Help wanted to remove membrane pore staining from image of nuclei

[Jacqueline Ross]

Hi everyone,

One of our graduate students is growing cells on membranes, cutting sections and then staining them.

We are imaging these sections with the confocal microscope.

Unfortunately, the membranes have pores and the Hoechst stain for nuclei seems to be sticking to the pores. Also, the sections aren't cut completely square so there's a gradient where the pores are visible in part of the image but not right across it due to the orientation.

I've done a lambda scan and it's definitely the same emission spectrum as Hoechst. The intensity is similar but I want to get rid of the pores.

I've tried a few background subtraction methods without success. I also tried FFT but the location of the pores isn't regular, it's random.

Can anyone help? I've attached a sample image as JPEG.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  [phone]

Website:    http://www.auckland.ac.nz/biru




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[Cammer, Michael]

Is this acceptable?


selectWindow("Hoechst_Nuclei_Pores_63x.jpg");
run("Duplicate...", "title=med");
run("Median...", "radius=16");
run("Subtract Background...", "rolling=127");
setAutoThreshold("Default dark");
//run("Threshold...");
//setThreshold(21, 255);
run("Convert to Mask");
imageCalculator("AND create", "Hoechst_Nuclei_Pores_63x.jpg","med");



Michael Cammer, Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

C: 914-309-3270  [hidden email]<mailto:[hidden email]>    http://microscopynotes.com/

https://med.nyu.edu/research/research-resources/scientific-cores-shared-resources/microscopy-laboratory

________________________________
From: Jacqui Ross <[hidden email]>
Sent: Monday, November 13, 2017 11:36:37 PM
To: [hidden email]
Subject: Help wanted to remove membrane pore staining from image of nuclei

Hi everyone,

One of our graduate students is growing cells on membranes, cutting sections and then staining them.

We are imaging these sections with the confocal microscope.

Unfortunately, the membranes have pores and the Hoechst stain for nuclei seems to be sticking to the pores. Also, the sections aren't cut completely square so there's a gradient where the pores are visible in part of the image but not right across it due to the orientation.

I've done a lambda scan and it's definitely the same emission spectrum as Hoechst. The intensity is similar but I want to get rid of the pores.

I've tried a few background subtraction methods without success. I also tried FFT but the location of the pores isn't regular, it's random.

Can anyone help? I've attached a sample image as JPEG.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  [phone]

Website:    https://urldefense.proofpoint.com/v2/url?u=http-3A__www.auckland.ac.nz_biru&d=DQIFAg&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Nv3lfOlW8c8BLzM6fwUbTpRb6jHbmVmHRN7tvaPb0FY&s=fK7W6vzW24bKMiRKiModI2FnuKGp8LfZ-v0R-qaxdh0&e=




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This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
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[Jacqueline Ross]

Hi Michael,

Just on my way home so reading this on my phone so can't view optimally but it looks good! More than acceptable. Will have a proper look when I'm back at work tomorrow.

Thanks!

Kind regards,

Jacqui
________________________________________
From: ImageJ Interest Group [[hidden email]] on behalf of Cammer, Michael [[hidden email]]
Sent: 14 November 2017 17:56
To: [hidden email]
Subject: Re: Help wanted to remove membrane pore staining from image of nuclei

Is this acceptable?


selectWindow("Hoechst_Nuclei_Pores_63x.jpg");
run("Duplicate...", "title=med");
run("Median...", "radius=16");
run("Subtract Background...", "rolling=127");
setAutoThreshold("Default dark");
//run("Threshold...");
//setThreshold(21, 255);
run("Convert to Mask");
imageCalculator("AND create", "Hoechst_Nuclei_Pores_63x.jpg","med");



Michael Cammer, Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

C: 914-309-3270  [hidden email]<mailto:[hidden email]>    http://microscopynotes.com/

https://med.nyu.edu/research/research-resources/scientific-cores-shared-resources/microscopy-laboratory

________________________________
From: Jacqui Ross <[hidden email]>
Sent: Monday, November 13, 2017 11:36:37 PM
To: [hidden email]
Subject: Help wanted to remove membrane pore staining from image of nuclei

Hi everyone,

One of our graduate students is growing cells on membranes, cutting sections and then staining them.

We are imaging these sections with the confocal microscope.

Unfortunately, the membranes have pores and the Hoechst stain for nuclei seems to be sticking to the pores. Also, the sections aren't cut completely square so there's a gradient where the pores are visible in part of the image but not right across it due to the orientation.

I've done a lambda scan and it's definitely the same emission spectrum as Hoechst. The intensity is similar but I want to get rid of the pores.

I've tried a few background subtraction methods without success. I also tried FFT but the location of the pores isn't regular, it's random.

