thresholding and detection issues when analyzing particles to obtain object counts in a petri dish

[Rich Pendleton]

Hey all,



I was wondering if someone might be able to provide some insight similar to
a question that was posted last month.  I am trying to use the analyze
particles function to count fish eggs in a petri dish.  Some eggs are more
mature than others and the smaller less mature eggs are rather
transparent.  Therefore when trying to adjust the threshold to detect the
less mature eggs, I get clumping of mature eggs that are in close proximity
to one another.  Despite using the watershed function in attempt to
separate eggs that are close, I still have many large clumps that have
numerous eggs that are being counted as one unit. As a result, the counts
are always much less than the true count (often over 1000 eggs less). If I
adjust the threshold in the other direction, I am unable to detect the less
mature eggs.  It is quite difficult to prevent eggs from clumping when the
petri dish is first scanned therefore was hoping I might be able to get
around it within ImageJ. I have attached a few pictures (from top to
bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
watershed, 4) outlines of counted particles.  The original image was
scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
provide would be greatly appreciated.



Cheers,



Rich Pendleton

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[ben_king]

Rich,

Try Process > Find Maxima. It's like a watershed separation, but for non-binary images. I got a count of 3050 for your image, and looks like most spots were identified. Playing with the noise tolerance might improve performance further.

[Aryeh Weiss]

On 08/12/2015 10:57 PM, Rich Pendleton wrote:

> Hey all,
>
>
>
> I was wondering if someone might be able to provide some insight similar to
> a question that was posted last month.  I am trying to use the analyze
> particles function to count fish eggs in a petri dish.  Some eggs are more
> mature than others and the smaller less mature eggs are rather
> transparent.  Therefore when trying to adjust the threshold to detect the
> less mature eggs, I get clumping of mature eggs that are in close proximity
> to one another.  Despite using the watershed function in attempt to
> separate eggs that are close, I still have many large clumps that have
> numerous eggs that are being counted as one unit. As a result, the counts
> are always much less than the true count (often over 1000 eggs less). If I
> adjust the threshold in the other direction, I am unable to detect the less
> mature eggs.  It is quite difficult to prevent eggs from clumping when the
> petri dish is first scanned therefore was hoping I might be able to get
> around it within ImageJ. I have attached a few pictures (from top to
> bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
> watershed, 4) outlines of counted particles.  The original image was
> scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
> provide would be greatly appreciated.
>

Try Process>Find Maxima..
For your image, a threshold of about 15 seems to work.
Process>Subtract Background may also help if you want to threshold, but
I dont think Find Maxima needs it.

However, I suggest that you not acquire your images as jpg -- use a
format that is not lossy ( or acquire uncompressed).
You resolution is currently about 20 micron/dot (1200 dots in 25.4 mm).  
You might do better if you can get a higher resolution scan.

--aryeh

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Rich Pendleton]

Thank you Ben and Aryeh.  At this point, total egg counts is the main
objective, but eventually we would like to use the area and perimeter
information from analyze particles to estimate egg size.  Do you have any
additional information that could lead to me still utilizing the analyze
particles function?

Thanks again,

Rich

On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]> wrote:

> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>
>> Hey all,
>>
>>
>>
>> I was wondering if someone might be able to provide some insight similar
>> to
>> a question that was posted last month.  I am trying to use the analyze
>> particles function to count fish eggs in a petri dish.  Some eggs are more
>> mature than others and the smaller less mature eggs are rather
>> transparent.  Therefore when trying to adjust the threshold to detect the
>> less mature eggs, I get clumping of mature eggs that are in close
>> proximity
>> to one another.  Despite using the watershed function in attempt to
>> separate eggs that are close, I still have many large clumps that have
>> numerous eggs that are being counted as one unit. As a result, the counts
>> are always much less than the true count (often over 1000 eggs less). If I
>> adjust the threshold in the other direction, I am unable to detect the
>> less
>> mature eggs.  It is quite difficult to prevent eggs from clumping when the
>> petri dish is first scanned therefore was hoping I might be able to get
>> around it within ImageJ. I have attached a few pictures (from top to
>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
>> watershed, 4) outlines of counted particles.  The original image was
>> scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
>> provide would be greatly appreciated.
>>
>>
> Try Process>Find Maxima..
> For your image, a threshold of about 15 seems to work.
> Process>Subtract Background may also help if you want to threshold, but I
> dont think Find Maxima needs it.
>
> However, I suggest that you not acquire your images as jpg -- use a format
> that is not lossy ( or acquire uncompressed).
> You resolution is currently about 20 micron/dot (1200 dots in 25.4 mm).
> You might do better if you can get a higher resolution scan.
>
> --aryeh
>
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph:  972-3-5317638
> FAX: [phone]
>
>

