How to measure grain size using ImageJ software

copy & paste from http://osdir.com/ml/java.imagej/2006-04/msg00010.html


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Anneliese writes,

>
> Date: Sat, 1 Apr 2006 14:46:34 +0200
> From: Klughammer GmbH
> Subject: ImageJ and Metallography
>
> Dear users,
>
> has anybody already made some experience with ImageJ and metallography?
>
> I am looking for
>
> - grain size measurement
> - graphite morphology
> - nodularity measurement
> - particle size distribution
>
> Anneliese
>
And Noel replies.
Refer to the following books.
Computer aided Microscopy by John Russ,
Quantitative Stereology by E. E. Underwood,
and the paper by D.C. Sterio 1984 Journal of Microscopy. (the unbiased
estimation of number and sizes of arbitrary particles using the disector, J
microscopy. 134, 127.

Grain size measurement in metallography proves to be difficult because it is
usually next door to impossible to produce a perfect polish and etch which
reveals all boundaries with sufficient difference to the matrix.
Thus thresholding will be incomplete.
Once you have perfect boundaries, then life is easier.
By perfect boundaries I mean black boundaries on a pure white background.
You may measure grain sizes using all three methods easily.
Linear intercept,
Triple point counting,
And area measurement.

Here are some formulae which are commonly used. (From Russ. J.C. Computer
Assisted Microscopy, Plenum Press 1990, isbn 0-306-43410-5.
P 225.
ASTM grain size G
Using intercept method

G=(-6.6457Log[base10](1/PL))-3.298

Where PL is the number of points per unit length, measured in millimeters.

Or if you measure areas of the grain bodies,

(From Russ. J.C. Computer Assisted Microscopy, Plenum Press 1990, isbn
0-306-43410-5.
P 225.
G=(3.22 Log[base10](NA.M^2))-2.95

Where NA is number of grains per unit area on a polished surface at a
magnification M.

If you count the nodes where triple points are then G is got from
(From Russ. J.C. Computer Assisted Microscopy, Plenum Press 1990, isbn
0-306-43410-5.
P 147.

G=(log[base e]((Nodes/2)-1)/Area)/(log[base e](2)) -2.95.

Some investigators have found it quicker to produce a photo, and to trace
the boundaries out manually using a transparent film and a felt pen. The
resulting drawing is then scanned into the computer and processed in ImageJ.
This will be black and white and easily thresholded into a perfectly
segmented binary image. Such an image is the ideal to which your preparation
must aspire.

Otherwise it may be more time consuming.

If your specimens are suitable, and your etching superb, then
it may be possible to produce a perfect image via the image processing tools
in ImageJ.
Techniques to investigate include, thresholding, subtract background, find
edges, skeletonize, erode, dilate, open, close, watershed and so on.
Also useful may be techniques which differentiate between rough and
smooth, or surfaces with different textures.
Each type of material will have its own behaviour, and you must discover
this for yourself.

I have just talked about grains here.
The particle size distribution will be more straightforward, so long as the
particles are easily discriminated from the matrix. Just be careful that the
smallest particles you need to measure are "larger" than the resolution
limit.
The analyze particles menu in ImageJ will be what you use here.

And for Graphite morphology and nodularity, I have no experience.
But I am sure the methods to be used will have the parameters you need and
these will easily be employed in a macro.

Regards
Noel Goldsmith

Noel Goldsmith
Aircraft Forensic Engineering
Air Vehicles Division
DSTO
506 Lorimer Street
Port Melbourne
Vic 3207
AUSTRALIA
Phone (613) 96267538
FAX (613) 96267089
Email noel.goldsmith@xxxxxxxxxxxxxxxxxxx

1 day training session on our new stereomicroscope & metallurgical microscope

Salam & selamat sejahtera,

Dear colleague, brothers & sisters

Please plan to be at the 1 day training session on our new stereomicroscope & metallurgical microscope (basic unit) that will be held on

Date:   16^th September 2009 (Wednesday)

Time:   10.00 am

Place: Makmal Kubang Gajah (MBG2).

A very experience application specialist from Crest Systems (M) Sdn Bhd will be the speaker and he will share his knowledge on microscope instruments, application using stereomicroscope & metallurgical microscope in material/ metallurgical research and other fields, basic/ advance techniques image analysis and etc.

The topic of training basically covers

i)     Streomicroscope (OLYMPUS SZ61TR) – low power scope

ii)    Metallurgical microscope (BX51M) – high power scope

iii)   CMOS Digital Camera (Moticam 2300)

iv)   Basic Image Analysis (Image + 2.0 & Image Adv.)

§         Counting particle and measuring function (linear, area, angle, perimeter, etc)

§         Multilayer version  of the same image (each focused at a different position, can be combined produce a single focused image)

§         Image Calibration

Who knows, some of these systems and equipment may be able to solve problems at our lab session, or provide good tools for metallurgical/ materials teaching subject or furthering your R&D activities. Hope to see you there.

