(no subject)

[Benjamin Grant]

HI All,
I am designing a cell phone microscope for *in vivo* fluorescence imaging.
I'm having a lot of trouble getting the contrast that I can get using a lab
grade camera + computer (not shocking, I know). I'm sure I could figure out
ways of better optimizing the system, but wanted to see what users thought
of current images in terms of usability. They're not sharp, I know and
respect that. What I use them for is calculating nuclear/cytoplasmic ratio.
The nuclei are the bright dots, everything else is the
background(cytoplasm).  Is the image quality too poor for effective
segmentation? Thanks for any thoughts
Ben

if image does not attach properly, also viewable
here<http://i.imgur.com/30tKBs9.jpg>
:

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[Unruh, Jay-2]

Benjamin,

I am quite impressed with the image.  Since I don't know the scaling or what you are looking at, I don't know how much to be impressed.  The image quality is definitely good enough for nuclear/cytoplasmic segmentation.  I attached my segmentation.  I was able to segment it quite well by Gaussian filtering with a radius of 2, background subtracting with a radius of 15 and using ImageJ's default autothreshold.  Finally I eliminated all of the spots with less than 25 pixels area.

Jay

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Grant
Sent: Thursday, June 13, 2013 4:08 PM
To: [hidden email]
Subject:

HI All,
I am designing a cell phone microscope for *in vivo* fluorescence imaging.
I'm having a lot of trouble getting the contrast that I can get using a lab grade camera + computer (not shocking, I know). I'm sure I could figure out ways of better optimizing the system, but wanted to see what users thought of current images in terms of usability. They're not sharp, I know and respect that. What I use them for is calculating nuclear/cytoplasmic ratio.
The nuclei are the bright dots, everything else is the background(cytoplasm).  Is the image quality too poor for effective segmentation? Thanks for any thoughts Ben

if image does not attach properly, also viewable here<http://i.imgur.com/30tKBs9.jpg>
:

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[Benjamin Grant]

Thanks Jay,
that looks wonderful - very impressed you can do that with such an uneven
background. Don't be too impressed, it's a cell phone with some optics
leading up to a fiber optic cable
 which is placed in direct contact with my lower lip - just some squamous
epithelium with a topical dye. It's essentially a flourescent microscope.
Thanks for your help!
Ben


On Fri, Jun 14, 2013 at 12:03 PM, Unruh, Jay <[hidden email]> wrote:

> Benjamin,
>
> I am quite impressed with the image.  Since I don't know the scaling or
> what you are looking at, I don't know how much to be impressed.  The image
> quality is definitely good enough for nuclear/cytoplasmic segmentation.  I
> attached my segmentation.  I was able to segment it quite well by Gaussian
> filtering with a radius of 2, background subtracting with a radius of 15
> and using ImageJ's default autothreshold.  Finally I eliminated all of the
> spots with less than 25 pixels area.
>
> Jay
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Benjamin Grant
> Sent: Thursday, June 13, 2013 4:08 PM
> To: [hidden email]
> Subject:
>
> HI All,
> I am designing a cell phone microscope for *in vivo* fluorescence imaging.
> I'm having a lot of trouble getting the contrast that I can get using a
> lab grade camera + computer (not shocking, I know). I'm sure I could figure
> out ways of better optimizing the system, but wanted to see what users
> thought of current images in terms of usability. They're not sharp, I know
> and respect that. What I use them for is calculating nuclear/cytoplasmic
> ratio.
> The nuclei are the bright dots, everything else is the
> background(cytoplasm).  Is the image quality too poor for effective
> segmentation? Thanks for any thoughts Ben
>
> if image does not attach properly, also viewable here<
> http://i.imgur.com/30tKBs9.jpg>
> :
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[Unruh, Jay-2]

Now I'm even more impressed!  I had thought that it was perhaps an oocyte with extra large cell bodies.  Can you reveal which topical dye you use for "live human imaging."

Jay

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Benjamin Grant
Sent: Friday, June 14, 2013 12:14 PM
To: [hidden email]
Subject:

Thanks Jay,
that looks wonderful - very impressed you can do that with such an uneven background. Don't be too impressed, it's a cell phone with some optics leading up to a fiber optic cable  which is placed in direct contact with my lower lip - just some squamous epithelium with a topical dye. It's essentially a flourescent microscope.
Thanks for your help!
Ben


On Fri, Jun 14, 2013 at 12:03 PM, Unruh, Jay <[hidden email]> wrote:

> Benjamin,
>
> I am quite impressed with the image.  Since I don't know the scaling
> or what you are looking at, I don't know how much to be impressed.  
> The image quality is definitely good enough for nuclear/cytoplasmic
> segmentation.  I attached my segmentation.  I was able to segment it
> quite well by Gaussian filtering with a radius of 2, background
> subtracting with a radius of 15 and using ImageJ's default
> autothreshold.  Finally I eliminated all of the spots with less than 25 pixels area.
>
> Jay
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Benjamin Grant
> Sent: Thursday, June 13, 2013 4:08 PM
> To: [hidden email]
> Subject:
>
> HI All,
> I am designing a cell phone microscope for *in vivo* fluorescence imaging.
> I'm having a lot of trouble getting the contrast that I can get using
> a lab grade camera + computer (not shocking, I know). I'm sure I could
> figure out ways of better optimizing the system, but wanted to see
> what users thought of current images in terms of usability. They're
> not sharp, I know and respect that. What I use them for is calculating
> nuclear/cytoplasmic ratio.
> The nuclei are the bright dots, everything else is the
> background(cytoplasm).  Is the image quality too poor for effective
> segmentation? Thanks for any thoughts Ben
>
> if image does not attach properly, also viewable here<
> http://i.imgur.com/30tKBs9.jpg>
> :
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[Benjamin Grant]

Thanks! And absolutely happy to share the dye's name. Just a note, I'm a
graduate student and didn't develop this approach.  We use proflavine, it's
a vital dye with a safe history of clinical use -historically as a topical
antiseptic. The inventor, Tim Muldoon (now a Professor at Arkansas), of the
original system deserves the most credit for this. This system is the one
that uses a research grade camera and a computer. His thesis can be found
here <http://goo.gl/JyV0q> . This is work done in Rebecca Richards-Kortum's
lab at rice university. If you use google scholar and type Richards-Kortum
and HRME (high-resolution microendoscope) you'll find many papers on the
subject. It's been used in clinical trials to identify cervical, oral and
esophogeal cancer/precancer.  One of the first papers can be viewed
here<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065245/>.
Please note, acriflavine<http://en.wikipedia.org/wiki/Acriflavinium_chloride>and
proflavine <http://en.wikipedia.org/wiki/Proflavine> are very similar -
proflavine is an acriflavine derivative so in some of the earlier papers
you may see acriflavine, but again, it is very similar.  The peak
excitation wavelength is close to 455nm, excites around 500+.  Thanks again
for the help - and hope the dye information was useful. Thanks!
Ben


On Fri, Jun 14, 2013 at 1:11 PM, Unruh, Jay <[hidden email]> wrote:

> Now I'm even more impressed!  I had thought that it was perhaps an oocyte
> with extra large cell bodies.  Can you reveal which topical dye you use for
> "live human imaging."
>
> Jay
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Benjamin Grant
> Sent: Friday, June 14, 2013 12:14 PM
> To: [hidden email]
> Subject:
>
> Thanks Jay,
> that looks wonderful - very impressed you can do that with such an uneven
> background. Don't be too impressed, it's a cell phone with some optics
> leading up to a fiber optic cable  which is placed in direct contact with
> my lower lip - just some squamous epithelium with a topical dye. It's
> essentially a flourescent microscope.
> Thanks for your help!
> Ben
>
>
> On Fri, Jun 14, 2013 at 12:03 PM, Unruh, Jay <[hidden email]> wrote:
>
> > Benjamin,
> >
> > I am quite impressed with the image.  Since I don't know the scaling
> > or what you are looking at, I don't know how much to be impressed.
> > The image quality is definitely good enough for nuclear/cytoplasmic
> > segmentation.  I attached my segmentation.  I was able to segment it
> > quite well by Gaussian filtering with a radius of 2, background
> > subtracting with a radius of 15 and using ImageJ's default
> > autothreshold.  Finally I eliminated all of the spots with less than 25
> pixels area.
> >
> > Jay
> >
> > -----Original Message-----
> > From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> > Benjamin Grant
> > Sent: Thursday, June 13, 2013 4:08 PM
> > To: [hidden email]
> > Subject:
> >
> > HI All,
> > I am designing a cell phone microscope for *in vivo* fluorescence
> imaging.
> > I'm having a lot of trouble getting the contrast that I can get using
> > a lab grade camera + computer (not shocking, I know). I'm sure I could
> > figure out ways of better optimizing the system, but wanted to see
> > what users thought of current images in terms of usability. They're
> > not sharp, I know and respect that. What I use them for is calculating
> > nuclear/cytoplasmic ratio.
> > The nuclei are the bright dots, everything else is the
> > background(cytoplasm).  Is the image quality too poor for effective
> > segmentation? Thanks for any thoughts Ben
> >
> > if image does not attach properly, also viewable here<
> > http://i.imgur.com/30tKBs9.jpg>
> > :
> >
> > --
> > ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> >
> > --
> > ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> >
>
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>
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