Did you use any other techniques suggested on this page? http://imagej.net/Particle_Analysis It might help if you post the image.

Yes, I have tried with these other techniques but the points seems to be too smal to be measured. Here's the picture. Even there's a background, you can see some small points that corresponde with our protein.

Thank you =)

Hello

First a remark. Using a channel's intensity for both detection and measurement introduces a bias. One usually detects something using some condition-independent staining (Like DAPI for nuclei) and then measures in the channel of interest (EdU for example). 

Second. OK you get point count and intensity. How are you going to relate these numbers between acquisitions and batches? Simple light microscopy is great for telling you where things are, not how much of it there is. Variations in staining protocol, light sources, microscope cleanliness, and such makes direct intensity measures difficult without internal controls (The equivalent of the actin band in a Western Blot).

I do not understand what you mean by "The points seem to small to be measured". Can the detection algorithms not detect them properly? If so, how about outlining them manually? Perhaps change the acquisition by using a larger objective so the objects cover a larger number of pixels? Without a serious image to look at, it seems rather difficult to provide help at this point. 

Best

HI,

First, I agree with Olivier on much of what he said. 2nd, Concerning the counting, what you need to use is a single molecule tracking software. You can try the Matlab-based free software called Fiesta. The learning curve is not that steep and you should be able to count "points" easily. Link is attached. I would also try another image analysis software called Fiji (imagej), there is a lot of work on tracking and there is a high chance you will find a Fiji plugin for tracking.

Be ware that you cant tell the number of proteins in each "point" unless you know the expected counts (intensity) of a single protein. This means that you will need to do bleaching as function of time. If all goes will, you will notice a step wise drop in the intensity signal from which you can tell the signal of one protein. (I advise reading about that before doing it)

Hope this helps

https://www.bcube-dresden.de/fiesta/wiki/FIESTA

well that is pritty easy with the right approach

a) the best:

see the following papers and the successor publications by Malte Wachsmuth

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging.

Weidemann T, Wachsmuth M, Knoch TA, Müller G, Waldeck W, Langowski J.

J Mol Biol. 2003 Nov 21;334(2):229-40.

b) make the right controls if you want to get it for entire cells - so you could use a flurimeter, or a FACS - but you need to have a good control and be very accurate...

so... 

You can give it a try to Process>Find Maxima... in Fiji or ImageJ. It ´s a pretty simple tool to use. I would recomend first to measure the STDV of the backgraound and to set a "noise value" >3-4 times higher.

I recommend also to read our paper: Endocytosis as a Biological Response in Receptor Pharmacology: Evaluation by Fluorescence Microscopy

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122604

We only count endosome number and no their intensity, but once you get the coordinates of your particle it is simple to measure the intensity.

Thank you all. I will try your advices =)

Hi Laura

I think, if you will follow the attached guide about counting fluorescence particles you can get something as I have done with your figure. I hope, it is meaningful image analysis

I think the ImageJ-plugin QuickPalm (already installed in recent versions) is exactly what you need. You find it via ImageJ->Analyze->QuickPalm->Analyse Particles

For help check out https://code.google.com/p/quickpalm/

on the analysis side, I agree with most of the comments above (although I usually use Imaris). On the imaging side, if the picture you posted is representative, you have a fairly low signal-to-noise ratio, with low signal in the first place, which question your ability to recognize the sites and quantify them. Second, there is limited spatial signal in this picture: i.e., it is unclear where you should quantify your points.

I am grateful for all your answers. I will try them and, if I definitively get it I will show you the result =)

FACS machines are pretty good quantifying machines, I don not remember right now an example of protein quantification, but we have been quaitifying  total DNA (c value) in cells

for protein, have a look

http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-259-9_7