http://imagej.273.s1.nabble.com/Focus-measurement-more-general-image-analysis-question-tp3700123p3700131.html
I don't know whether I understood... (maybe I made a diffuse
description)... I don't want to filter objects within one image, I would
like to judge whether the majority of objects is in focus or not. In
focus over the whole area. I just would like to analyze the images which
have well focused objects. And I have to automate this. That means, I'd
> Ok, now we come to some progress, hopefully.
>
> You have the detected cell/nuclei, than
> -apply the already recommended Sobel or any other edge detector to the
> vesicle fluorescence image,
> -Measure SD and mean Gray Value (Redirect To the filtered image) (don't
> ask me how that is done, but I know it is possible)
> -find the threshold(s) for which YOU consider the vesicle as bad focussed
> -throw away the objects falling under the criteria
> typically SD or SD/Mean can discriminate unfocussed (smoothed) areas.
>
> Regards
> Karsten
>
> Am 06.03.2007 um 11:42 schrieb Antje:
>
>> Hi Karsten,
>>
>> okay, I try to explain what I would like to do.
>> Let's assume I have 2 images (gray-values) of different fluorochromes
>> of the same objects. On is representing the nucleus and the cell, the
>> other represents a dotty structure within the cell (vesicles).
>> Now, I would like to detect the cell, the nucleus and the vesicles, so
>> that I can measure them (e.g. size of the vesicles).
>> But I would like to exclude that I get biased data from objects which
>> are bad focused. A blurred vesicle then is much larger, than a sharp
>> one, though they might have the same size in reality...
>> My aim is to do high content analysis in an automated way (maybe also
>> extract further parameters of the vesicles and to feed some kind of
>> classifier with the data).
>>
>> Ciao,
>> Antje
>>
>>
>>
>> Karsten Rodenacker schrieb:
>>> Hi Antje,
>>> a quite difficult discussion track. Perhaps you should explain what
>>> you would like to do. I tried to say that there is no focus per se.
>>> You should describe for what which focus is necessary.
>>> If you like to estimate maker density per cell number or cell volume
>>> the focus might be not so important. Since number estimation is
>>> expensive (time consuming) possibly the density per volume is
>>> sufficient. I have had very often discussions about necessity!
>>> Unbiased Stereology from Howard and Reed might help.
>>> Regards
>>> Karsten
>>> Am 06.03.2007 um 10:51 schrieb Antje:
>>>> Hi Jan,
>>>>
>>>> if I take a Sobel filter, it'll be dependent of the amount of
>>>> objects within the image. Mean, Median or sum of the gradient values
>>>> over the whole image will be influenced by the number of objects
>>>> (density), I guess. Further, object detection before, might be
>>>> dependent on the focus so that you could end up with values you
>>>> cannot compare.
>>>> Is there any other way to use the gradient information?
>>>>
>>>> Ciao,
>>>> Antje
>>>>
>>>>
>>>>
>>>> Jan Eglinger schrieb:
>>>>> Dear Antje,
>>>>> I'd suggest to use an edge detection filter (e.g. Sobel filter
>>>>> "Find Edges") and to take the brightness of the result as a score.
>>>>> Images with sharp edges should produce a high score that permits
>>>>> you to sort your images.
>>>>> I don't know if this will work with your set of images, but it
>>>>> should be a quick way to get some idea of the "focus" quality
>>>>> (provided your objects are mainly in a single plane of focus).
>>>>> Best,
>>>>> Jan
>>>>> -------- Original Message --------
>>>>> From: Antje <
[hidden email]>
>>>>> To:
[hidden email]
>>>>> Subject: Re:Focus measurement (more general image analysis question)
>>>>> Date: 06.03.2007 10:04
>>>>>> Hi Karsten,
>>>>>>
>>>>>> you're right confocal microscopic images should be in focus by
>>>>>> definition. It's for sure that our microscope needs to undergo
>>>>>> improvements...
>>>>>> Anyhow, due to a large screening mode of this microscope, I have
>>>>>> to cope with out of focus images (unfortunately, I'm not that deep
>>>>>> into physics to be able to explain the reason for this focus
>>>>>> problems).
>>>>>> I don't have stacks.
>>>>>>
>>>>>> Ciao,
>>>>>> Antje
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Karsten Rodenacker schrieb:
>>>>>>> You are tackling an interesting question. To be in focus is
>>>>>>> always meant for an object, adjust focus is meant for a whole
>>>>>>> image. Hence the problem with focus is the observer, a cell of
>>>>>>> certain thickness might be in focus at the cell border or at the
>>>>>>> cell center.
>>>>>>>
>>>>>>> However, still there is a more important question. Images from
>>>>>>> confocal microscopes are by definition in focus of course related
>>>>>>> to the 'pin-hole' size. If you call such images in 'bad focus'
>>>>>>> maybe there is some help in microscope adjustment necessary?
>>>>>>> If you have stacks of images, images taken at different focal
>>>>>>> adjustments there is a program to merge such stacks into one
>>>>>>> image (e.g. plugin Exteded depth of field). However, my first
>>>>>>> remark is still valid, meaning the outcome of such a method is
>>>>>>> possibly far away from expectation.
>>>>>>> Regards
>>>>>>> Karsten
>>>>>>>
>>>>>>> Am 06.03.2007 um 09:17 schrieb Antje:
>>>>>>>
>>>>>>>> Hello,
>>>>>>>>
>>>>>>>> sorry, the question is not that strongly related with ImageJ but
>>>>>>>> maybe there is still someone who can help?
>>>>>>>>
>>>>>>>> I have a huge set of microscopic images from a confocal
>>>>>>>> microscope (cells/nuclei + marker) and I'd like to automatically
>>>>>>>> filter images which have a bad focus. First, I was thinking
>>>>>>>> about some gradient values to judge but they are so dependent of
>>>>>>>> the content (number of cells... , presence of the marker). Is
>>>>>>>> there any reliable method known to measure and compare the focus
>>>>>>>> of images??? Or does anybody know papers dealing with this issue???
>>>>>>>> (If there is something available in ImageJ it would be fine but
>>>>>>>> I could not find anything...)
>>>>>>>>
>>>>>>>> Antje
>>>>>>>>
>>>>>>>>
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