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Aryeh Weiss]

On 09/12/2015 3:39 PM, Rich Pendleton wrote:
> Thank you Ben and Aryeh.  At this point, total egg counts is the main
> objective, but eventually we would like to use the area and perimeter
> information from analyze particles to estimate egg size.  Do you have
> any additional information that could lead to me still utilizing the
> analyze particles function?
>

The find maxima command has a segmentation option that segments your
image into regions, each (hopefully) with a single object.
Also, after doing background subtraction, you might try  hysteresis
segmentation.
You can find this in the  3D ImageJ Suite
http://imagejdocu.tudor.lu/doku.php?id=plugin:stacks:3d_ij_suite:start
They have a Fiji update site.
This allows you to set a low enough threshold that you can include the
entire egg, but only if the object includes a peak higher than the upper
threshold.

I think you will really need higher resolution. I do not know what
constraints you have, but there are scanners
with 6400 DPI optical resolution and even higher, so that you can get
more than a few pixels across the eggs.

Best regards
--aryeh

> Thanks again,
>
> Rich
>
> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]
> <mailto:[hidden email]>> wrote:
>
>     On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>
>         Hey all,
>
>
>
>         I was wondering if someone might be able to provide some
>         insight similar to
>         a question that was posted last month.  I am trying to use the
>         analyze
>         particles function to count fish eggs in a petri dish.  Some
>         eggs are more
>         mature than others and the smaller less mature eggs are rather
>         transparent.  Therefore when trying to adjust the threshold to
>         detect the
>         less mature eggs, I get clumping of mature eggs that are in
>         close proximity
>         to one another.  Despite using the watershed function in
>         attempt to
>         separate eggs that are close, I still have many large clumps
>         that have
>         numerous eggs that are being counted as one unit. As a result,
>         the counts
>         are always much less than the true count (often over 1000 eggs
>         less). If I
>         adjust the threshold in the other direction, I am unable to
>         detect the less
>         mature eggs.  It is quite difficult to prevent eggs from
>         clumping when the
>         petri dish is first scanned therefore was hoping I might be
>         able to get
>         around it within ImageJ. I have attached a few pictures (from
>         top to
>         bottom) 1) original image set to 8 bit, 2) after thresholding,
>         3) after
>         watershed, 4) outlines of counted particles.  The original
>         image was
>         scanned in as a jpeg with 1200 dpi.  Any help that you may be
>         able to
>         provide would be greatly appreciated.
>
>
>     Try Process>Find Maxima..
>     For your image, a threshold of about 15 seems to work.
>     Process>Subtract Background may also help if you want to
>     threshold, but I dont think Find Maxima needs it.
>
>     However, I suggest that you not acquire your images as jpg -- use
>     a format that is not lossy ( or acquire uncompressed).
>     You resolution is currently about 20 micron/dot (1200 dots in 25.4
>     mm).  You might do better if you can get a higher resolution scan.
>
>     --aryeh
>
>     --
>     Aryeh Weiss
>     Faculty of Engineering
>     Bar Ilan University
>     Ramat Gan 52900 Israel
>
>     Ph:  972-3-5317638
>     FAX: [phone]
>
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]


--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Herbie]

Rich,

as others have pointed out already, the spatial resolution of the shown
image is generally _too small_, and especially if you
"would like to use the area and perimeter information from analyze
particles to estimate egg size."

Maybe scans of parts of your dish may help but this depends on the
optics of your scanner.

Please note that it is important to consider the _optical_ resolution of
scanners. (Interpolated resolution is for selling not for science.)

Again, don't use compressed image formats! In case your scanner outputs
JPEG-images only, then it is _not_ suited for scientific purposes.

Have success

Herbie

::::::::::::::::::::::::::::::::::::::::::::
Am 09.12.15 um 14:39 schrieb Rich Pendleton:

> Thank you Ben and Aryeh.  At this point, total egg counts is the main
> objective, but eventually we would like to use the area and perimeter
> information from analyze particles to estimate egg size.  Do you have any
> additional information that could lead to me still utilizing the analyze
> particles function?
>
> Thanks again,
>
> Rich
>
> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]> wrote:
>
>> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>>
>>> Hey all,
>>>
>>>
>>>
>>> I was wondering if someone might be able to provide some insight similar
>>> to
>>> a question that was posted last month.  I am trying to use the analyze
>>> particles function to count fish eggs in a petri dish.  Some eggs are more
>>> mature than others and the smaller less mature eggs are rather
>>> transparent.  Therefore when trying to adjust the threshold to detect the
>>> less mature eggs, I get clumping of mature eggs that are in close
>>> proximity
>>> to one another.  Despite using the watershed function in attempt to
>>> separate eggs that are close, I still have many large clumps that have
>>> numerous eggs that are being counted as one unit. As a result, the counts
>>> are always much less than the true count (often over 1000 eggs less). If I
>>> adjust the threshold in the other direction, I am unable to detect the
>>> less
>>> mature eggs.  It is quite difficult to prevent eggs from clumping when the
>>> petri dish is first scanned therefore was hoping I might be able to get
>>> around it within ImageJ. I have attached a few pictures (from top to
>>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
>>> watershed, 4) outlines of counted particles.  The original image was
>>> scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
>>> provide would be greatly appreciated.
>>>
>>>
>> Try Process>Find Maxima..
>> For your image, a threshold of about 15 seems to work.
>> Process>Subtract Background may also help if you want to threshold, but I
>> dont think Find Maxima needs it.
>>
>> However, I suggest that you not acquire your images as jpg -- use a format
>> that is not lossy ( or acquire uncompressed).
>> You resolution is currently about 20 micron/dot (1200 dots in 25.4 mm).
>> You might do better if you can get a higher resolution scan.
>>
>> --aryeh
>>
>> --
>> Aryeh Weiss
>> Faculty of Engineering
>> Bar Ilan University
>> Ramat Gan 52900 Israel
>>
>> Ph:  972-3-5317638
>> FAX: [phone]

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Rich Pendleton]

Thanks Herbie and Aryeh.  Unfortunately, we are scanner limited (and only
saves a jpeg) and do not currently have funding for this project to buy one
suited for higher quality images.  I did reduce the size of the images I
sent to be able to fit in emails but agree they still lack optimal
resolution.  I know you can use other programs such as photoshop to run a
scanner with options other than the scanner's default options.  Do you know
of any free programs that would give me this option? Thanks again for
everyone input.

On Wed, Dec 9, 2015 at 9:16 AM, Herbie <[hidden email]> wrote:

> Rich,
>
> as others have pointed out already, the spatial resolution of the shown
> image is generally _too small_, and especially if you
> "would like to use the area and perimeter information from analyze
> particles to estimate egg size."
>
> Maybe scans of parts of your dish may help but this depends on the optics
> of your scanner.
>
> Please note that it is important to consider the _optical_ resolution of
> scanners. (Interpolated resolution is for selling not for science.)
>
> Again, don't use compressed image formats! In case your scanner outputs
> JPEG-images only, then it is _not_ suited for scientific purposes.
>
> Have success
>
> Herbie
>
> ::::::::::::::::::::::::::::::::::::::::::::
> Am 09.12.15 um 14:39 schrieb Rich Pendleton:
>
> Thank you Ben and Aryeh.  At this point, total egg counts is the main
>> objective, but eventually we would like to use the area and perimeter
>> information from analyze particles to estimate egg size.  Do you have any
>> additional information that could lead to me still utilizing the analyze
>> particles function?
>>
>> Thanks again,
>>
>> Rich
>>
>> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]> wrote:
>>
>> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>>>
>>> Hey all,
>>>>
>>>>
>>>>
>>>> I was wondering if someone might be able to provide some insight similar
>>>> to
>>>> a question that was posted last month.  I am trying to use the analyze
>>>> particles function to count fish eggs in a petri dish.  Some eggs are
>>>> more
>>>> mature than others and the smaller less mature eggs are rather
>>>> transparent.  Therefore when trying to adjust the threshold to detect
>>>> the
>>>> less mature eggs, I get clumping of mature eggs that are in close
>>>> proximity
>>>> to one another.  Despite using the watershed function in attempt to
>>>> separate eggs that are close, I still have many large clumps that have
>>>> numerous eggs that are being counted as one unit. As a result, the
>>>> counts
>>>> are always much less than the true count (often over 1000 eggs less).
>>>> If I
>>>> adjust the threshold in the other direction, I am unable to detect the
>>>> less
>>>> mature eggs.  It is quite difficult to prevent eggs from clumping when
>>>> the
>>>> petri dish is first scanned therefore was hoping I might be able to get
>>>> around it within ImageJ. I have attached a few pictures (from top to
>>>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
>>>> watershed, 4) outlines of counted particles.  The original image was
>>>> scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
>>>> provide would be greatly appreciated.
>>>>
>>>>
>>>> Try Process>Find Maxima..
>>> For your image, a threshold of about 15 seems to work.
>>> Process>Subtract Background may also help if you want to threshold, but I
>>> dont think Find Maxima needs it.
>>>
>>> However, I suggest that you not acquire your images as jpg -- use a
>>> format
>>> that is not lossy ( or acquire uncompressed).
>>> You resolution is currently about 20 micron/dot (1200 dots in 25.4 mm).
>>> You might do better if you can get a higher resolution scan.
>>>
>>> --aryeh
>>>
>>> --
>>> Aryeh Weiss
>>> Faculty of Engineering
>>> Bar Ilan University
>>> Ramat Gan 52900 Israel
>>>
>>> Ph:  972-3-5317638
>>> FAX: [phone]
>>>
>>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Herbie]

Rich,

I doubt that a scanner needs another software in order to output other
image formats than JPEG. In any case have a look at the scanner-manual
that will tell you about its optical resolution as well.