Thanks

Note: Postgraduate students or research asst. are also invited

Common Etchants for Copper, Nickel and Cobalt: Copper & Alloys

Composition	Comments
2 5 mL NH4OH 25 mL water (optional) 25-50 mL H2O2 (3%)	General purpose grain contrasts etch for Cu and alloys (produces a flat etch for some alloys). Use fresh, add peroxide last. Use under a hood. Swab specimen 5-45 seconds.
100 mL water 10 g ammonium persulfate orientation.	General purposes etch for Cu and alloys. Immerse or swab for 3-60 seconds. Reveals grain boundaries but is sensitive to crystallographic
100 mL water 3g ammonium persulfate 1mL NH4OH	General purpose etch for Cu and alloys, particularly Cu-Be alloys.
70 mL water 5 g Fe(NO3)3 25 mL HCI	Excellent general purpose etch, reveals grain boundaries well. Immerse specimen 10-30 seconds

Common Etchants for Iron and Steel

Composition	Comments
90-99 mL methanol or ethanol 1-10 mL HNO3	Nital. Most common etchant for Fe, carbon and alloy steels, cast iron. Reveals alpha grain boundaries and constituents. Excellent for martensitic structures. The 2% solution is most common, 5-10% used for high alloy steels (do not store). Use by immersion or swabbing of sample for up to bout 60 seconds.
1 00 mL ethanol 4 g picric acid	Picral. Recommended for structures consisting of ferrite and carbide. Does not reveal ferrite grain boundaries. Addition of about 0.5-1% zephiran chloride improves etch rate and uniformity.
100 mL ethanol 5 mL HCI 1 g picric acid	Vilella’s reagent. Good for ferrite-carbide structures. Produces grain contrast for estimating prior austenite grain size. Results best on martensite tempered at 572-932 °F (300-500 °C). Occasionally reveals prior-austenite grain boundaries in high alloy steels. Outlines constituents in stainless steels. Good for tool steels and martensitic stainless steels.
Saturated aqueous picric acid solution grain plus small amount of a wetting agent	Bechet and Beaujard’s etch, Most successful etchant for prior-austenite boundaries. Good for martensitic and bainitic steels. Many wetting agents have been used, sodium tridecylbenzene sulfonate is one of most successful (the dodecyl version is easier to obtain and works as well). Use at 20-100 °C. Swab or immerse sample for 2-60 minutes. Etch in ultrasonic cleaner Additions of 0.5g CuCl2 per 100mL solution or about 1% HCI have been used for higher alloy steels to produce etching. Room temperature etching most common. Lightly back polish to remove surface smut.
150 mL water 50 mL HCI 25 mL HNO3 1 g CuCl2	Modified Fry’s reagent. Used for 18% Ni maraging steels, martensitic and PH stainless steels.
1 00 mL water 25 g NaOH 2 g picric acid	Alkaline sodium picrate. Best etch for McQuaid-Ehn carburized samples. Darkens cementite. Use boiling for 1-15 minutes or electrolytic at 6 V dc, 0.5 A/in2, 30-120 seconds. May reveal prior-austenite grain boundaries in high carbon steels when no apparent grain boundary film is present.
1 00 mL ethanol 100 mL HCI 5 g CuCl2	Kalling’s no. 2 (“waterless” Kalling’s) Etch for austenitic and duplex stainless steels. Ferrite attacked readily, carbides unattacked, austenite slightly attacked. Use at 20 °C by immersion or swabbing. Can be stored.
1 5 mL HCI 10 mL acetic acid 5 mL HNO3 2 drops glycerol	Acetic glyceregia. Mix fresh; do not store. Use for high alloy stainless steels.
100 mL water 10 g K2Fe(CN)6 10 g KOH or NaOH	Murakami’s reagent. Usually works better on ferritic stainless grades than on austenitic grades. Use at 20 °C for 7-60 seconds: reveals carbides sigma faintly attacked with etching up to 3 minutes. Use at 80°C (176°F) to boiling for 2-60 minutes: carbides dark, sigma blue (not always attacked), ferrite yellow to yellow-brown, austenite unattacked. Do not always get uniform etching.
100 mL water 1 0 g oxalic acid	Use for stainless steels at 6 V dc. Carbides revealed by etching for 15-30 seconds, grain boundaries after 45-60 seconds, sigma outlined after 6 seconds. 1-3 V also used. Dissolves carbides, sigma strongly attacked, austenite moderately attacked, ferrite unattacked.
100 mL water 20 g NaOH	Used to color ferrite in martensitic, PH or dual-phase stainless steels. Use at 3-5 V dc, 20°C, 5 seconds, stainless steel cathode. Ferrite outlined and colored tan.
40 mL water 60 mL HNO3	Electrolytic etch to reveal austenite boundaries but not twin boundaries in austenitic stainless steels (304, 316, etc.). Voltage is critical. Pt cathode preferred to stainless steel. Use at 1.4 V dc, 2 minutes.

