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Image Analysis:

To utilize the information contained in the

digital image data matrix for the purpose
of quantification.

1) Particle Counts
2) Area measurements
3) Mean particle diameter
4) Length or Size distribution
Image Analysis:
Underlying the principles of image analysis
the operator must remember one essential
Image Analysis:
Underlying the principles of image analysis
the operator must remember one essential

Computers are STUPID!

Image Analysis:

Dog or Cat?
Any four year old can tell the difference but even
the most sophisticated computers would have
difficulty making the distinction.
First step in image analysis is to define
those features that you wish to analyze so
that the computer can know what data
in the image is significant.
1. By grey level (pixel value)
2. By size (# of contiguous pixels within a
certain value range)
3. By shape (round vs. elongate)
First step is create a
binary image based
on some cut off value
for pixel intensity.
First step is create a
binary image based
on some cut off value
for pixel intensity.
A binary image can
also be adjusted to
cover a subset of pixel
In cases where the objects of interest are quite
distinct it can be relatively straightforward to
distinguish them based on pixel value alone.
Sometimes simple
thresholding is insufficient
in defining those features
one wishes to count,
especially if the objects
are touching each other
making it difficult to
distinguish one object
from another.
Adjusting the contrast of
the image may help the
operator identify the
objects of interest but the
computer would still have
difficulty identifying the
The operator can use a
marking tool to identify
the objects of interest.
This image may be easily
thresholded and made
into a binary image in
which the number of
objects may be easily
The creation of a binary image is only part of
what needs to be done.
If Objects appear to be connected, even by a
narrow bridge, then those two objects will be
considered as a single object by the computer.
The connections can be dissolved by
performing an erosion operation which will
remove the peripheral pixels in a pixel by
pixel manner.
This will reduce the area occupied by the objects
but pixels can be added back by a process known as
dilation so that objects are restored to nearly their
original size without reconnection.
Free image analysis programs are
available for downloading from the
National Institutes of Health. They include
many basic and some sophisticated

rsb.info.nih.gov/nih-image NIH Image (Mac)

rsb.info.nih.gov/ij ImageJ (PC)

Both NIH-Image and ImageJ have similar
capabilities although the layouts are quite
First step is to crop the image so that only the
area of interest is used.
Sometimes the image contrast must be inverted
if the objects of interest are dark.
Next the image must be thresholded to create a
binary image.
Using the Analyze Particles under the
Analyze window all particles greater than 1
pixel and smaller than 999999 will be counted
Over 1300 particles are counted, most of
which are only 1 pixel in size
If we raise the minimum size to 5 adjacent
pixels and rerun the analysis
If we raise the minimum size to 5 adjacent
pixels and rerun the analysis we get many fewer
particles, only the 166 ones of interest
The output of the
particle analysis can be
exported as a file that
can be uploaded into a
spreadsheet program
such as Excel and
When placed in a
spreadsheet the data
can be analyzed in
many different ways
including size
distribution, average
size, percent area, etc.
Using this information one can refine the
analysis looking for a way to distinguish single
pores (72-92) vs. double pores (100-152)
An average area
measurement can now
be calculated for each
single pore (85.5) and a
ratio of single vs.
double pores (5:2) can
be determined.
Depending on how the
image is to be used the
operator can choose to
collect the image in
very high contrast.
This will make the
thresholding of the
image much easier.
Side SE detector

The choice of
detector can also
affect the image

In-lens SE detector
Side SE detector

Especially after the

image is

In-lens SE detector
If one can calculate the pixel size, then
accurate size and area measurements are
Sophisticated image analysis can recognize patterns
and detect and identify such complex patterns as
those contained in diffraction patterns.
Measurements in Three Dimensions:

What if we wanted to measure the precise

height difference between two objects in an
SEM image?
Measurements in Three Dimensions:

First we must create

a stereo pair image.
Even though a
conventional SEM
image has a great
depth of field it is
still a two
dimensional image.
Measurements in Three Dimensions:

Steps in creating a stereo pair image

(Bozzola & Russell p. 221-224)

True 3-D imaging requires that the object be

viewed from two different angles at the same
time. A person with sight in only one eye can
have excellent depth perception but cannot
see something in 3-D.
Measurements in Three Dimensions:

The first step is to create a stereo pair of

images in which the specimen is
eucentricaly tilted 8-12 degrees between
Measurements in Three Dimensions:
To aid the observer in visualizing the stereo
pair the left hand view can be colorized
blue and the right hand view made red and
superimposed on one another
Measurements in Three Dimensions:
These Red/Blue images are known as anaglyph
projections and can be quite dramatic
Measurements in Three Dimensions:
Measurements in Three Dimensions:

MeX is a software product to compute and analyze

digital elevation models (DEMs) from stereoscopic
scanning electron microscope (SEM) images.
MeX opens up the third dimension to the SEM
users. In order to determine the topography of
microstructures MeX is the ultimate tool when
other means have come to an end.
Using MeX you can measure profiles, roughness
values, area parameters and even volumes of your
specimen from SEM images.
Measurements in Three Dimensions:

First one defines

an area for a
digital elevation
map (DEM).

Measurements in Three Dimensions:
The creation of a
DEM is
intensive and starts
by building a wire-
frame model which
can then be
surfaced rendered

Measurements in Three Dimensions:

Once created the DEM

can be viewed from
many angles and color
coded to emphasize
features such as height.

Measurements in Three Dimensions:

Compare the DEM with an anaglyph projection

Measurements in Three Dimensions:

Profiles can
for objects that
vary in height

Measurements in Three Dimensions:
One can even
measure volumes
using this

Unlike ImageJ
MeX is not free!