What you could try is use a digital camera but one that
1.
is able to output uncompressed raw-images
2.
has decent optics that give you good image quality for the given object
distance.

If you can find and use such a camera, then try for optimum illumination
of the dish, i.e. one that results in good contrast of the eggs (little
stray light) without reflections. You may also experiment with color
filters.

The settings of the camera are of utmost importance. If possible use the
"gamma = 1" mode, deselect all preprocessing options, such as sharpening
(contrast enhancement), and don't use a high ASA-sensitivity setting
(just one that fits your illumination at reasonable stops [watch for the
depth of focus] and exposure times). Don't use the flash.

HTH

Herbie

::::::::::::::::::::::::::::::::::::::::::::
Am 09.12.15 um 16:42 schrieb Rich Pendleton:

> Thanks Herbie and Aryeh.  Unfortunately, we are scanner limited (and only
> saves a jpeg) and do not currently have funding for this project to buy one
> suited for higher quality images.  I did reduce the size of the images I
> sent to be able to fit in emails but agree they still lack optimal
> resolution.  I know you can use other programs such as photoshop to run a
> scanner with options other than the scanner's default options.  Do you know
> of any free programs that would give me this option? Thanks again for
> everyone input.
>
> On Wed, Dec 9, 2015 at 9:16 AM, Herbie <[hidden email]> wrote:
>
>> Rich,
>>
>> as others have pointed out already, the spatial resolution of the shown
>> image is generally _too small_, and especially if you
>> "would like to use the area and perimeter information from analyze
>> particles to estimate egg size."
>>
>> Maybe scans of parts of your dish may help but this depends on the optics
>> of your scanner.
>>
>> Please note that it is important to consider the _optical_ resolution of
>> scanners. (Interpolated resolution is for selling not for science.)
>>
>> Again, don't use compressed image formats! In case your scanner outputs
>> JPEG-images only, then it is _not_ suited for scientific purposes.
>>
>> Have success
>>
>> Herbie
>>
>> ::::::::::::::::::::::::::::::::::::::::::::
>> Am 09.12.15 um 14:39 schrieb Rich Pendleton:
>>
>> Thank you Ben and Aryeh.  At this point, total egg counts is the main
>>> objective, but eventually we would like to use the area and perimeter
>>> information from analyze particles to estimate egg size.  Do you have any
>>> additional information that could lead to me still utilizing the analyze
>>> particles function?
>>>
>>> Thanks again,
>>>
>>> Rich
>>>
>>> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]> wrote:
>>>
>>> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>>>>
>>>> Hey all,
>>>>>
>>>>>
>>>>>
>>>>> I was wondering if someone might be able to provide some insight similar
>>>>> to
>>>>> a question that was posted last month.  I am trying to use the analyze
>>>>> particles function to count fish eggs in a petri dish.  Some eggs are
>>>>> more
>>>>> mature than others and the smaller less mature eggs are rather
>>>>> transparent.  Therefore when trying to adjust the threshold to detect
>>>>> the
>>>>> less mature eggs, I get clumping of mature eggs that are in close
>>>>> proximity
>>>>> to one another.  Despite using the watershed function in attempt to
>>>>> separate eggs that are close, I still have many large clumps that have
>>>>> numerous eggs that are being counted as one unit. As a result, the
>>>>> counts
>>>>> are always much less than the true count (often over 1000 eggs less).
>>>>> If I
>>>>> adjust the threshold in the other direction, I am unable to detect the
>>>>> less
>>>>> mature eggs.  It is quite difficult to prevent eggs from clumping when
>>>>> the
>>>>> petri dish is first scanned therefore was hoping I might be able to get
>>>>> around it within ImageJ. I have attached a few pictures (from top to
>>>>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
>>>>> watershed, 4) outlines of counted particles.  The original image was
>>>>> scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
>>>>> provide would be greatly appreciated.
>>>>>
>>>>>
>>>>> Try Process>Find Maxima..
>>>> For your image, a threshold of about 15 seems to work.
>>>> Process>Subtract Background may also help if you want to threshold, but I
>>>> dont think Find Maxima needs it.
>>>>
>>>> However, I suggest that you not acquire your images as jpg -- use a
>>>> format
>>>> that is not lossy ( or acquire uncompressed).
>>>> You resolution is currently about 20 micron/dot (1200 dots in 25.4 mm).
>>>> You might do better if you can get a higher resolution scan.
>>>>
>>>> --aryeh
>>>>
>>>> --
>>>> Aryeh Weiss
>>>> Faculty of Engineering
>>>> Bar Ilan University
>>>> Ramat Gan 52900 Israel
>>>>
>>>> Ph:  972-3-5317638
>>>> FAX: [phone]