Commonly Used Etchants for Magnesium and Alloys

Composition	Comments
25 mL water 75 mL 3-5 ethylene glycol 1 mL HNO3	Glycol etch, general purpose etch for pure Mg and alloys. Swab specimen seconds for F and T6 temper alloys, 1-2 minutes for T4 and 0 temper alloys.
19 mL water 60 mL ethylene glycol 20 mL acetic acid 1 mL HNO3	Acetic glycol etchant for pure Mg and alloys. Swab specimen 1-3 seconds for F and T6 temper alloys, 10 seconds for T4 and 0 temper alloys. Reveals grain boundaries in solution-treated castings and most wrought alloys.
100 mL ethanol 10 mL water 5 g picric acid	For Mg and alloys. Use fresh. Immerse specimen for 15-30 seconds. Produces grain contrast.

Commonly Used Etchants for Aluminum and Alloys

Composition	Comments
95 mL water 2.5 mL HNO3 1.5 mL HCI 1.0 mL HF	Keller’s reagent, very popular general purpose reagent for Al and Al alloys, except high-Si alloys. Immerse sample 10-20 seconds, wash in warm water. Can follow with a dip in conc. HNO3. Outlines all common constituents, reveals grain structure in certain alloys when used by immersion.
90-100 mL water 0.1-10 mL HF	General-purpose reagent. Attacks FeAl3, other constituents outlined. The 0.5% concentration of HF is very popular.
84 mL water 15.5 mL HNO3 0.5 mL HF 3g CrO3	Graff and Sargent’s etchant, for grain size of 2XXX, 3XXX, 6XXX, and 7XXX wrought alloys. Immerse specimen 20-60 seconds with mild agitation.
1.8% fluoboric acid in water	Barker’s anodizing method for grain structure. Use 0.5-1.5 A/in2, 30-45 V dc. For most alloys and tempers, 20 seconds at 1 A/in2 and 30 V dc at 20 °C is sufficient. Stirring not needed. Rinse in warm water, dry. Use polarized light; sensitive tint helpful.

Etching Procedures

Microscopic examination is usually limited to a maximum magnification of 1000X — the approximate useful limit of the light microscope, unless oil immersion objectives are used. Many image analysis systems use relay lenses that yield higher screen magnifications that may make detection of fine structures easier. However, resolution is not improved beyond the limit of 0.2-0.3-um for the light microscope. Microscopic examination of a properly prepared specimen will clearly reveal structural characteristics such as grain size, segregation, and the shape, size, and distribution of the phases and inclusions that are present. Examination of the microstructure will reveal prior mechanical and thermal treatments give the metal. Many of these microstructural features are measured either according to established image analysis procedures, e.g., ASTM standards, or internally developed methods.

Etching is done by immersion or by swabbing (or electrolytically) with a suitable chemical solution that essentially produces selective corrosion. Swabbing is preferred for those metals and alloys that form a tenacious oxide surface layer with atmospheric exposure such as stainless steels, aluminum, nickel, niobium, and titanium and their alloys. It is best to use surgical grade cotton that will not scratch the polished surface. Etch time varies with etch strength and can only be determined by experience. In general, for high magnification examination the etch depth should be shallow; while for low magnification examination a deeper etch yields better image contrast. Some etchants produce selective results in that only one phase will be attacked or colored. Etchants that reveal grain boundaries are very important for successful determination of the grain size. A vast number of common etchants have been developed and it is displayed in Table 1 after this section.

ETCHING

Metallographic etching encompasses all processes used to reveal particular structural characteristics of a metal that are not evident in the as-polished condition. Examination of a properly polished specimen before etching may reveal structural aspects such as porosity, cracks, and nonmetallic inclusions. Indeed, certain constituents are best measured by image analysis without etching, because etching will reveal additional, unwanted detail and make detection difficult or impossible. The classic examples are the measurement of inclusions in steels and graphite in cast iron. Of course, inclusions are present in all metals, not just steels. Many intermetallic precipitates and nitrides can be measured effectively in the as-polished condition.

In certain nonferrous alloys that have non-cubic crystallographic structures (such as beryllium, hafnium, magnesium, titanium, uranium and zirconium), grain size can be revealed adequately in the as polished condition using polarized light. Figure (1) shows the microstructure of cold-drawn zirconium viewed in cross-polarized light. This produces grain coloration, rather than a “flat etched” appearance where only the grain boundaries are dark.

Figure 1: Mechanical twins at the surface of hot worked and cold drawn high-purity zirconium viewed with polarized light (200X).