Analysis

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Track Objects


Do Tracking Exercise 5
-
8


©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Motion analysis


When would you use Kymograph, Track Points, Track Objects or
nD

Motion?


Kymograph


Visualization of time vs. space can aid in locating faint objects


Useful for objects traveling along known one
-
dimensional paths (e.g.
leukocyte rolling along vessel wall)


Track Points


Manual for difficult samples and/or greatest accuracy


Validation of automated methods


Track Objects


Automated search


Saves time


Avoids human bias


Multi
-
dimensional Motion Analysis


Automated object identification


nD

data sets

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Advanced segmentation &
analysis

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.


Intuitive, dedicated dialog boxes for biology
-
specific applications,
consistent basic set of input parameters, interactive data and graphics


Adaptive Background Correction™: Improved segmentation through
adaptation to local content


Field and cell
-
by
-
cell data logging


“Canned,” walk
-
away automation


Validated results in
-
house and with third
-
party


Can be incorporated into a journal for increased customization and
further automation of your analysis

What are Application Modules?

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Available modules for version 7.7

1.
Angiogenesis Tube Formation

2.
Cell Cycle

3.
Cell Health*

4.
Cell Scoring

5.
Count Nuclei

6.
Granularity

7.
Live/Dead

8.
Mitotic Index

9.
Monopole Detection

10.
Multi Wavelength Cell Scoring

11.
Neurite Outgrowth

12.
Micronuclei




©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Adaptive Background Correction: robust algorithms built
-
in


Splitting of touching cells


Detection even in noisy and poorly stained images


Stable parameters perform consistently across entire experiment set

Analysis
automatically
adjusts for
uneven image
background

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules


Modules require grayscale 16 bit fluorescent
*

images (one for each
wavelength analyzed)


Modules require a set of size and contrast parameters to adjust the
processing the image(s)


Approximate object size range


Intensity above local background levels


The measurements typically include


Object counts and normalized counts


Areas


Intensity, average and total


Cell classifications

*some modules can be used with transmitted light also

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules: preview each wavelength

Each wavelength
configured
independently
with quick
Preview

of
results

Consistent set of
segmentation
parameters: size
range and
staining
intensity

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules:

determining size parameters


Select the Line tool from the Region toolbar


Move the mouse pointer the edge of a small
object and click


A tool
-
tip appears showing the current X and Y
values of the pointer, as well as the length



Move the mouse pointer to the opposite edge of the object and note the
Length value


This number represents the cell width in pixels


If the image is calibrated, the length is in both pixels and calibrated units


Type or select the value in microns as “Approximate min width”


Repeat steps for a large object

values are approximate to control filtering
during segmentation

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules: intensity parameters


The “Intensity above local background” field is common for all application
modules


Part of automatic
Adaptive Background Correction

system


Specifies the intensity threshold
relative

to nearby background values


Controls the sensitivity of object detection



Estimate the difference between the object’s signal and background


Hint: gray
-
level values of image pixels under the mouse cursor are
shown in the main MetaMorph status bar (bottom of main window)



Enter this number in the “Intensity above local background” field

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules: logging measurements


Summary Log Parameters


Data Log (Cell) Parameters



Neurite
Outgrowth




Neurite
Outgrowth

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules: interactive graphics

Graphic overlays of
segmentation results
interact with cell
-
by
-
cell
spreadsheet views of
measurement data

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Running the Application Modules: interactive data table

Cellular Results Table: click on objects in the table and the corresponding
cells in the source images are highlighted in yellow. Click on a cell in the
source images and the corresponding data is highlighted in the Cellular
Results Table.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Count Nuclei Application Module


Single
-
wavelength segmentation and
counting


Building block for multi
-
wavelength
applications


Applications


Cell Proliferation


Cell Counting


Cell Migration



Note: Grey value in result image = cell
assigned label #

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devi
ces
, LLC or their respective owners.

Angiogenesis Tube Formation Application Module


Applications


Measuring and characterizing endothelial tube formation (a model
system for angiogenesis)


Research focused on the promotion or inhibition of blood vessel
formation

Important in cancer, diabetes and other vascular disease research


Specific kits available from leading manufacturers


Area of research increasing in popularity with success of drugs such as
Genentech’s
Avastin

which blocks VEGF activity