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Aryeh Weiss]

Hi Rich

On 09/12/2015 5:42 PM, Rich Pendleton wrote:
> Thanks Herbie and Aryeh.  Unfortunately, we are scanner limited (and only
> saves a jpeg) and do not currently have funding for this project to buy one
> suited for higher quality images.  I did reduce the size of the images I
> sent to be able to fit in emails but agree they still lack optimal
> resolution.  I know you can use other programs such as photoshop to run a
> scanner with options other than the scanner's default options.  Do you know
> of any free programs that would give me this option? Thanks again for
> everyone input.

I saw scanners with much higher optical resolution for under $100, and
many options under $200.
Considering how much experiments cost  (even just the consumables, let
alone time, even for grad students),
you may find that being scanner limited is penny-wise and pound-foolish.

You can try posting you original images in a shared folder and post a
link, if you want to share a full size image.

Best regards
--aryeh

> On Wed, Dec 9, 2015 at 9:16 AM, Herbie <[hidden email]> wrote:
>
>> Rich,
>>
>> as others have pointed out already, the spatial resolution of the shown
>> image is generally _too small_, and especially if you
>> "would like to use the area and perimeter information from analyze
>> particles to estimate egg size."
>>
>> Maybe scans of parts of your dish may help but this depends on the optics
>> of your scanner.
>>
>> Please note that it is important to consider the _optical_ resolution of
>> scanners. (Interpolated resolution is for selling not for science.)
>>
>> Again, don't use compressed image formats! In case your scanner outputs
>> JPEG-images only, then it is _not_ suited for scientific purposes.
>>
>> Have success
>>
>> Herbie
>>
>> ::::::::::::::::::::::::::::::::::::::::::::
>> Am 09.12.15 um 14:39 schrieb Rich Pendleton:
>>
>> Thank you Ben and Aryeh.  At this point, total egg counts is the main
>>> objective, but eventually we would like to use the area and perimeter
>>> information from analyze particles to estimate egg size.  Do you have any
>>> additional information that could lead to me still utilizing the analyze
>>> particles function?
>>>
>>> Thanks again,
>>>
>>> Rich
>>>
>>> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]> wrote:
>>>
>>> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>>>> Hey all,
>>>>>
>>>>>
>>>>> I was wondering if someone might be able to provide some insight similar
>>>>> to
>>>>> a question that was posted last month.  I am trying to use the analyze
>>>>> particles function to count fish eggs in a petri dish.  Some eggs are
>>>>> more
>>>>> mature than others and the smaller less mature eggs are rather
>>>>> transparent.  Therefore when trying to adjust the threshold to detect
>>>>> the
>>>>> less mature eggs, I get clumping of mature eggs that are in close
>>>>> proximity
>>>>> to one another.  Despite using the watershed function in attempt to
>>>>> separate eggs that are close, I still have many large clumps that have
>>>>> numerous eggs that are being counted as one unit. As a result, the
>>>>> counts
>>>>> are always much less than the true count (often over 1000 eggs less).
>>>>> If I
>>>>> adjust the threshold in the other direction, I am unable to detect the
>>>>> less
>>>>> mature eggs.  It is quite difficult to prevent eggs from clumping when
>>>>> the
>>>>> petri dish is first scanned therefore was hoping I might be able to get
>>>>> around it within ImageJ. I have attached a few pictures (from top to
>>>>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3) after
>>>>> watershed, 4) outlines of counted particles.  The original image was
>>>>> scanned in as a jpeg with 1200 dpi.  Any help that you may be able to
>>>>> provide would be greatly appreciated.
>>>>>
>>>>>
>>>>> Try Process>Find Maxima..
>>>> For your image, a threshold of about 15 seems to work.
>>>> Process>Subtract Background may also help if you want to threshold, but I
>>>> dont think Find Maxima needs it.
>>>>
>>>> However, I suggest that you not acquire your images as jpg -- use a
>>>> format
>>>> that is not lossy ( or acquire uncompressed).
>>>> You resolution is currently about 20 micron/dot (1200 dots in 25.4 mm).
>>>> You might do better if you can get a higher resolution scan.
>>>>
>>>> --aryeh
>>>>
>>>> --
>>>> Aryeh Weiss
>>>> Faculty of Engineering
>>>> Bar Ilan University
>>>> Ramat Gan 52900 Israel
>>>>
>>>> Ph:  972-3-5317638
>>>> FAX: [phone]
>>>>
>> --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Olivier FRANCOIS]