Can anyone help? I've attached a sample image as JPEG.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  [phone]

Website:    https://urldefense.proofpoint.com/v2/url?u=http-3A__www.auckland.ac.nz_biru&d=DQIFAg&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Nv3lfOlW8c8BLzM6fwUbTpRb6jHbmVmHRN7tvaPb0FY&s=fK7W6vzW24bKMiRKiModI2FnuKGp8LfZ-v0R-qaxdh0&e=




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This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
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[Jim Peterson]

You might also want to erode or dilate the mask (depending on the
assignment of 0 and 255) to enlarge the mask around the objects of
interest prior to combining the original image and the mask.  That will
help preserve the shape of some of the very low contrast objects.

- Jim

On 11/14/17, 12:21 AM, "ImageJ Interest Group on behalf of Jacqui Ross"
<[hidden email] on behalf of [hidden email]> wrote:

>Hi Michael,
>
>Just on my way home so reading this on my phone so can't view optimally
>but it looks good! More than acceptable. Will have a proper look when I'm
>back at work tomorrow.
>
>Thanks!
>
>Kind regards,
>
>Jacqui
>________________________________________
>From: ImageJ Interest Group [[hidden email]] on behalf of Cammer,
>Michael [[hidden email]]
>Sent: 14 November 2017 17:56
>To: [hidden email]
>Subject: Re: Help wanted to remove membrane pore staining from image of
>nuclei
>
>Is this acceptable?
>
>
>selectWindow("Hoechst_Nuclei_Pores_63x.jpg");
>run("Duplicate...", "title=med");
>run("Median...", "radius=16");
>run("Subtract Background...", "rolling=127");
>setAutoThreshold("Default dark");
>//run("Threshold...");
>//setThreshold(21, 255);
>run("Convert to Mask");
>imageCalculator("AND create", "Hoechst_Nuclei_Pores_63x.jpg","med");
>
>
>
>Michael Cammer, Research Scientist, DART Microscopy Laboratory
>
>NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
>10016
>
>C: 914-309-3270  
>[hidden email]<mailto:[hidden email]>
>http://microscopynotes.com/
>
>https://med.nyu.edu/research/research-resources/scientific-cores-shared-re
>sources/microscopy-laboratory
>
>________________________________
>From: Jacqui Ross <[hidden email]>
>Sent: Monday, November 13, 2017 11:36:37 PM
>To: [hidden email]
>Subject: Help wanted to remove membrane pore staining from image of nuclei
>
>Hi everyone,
>
>One of our graduate students is growing cells on membranes, cutting
>sections and then staining them.
>
>We are imaging these sections with the confocal microscope.
>
>Unfortunately, the membranes have pores and the Hoechst stain for nuclei
>seems to be sticking to the pores. Also, the sections aren't cut
>completely square so there's a gradient where the pores are visible in
>part of the image but not right across it due to the orientation.
>
>I've done a lambda scan and it's definitely the same emission spectrum as
>Hoechst. The intensity is similar but I want to get rid of the pores.
>
>I've tried a few background subtraction methods without success. I also
>tried FFT but the location of the pores isn't regular, it's random.
>
>Can anyone help? I've attached a sample image as JPEG.
>
>Kind regards,
>
>Jacqui
>
>Jacqueline Ross
>Biomedical Imaging Microscopist
>Biomedical Imaging Research Unit (BIRU)
>School of Medical Sciences
>Faculty of Medical & Health Sciences
>The University of Auckland
>Private Bag 92019
>Auckland 1142, NEW ZEALAND
>
>Telephone: Ext 87438; DDI:  [phone]
>
>Website:    
>https://urldefense.proofpoint.com/v2/url?u=http-3A__www.auckland.ac.nz_bir
>u&d=DQIFAg&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydl
>bm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Nv3lfOlW8c8BLzM6fwUbTpRb6jHbmVmHRN7tvaPb0FY
>&s=fK7W6vzW24bKMiRKiModI2FnuKGp8LfZ-v0R-qaxdh0&e=
>
>
>
>
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>ImageJ mailing list:
>https://urldefense.proofpoint.com/v2/url?u=http-3A__imagej.nih.gov_ij_list
>.html&d=DQIFAg&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNst
>Aydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Nv3lfOlW8c8BLzM6fwUbTpRb6jHbmVmHRN7tvaP
>b0FY&s=wIqsiCO1nFwmHqr-OVAxf3-__T37TJqAxLxDUNNH3uo&e=
>
>------------------------------------------------------------
>This email message, including any attachments, is for the sole use of the
>intended recipient(s) and may contain information that is proprietary,
>confidential, and exempt from disclosure under applicable law. Any
>unauthorized review, use, disclosure, or distribution is prohibited. If
>you have received this email in error please notify the sender by return
>email and delete the original message. Please note, the recipient should
>check this email and any attachments for the presence of viruses. The
>organization accepts no liability for any damage caused by any virus
>transmitted by this email.
>=================================
>
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>
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