Generally speaking, in terms of image analysis the magnification is irrelevant, especially once you are looking at something on the screen.

What you need to know is how many pixels represent a known unit of length.

In your image, this information is conveyed by the scale bar.

Open the image in ImageJ (or Fiji, etc) and use the line tool (5th from the right on the toolbar) to mark a line over the length of the scale bar: click on one side and then the other. Now go to the menu: Analyze > Set Scale...

The first field, Distance in pixels should be filled for you (from the use of the line tool). In the 2nd field, enter the known distance: 50

In the last field, Unit enter: um for micrometers.

If you plan on analyzing several images with the same scale, check the box "Global" else just hit OK. You now have a calibrated image: at the top of your image you should see the dimensions in microns and not pixels.

Now using the same line tool you can now measure distances. Click on either side of your object and then go to Analyze > Measure (the keyboard shortcut is useful). You should get a window to pop up with the Results. You can adjust what is in the Results in the menu Analyze > Set Measurements...

I follow the question.

Hi @Na Le,

You can only know the size of this object by taking the measurement in microscopy. Then, when you import the image to ImageJ, you already know the exact length of object and the corresponding length in pixel given by ImageJ. Next, you should Set Scale i.e. 1 um corresponds to how many pixels. I assume that you want to measure the in situ image taken in experiment, then with the length in pixels, you can get the length in um.

Generally speaking, in terms of image analysis the magnification is irrelevant, especially once you are looking at something on the screen.

What you need to know is how many pixels represent a known unit of length.

In your image, this information is conveyed by the scale bar.

Open the image in ImageJ (or Fiji, etc) and use the line tool (5th from the right on the toolbar) to mark a line over the length of the scale bar: click on one side and then the other. Now go to the menu: Analyze > Set Scale...

The first field, Distance in pixels should be filled for you (from the use of the line tool). In the 2nd field, enter the known distance: 50

In the last field, Unit enter: um for micrometers.

If you plan on analyzing several images with the same scale, check the box "Global" else just hit OK. You now have a calibrated image: at the top of your image you should see the dimensions in microns and not pixels.

Now using the same line tool you can now measure distances. Click on either side of your object and then go to Analyze > Measure (the keyboard shortcut is useful). You should get a window to pop up with the Results. You can adjust what is in the Results in the menu Analyze > Set Measurements...

Peter Sobolewski

Thank you very much for your answer. I did the same as you have mentioned. However, I think I need to also consider about the magnification. Because the distance I measured by this method is different from the actual size of this rectangular.

So I did like this: Firstly, I took an image of a line with known length. then I calculated the ratio of distance (micron) to pixels.

secondly, I took an image of the rectangular that I need to known the size with the same objective lens, same magnification. Then I counted the pixels and used the ratio above to calculate the distance( in micron)

Vinh H. Nguyen Anh Vinh

e tính được rồi, phương pháp giống anh nói

Ok Na, hehe

Based on the scale bar in the image, the object is ~450 micron wide.

You know the dimensions of the square object and this is not correct? If so, that means the scale bar present in the image right now is simply incorrect and you need to calibrate the software you used to generate it.

Again, it's not magnification that is important but the relationship between pixels and known distance. For a given objective *and* camera resolution, this is a constant, so you can use the image of a known object—ideally a micrometer slide—to calibrate. If you open the image of the known object, mark the known distance, and follow my instructions above, just check the Global box. Then open the images you want to measure that were captured with the same objective and resolution. If you change the resolution or objective you need to recalibrate.

Listen to Peter Sobolewski. Magnification is irrelevant, its value is always wrong for reproduced image, Only scale bar can help you.

Peter Sobolewski thank you for your reply.

I have one more question.

AS you can see the scale bar from the image. Is this true that I can always use the scale bar to measure the length without consider any magnification?

Thank you.

yes, you are correct.

Suppose you have this picture on a monitor of your computer. Then you drag its corner so that to make its sides twice as long. It means you magnified it x2 compared to its previous state (on your monitor). The value of magnification in your records stay the same (and it is wrong), but the length of a micron bar was increased.

You can change the size of your picture as you wish, the length of micron bar will be changed as well and always will give the right value.

thank you Vladimir Dusevich

I know what you mean.

I mean that the magnification of camera, lens,....I dont have to care about it. Is it true that I dont have to care about them?

I just use the scale bar to calibrate and then calculate the length?

N. T. L. , as I understand the issue, you only can 'trust' the scale bar in your imaging system

AFTER you have CALIBRATED*) the imaging system (= digital camera) for "true" length distances in your sections....

that usually has to be done FIRST (and usually only once for any microscopic objective**) you use) by means of an "object-micrometer slide" (one can get from relevant microscopy suppliers).

*) cf. Reply by Peter Sobolewski 8 days ago: "...you need to calibrate the software you used to generate it [NB: i.e., the digital image you capture and finally will access and make visible on your monitor)] .

**)As you know for sure that the engraved "magnification number" [e.g. x10, x25, x40, Oil x50 or Oil x100) on a certain objective of a microscope only is a rough measure information. The "true magnification power" of a microscopic objective always has to be established by means of an 'eye-piece micrometer' in connection with an 'objective micrometer'

(for a view online cf. e.g. https://www.shibuya-opt.co.jp/eng/stage_top.html, or: https://www.shibuya-opt.co.jp/eng/stage.html; more information on the handling too: e.g. cf.: https://www.preiser.com/objectivemicrometer001mmrulings.aspx.

and/or: http://www.mecanusa.com/microscope/micrometer/Calibration.htm

I am sure that anywhere in your lab - or at least the 'vicinity of your lab' - you might find an S. O. P. or an operation manual about the Digital Imaging System (product information) or - if combined with the microscope you use, from the microscope manufacturer. If you can't find yourself, ask your colleagues, the lab-supervisor, or try to get manuals ('instruction for use', 'How to...', etc) from the supplier / commercial provider of the instruments you use, the last resource always would be the Manufacturing Company of your Digital Imaging System or (Light) Microscope.

Best of luck, regards W. H. M.

[Disclaimer: no affiliation with or any financial interest in - regarding the mentioning of company names here.]

Magnification in microscopy is calculating by multiplying the power of the ocular x objective. Software are good for calculating complex images, for a square shape you can go with a grid. https://www.microscopeworld.com/c-243-reticles-and-stage-micrometers.aspx#/?Category1=Accessories&Category2=Reticles+and+Stage+Micrometers&search_return=all&Category3=Grid+Reticles

@ Na Le

Only scale bar. But, as pointed by Wolfgang H. Muss your system should be properly calibrated (do display a scale bar of the right size).

Dear Na Le,

I cam late to your question. By reviewing the scale bar length and the magnification i found that both are correct.

The proof:

The length of the scale bar in the image = 10 mm= 1 cm,

The magnification= 200,

Then the corresponding object length= 10 mm/200= 50 um,

Which is the value indicated on the scale bar for the object.

Where is then the problem, you can use both to get the object length.

The value is the scale bar is already calculated based on the magnification.

Best wishes

@ Abdelhalim Zekry

You found out that magnification is correct? Remarkable...