Rich,

If you're still looking for another software, with a linux environnement
you can use either the graphical tool named xsane or the command line
tool named scanimage (i'm using it for my project).
The both can perform image acquisition and you can get the format you
want (*.pnm for no-compressed image for example). If you need more
options than xsane provide, scanimage is the way to do it.

If you don't have a linux, you can simply try it with the free software
virtualBox (https://www.virtualbox.org/) wich is a virtual machine and
then install any linux distribution you like, it's quite easy.

Tell me if you want more informations about this.

Olivier FRANCOIS- [hidden email]
Développeur projets numériques
Universit´ d'Auvergne
<http://www.u-clermont1.fr>
CROC EA 4847
Centre de Recherche en Odontologie Clinique
Université d'Auvergne
2 Rue de Braga - 63100 CLERMONT-FERRAND
tél: [phone]
www.u-clermont1.fr <http://www.u-clermont1.fr> - www.udapro.fr
<http://www.udapro.fr>

Le 09/12/2015 17:09, Herbie a écrit :

> Rich,
>
> I doubt that a scanner needs another software in order to output other
> image formats than JPEG. In any case have a look at the scanner-manual
> that will tell you about its optical resolution as well.
>
> What you could try is use a digital camera but one that
> 1.
> is able to output uncompressed raw-images
> 2.
> has decent optics that give you good image quality for the given
> object distance.
>
> If you can find and use such a camera, then try for optimum
> illumination of the dish, i.e. one that results in good contrast of
> the eggs (little stray light) without reflections. You may also
> experiment with color filters.
>
> The settings of the camera are of utmost importance. If possible use
> the "gamma = 1" mode, deselect all preprocessing options, such as
> sharpening (contrast enhancement), and don't use a high
> ASA-sensitivity setting (just one that fits your illumination at
> reasonable stops [watch for the depth of focus] and exposure times).
> Don't use the flash.
>
> HTH
>
> Herbie
>
> ::::::::::::::::::::::::::::::::::::::::::::
> Am 09.12.15 um 16:42 schrieb Rich Pendleton:
>> Thanks Herbie and Aryeh.  Unfortunately, we are scanner limited (and
>> only
>> saves a jpeg) and do not currently have funding for this project to
>> buy one
>> suited for higher quality images.  I did reduce the size of the images I
>> sent to be able to fit in emails but agree they still lack optimal
>> resolution.  I know you can use other programs such as photoshop to
>> run a
>> scanner with options other than the scanner's default options. Do you
>> know
>> of any free programs that would give me this option? Thanks again for
>> everyone input.
>>
>> On Wed, Dec 9, 2015 at 9:16 AM, Herbie <[hidden email]> wrote:
>>
>>> Rich,
>>>
>>> as others have pointed out already, the spatial resolution of the shown
>>> image is generally _too small_, and especially if you
>>> "would like to use the area and perimeter information from analyze
>>> particles to estimate egg size."
>>>
>>> Maybe scans of parts of your dish may help but this depends on the
>>> optics
>>> of your scanner.
>>>
>>> Please note that it is important to consider the _optical_
>>> resolution of
>>> scanners. (Interpolated resolution is for selling not for science.)
>>>
>>> Again, don't use compressed image formats! In case your scanner outputs
>>> JPEG-images only, then it is _not_ suited for scientific purposes.
>>>
>>> Have success
>>>
>>> Herbie
>>>
>>> ::::::::::::::::::::::::::::::::::::::::::::
>>> Am 09.12.15 um 14:39 schrieb Rich Pendleton:
>>>
>>> Thank you Ben and Aryeh.  At this point, total egg counts is the main
>>>> objective, but eventually we would like to use the area and perimeter
>>>> information from analyze particles to estimate egg size.  Do you
>>>> have any
>>>> additional information that could lead to me still utilizing the
>>>> analyze
>>>> particles function?
>>>>
>>>> Thanks again,
>>>>
>>>> Rich
>>>>
>>>> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]>
>>>> wrote:
>>>>
>>>> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>>>>>
>>>>> Hey all,
>>>>>>
>>>>>>
>>>>>>
>>>>>> I was wondering if someone might be able to provide some insight
>>>>>> similar
>>>>>> to
>>>>>> a question that was posted last month.  I am trying to use the
>>>>>> analyze
>>>>>> particles function to count fish eggs in a petri dish. Some eggs are
>>>>>> more
>>>>>> mature than others and the smaller less mature eggs are rather
>>>>>> transparent.  Therefore when trying to adjust the threshold to
>>>>>> detect
>>>>>> the
>>>>>> less mature eggs, I get clumping of mature eggs that are in close
>>>>>> proximity
>>>>>> to one another.  Despite using the watershed function in attempt to
>>>>>> separate eggs that are close, I still have many large clumps that
>>>>>> have
>>>>>> numerous eggs that are being counted as one unit. As a result, the
>>>>>> counts
>>>>>> are always much less than the true count (often over 1000 eggs
>>>>>> less).
>>>>>> If I
>>>>>> adjust the threshold in the other direction, I am unable to
>>>>>> detect the
>>>>>> less
>>>>>> mature eggs.  It is quite difficult to prevent eggs from clumping
>>>>>> when
>>>>>> the
>>>>>> petri dish is first scanned therefore was hoping I might be able
>>>>>> to get
>>>>>> around it within ImageJ. I have attached a few pictures (from top to
>>>>>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3)
>>>>>> after
>>>>>> watershed, 4) outlines of counted particles.  The original image was
>>>>>> scanned in as a jpeg with 1200 dpi.  Any help that you may be
>>>>>> able to
>>>>>> provide would be greatly appreciated.
>>>>>>
>>>>>>
>>>>>> Try Process>Find Maxima..
>>>>> For your image, a threshold of about 15 seems to work.
>>>>> Process>Subtract Background may also help if you want to
>>>>> threshold, but I
>>>>> dont think Find Maxima needs it.
>>>>>
>>>>> However, I suggest that you not acquire your images as jpg -- use a
>>>>> format
>>>>> that is not lossy ( or acquire uncompressed).
>>>>> You resolution is currently about 20 micron/dot (1200 dots in 25.4
>>>>> mm).
>>>>> You might do better if you can get a higher resolution scan.
>>>>>
>>>>> --aryeh
>>>>>
>>>>> --
>>>>> Aryeh Weiss
>>>>> Faculty of Engineering
>>>>> Bar Ilan University
>>>>> Ramat Gan 52900 Israel
>>>>>
>>>>> Ph:  972-3-5317638
>>>>> FAX: [phone]
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Olivier FRANCOIS]

My apologies for the blank message  !


Rich,

If you're still looking for another software, with a linux environnement
you can use either the graphical tool named xsane or the command line
tool named scanimage (i'm using it for my project).
The both can perform image acquisition and you can get the format you
want (*.pnm for no-compressed image for example). If you need more
options than xsane provide, scanimage is the way to do it.

If you don't have a linux, you can simply try it with the free software
virtualBox (https://www.virtualbox.org/) wich is a virtual machine and
then install any linux distribution you like, it's quite easy.

Tell me if you want more informations about this.



Le 09/12/2015 17:09, Herbie a écrit :

> Rich,
>
> I doubt that a scanner needs another software in order to output other
> image formats than JPEG. In any case have a look at the scanner-manual
> that will tell you about its optical resolution as well.
>
> What you could try is use a digital camera but one that
> 1.
> is able to output uncompressed raw-images
> 2.
> has decent optics that give you good image quality for the given
> object distance.
>
> If you can find and use such a camera, then try for optimum
> illumination of the dish, i.e. one that results in good contrast of
> the eggs (little stray light) without reflections. You may also
> experiment with color filters.
>
> The settings of the camera are of utmost importance. If possible use
> the "gamma = 1" mode, deselect all preprocessing options, such as
> sharpening (contrast enhancement), and don't use a high
> ASA-sensitivity setting (just one that fits your illumination at
> reasonable stops [watch for the depth of focus] and exposure times).
> Don't use the flash.
>
> HTH
>
> Herbie
>
> ::::::::::::::::::::::::::::::::::::::::::::
> Am 09.12.15 um 16:42 schrieb Rich Pendleton:
>> Thanks Herbie and Aryeh.  Unfortunately, we are scanner limited (and
>> only
>> saves a jpeg) and do not currently have funding for this project to
>> buy one
>> suited for higher quality images.  I did reduce the size of the images I
>> sent to be able to fit in emails but agree they still lack optimal
>> resolution.  I know you can use other programs such as photoshop to
>> run a
>> scanner with options other than the scanner's default options. Do you
>> know
>> of any free programs that would give me this option? Thanks again for
>> everyone input.
>>
>> On Wed, Dec 9, 2015 at 9:16 AM, Herbie <[hidden email]> wrote:
>>
>>> Rich,
>>>
>>> as others have pointed out already, the spatial resolution of the shown
>>> image is generally _too small_, and especially if you
>>> "would like to use the area and perimeter information from analyze
>>> particles to estimate egg size."
>>>
>>> Maybe scans of parts of your dish may help but this depends on the
>>> optics
>>> of your scanner.
>>>
>>> Please note that it is important to consider the _optical_
>>> resolution of
>>> scanners. (Interpolated resolution is for selling not for science.)
>>>
>>> Again, don't use compressed image formats! In case your scanner outputs
>>> JPEG-images only, then it is _not_ suited for scientific purposes.
>>>
>>> Have success
>>>
>>> Herbie
>>>
>>> ::::::::::::::::::::::::::::::::::::::::::::
>>> Am 09.12.15 um 14:39 schrieb Rich Pendleton:
>>>
>>> Thank you Ben and Aryeh.  At this point, total egg counts is the main
>>>> objective, but eventually we would like to use the area and perimeter
>>>> information from analyze particles to estimate egg size.  Do you
>>>> have any
>>>> additional information that could lead to me still utilizing the
>>>> analyze
>>>> particles function?
>>>>
>>>> Thanks again,
>>>>
>>>> Rich
>>>>
>>>> On Tue, Dec 8, 2015 at 11:28 PM, Aryeh Weiss <[hidden email]>
>>>> wrote:
>>>>
>>>> On 08/12/2015 10:57 PM, Rich Pendleton wrote:
>>>>>
>>>>> Hey all,
>>>>>>
>>>>>>
>>>>>>
>>>>>> I was wondering if someone might be able to provide some insight
>>>>>> similar
>>>>>> to
>>>>>> a question that was posted last month.  I am trying to use the
>>>>>> analyze
>>>>>> particles function to count fish eggs in a petri dish. Some eggs are
>>>>>> more
>>>>>> mature than others and the smaller less mature eggs are rather
>>>>>> transparent.  Therefore when trying to adjust the threshold to
>>>>>> detect
>>>>>> the
>>>>>> less mature eggs, I get clumping of mature eggs that are in close
>>>>>> proximity
>>>>>> to one another.  Despite using the watershed function in attempt to
>>>>>> separate eggs that are close, I still have many large clumps that
>>>>>> have
>>>>>> numerous eggs that are being counted as one unit. As a result, the
>>>>>> counts
>>>>>> are always much less than the true count (often over 1000 eggs
>>>>>> less).
>>>>>> If I
>>>>>> adjust the threshold in the other direction, I am unable to
>>>>>> detect the
>>>>>> less
>>>>>> mature eggs.  It is quite difficult to prevent eggs from clumping
>>>>>> when
>>>>>> the
>>>>>> petri dish is first scanned therefore was hoping I might be able
>>>>>> to get
>>>>>> around it within ImageJ. I have attached a few pictures (from top to
>>>>>> bottom) 1) original image set to 8 bit, 2) after thresholding, 3)
>>>>>> after
>>>>>> watershed, 4) outlines of counted particles.  The original image was
>>>>>> scanned in as a jpeg with 1200 dpi.  Any help that you may be
>>>>>> able to
>>>>>> provide would be greatly appreciated.
>>>>>>
>>>>>>
>>>>>> Try Process>Find Maxima..
>>>>> For your image, a threshold of about 15 seems to work.
>>>>> Process>Subtract Background may also help if you want to
>>>>> threshold, but I
>>>>> dont think Find Maxima needs it.
>>>>>
>>>>> However, I suggest that you not acquire your images as jpg -- use a
>>>>> format
>>>>> that is not lossy ( or acquire uncompressed).
>>>>> You resolution is currently about 20 micron/dot (1200 dots in 25.4
>>>>> mm).
>>>>> You might do better if you can get a higher resolution scan.
>>>>>
>>>>> --aryeh
>>>>>
>>>>> --
>>>>> Aryeh Weiss
>>>>> Faculty of Engineering
>>>>> Bar Ilan University
>>>>> Ramat Gan 52900 Israel
>>>>>
>>>>> Ph:  972-3-5317638
>>>>> FAX: [phone]
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Bethany Uttaro]

Hi Rich,

Have you tried photographing the dish instead of scanning it? Use a digital camera with an option for RAW images, and if you are shooting close up or in macro, check for distortion using a grid. The distortion isn't a problem for counting, of course, but will cause problems if the same images are later used for dimensions. Oh, and include a scale/ruler in the image, of course.

Bethany

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