getDirectory() leaves me within the found directory

[Bill Christens-Barry]

In ImageJ, if I run a macro with the following lines:

    dir = getDirectory("Choose a directory:");
    dir = getDirectory("Choose a directory:");

after execution of the first line, the second line starts execution within the directory chosen by the user in the first line. Is there a simple way to have the execution of the first line not influence where the second run begins execution? My inspection of the online macro language doc didn't suggest a simple solution. I'm trying to avoid using commands such as File.getParent(pathOfSomeFileWithinWhereTheFirstLinePutMe) and parsing my way up, or setting up some sort of preference tailored to what I want. More generally, is there a way in the macro language to specify the initial path used by getDirectory(), or some way of specifying what should be used as the current directory for this and perhaps other commands?

Thanks.

Bill Christens-Barry

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[Angeline Lim]

Hi,

I used RGB split on a RGB image -- however i notice there is some blue  
in the green.
Any idea why this is happening and how to fix it?

(i checked the raw images and the blue does not show up in the green  
channel at all, so its not the microscope...)

Thanks

Angeline

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[Jacqueline Ross]

Hi Angeline,

What are you imaging? Fluorescence? In which case depending on the fluorophore, I wouldn't be surprised to find blue and red as well as green. You often get some autofluorescence that can show up in the blue channel and most "green-emitting" fluorophores like FITC have a spectrum that extends in the "yellow" range resulting in this coming into the "red" channel.

A way out of this is to use your camera in monochrome mode if it has that available. Otherwise, I would just split the channels and use the green one only since this preserves the intensity better than converting to 8bit.

Also, the colour cameras have the Bayer mask so this probably also contributes to the problem.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Angeline Lim
Sent: Tuesday, 26 June 2012 10:40 a.m.
To: [hidden email]
Subject: RGB split -- blue shows up in green?

Hi,

I used RGB split on a RGB image -- however i notice there is some blue  
in the green.
Any idea why this is happening and how to fix it?

(i checked the raw images and the blue does not show up in the green  
channel at all, so its not the microscope...)

Thanks

Angeline

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[Angeline Lim]

Hi,

I thought it had to do with the image acquisition too -- But
I use the Volocity system to acquire the images (yes they are  
fluorescence, DAPI, eGFP and Alexafluo 594), i then did a max.  
projection with all 3 channels, and exported that as a tiff file.
open the tiff file in imageJ, did an RGB split -- that is when the  
blue shows up in the green...
On Volocity, when i looked at each channels, i don't see this. So it  
appears to be something that is software/post processing related, not  
in the raw data.

thanks

Angeline


On Jun 25, 2012, at 10:29 PM, Jacqui Ross wrote:

> Hi Angeline,
>
> What are you imaging? Fluorescence? In which case depending on the  
> fluorophore, I wouldn't be surprised to find blue and red as well as  
> green. You often get some autofluorescence that can show up in the  
> blue channel and most "green-emitting" fluorophores like FITC have a  
> spectrum that extends in the "yellow" range resulting in this coming  
> into the "red" channel.
>
> A way out of this is to use your camera in monochrome mode if it has  
> that available. Otherwise, I would just split the channels and use  
> the green one only since this preserves the intensity better than  
> converting to 8bit.
>
> Also, the colour cameras have the Bayer mask so this probably also  
> contributes to the problem.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
>
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland, NEW ZEALAND
>
> Tel: [phone] Ext 87438
> Fax: [phone]
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf  
> Of Angeline Lim
> Sent: Tuesday, 26 June 2012 10:40 a.m.
> To: [hidden email]
> Subject: RGB split -- blue shows up in green?
>
> Hi,
>
> I used RGB split on a RGB image -- however i notice there is some blue
> in the green.
> Any idea why this is happening and how to fix it?
>
> (i checked the raw images and the blue does not show up in the green
> channel at all, so its not the microscope...)
>
> Thanks
>
> Angeline
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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[Theresa Swayne]

Hi Angeline,

Could it be that Volocity is using a wavelength-based LUT to encode the channels, rather than pure red, green, and blue?

You can check this by looking at Tools > Change Colors.

Good luck,
Theresa


On Jun 26, 2012, at 2:34 AM, Angeline Lim wrote:

> I thought it had to do with the image acquisition too -- But
> I use the Volocity system to acquire the images (yes they are fluorescence, DAPI, eGFP and Alexafluo 594), i then did a max. projection with all 3 channels, and exported that as a tiff file.
> open the tiff file in imageJ, did an RGB split -- that is when the blue shows up in the green...
> On Volocity, when i looked at each channels, i don't see this. So it appears to be something that is software/post processing related, not in the raw data.

------------------------------------
Theresa C. Swayne, Ph.D.
Manager, Confocal and Specialized Microscopy Shared Resource
Herbert Irving Comprehensive Cancer Center, Columbia University
1130 Saint Nicholas Ave, 222A
New York, NY 10032

212-851-4613

[hidden email]
http://hiccc.columbia.edu/research/sharedresources/confocal


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[Angeline Lim]

HI,
I just checked it and you were right. I had to change the channels and  
its fine now.
Thanks!

Angeline

On Jun 26, 2012, at 8:32 AM, Theresa Swayne wrote:

>
> Hi Angeline,
>
> Could it be that Volocity is using a wavelength-based LUT to encode  
> the channels, rather than pure red, green, and blue?
>
> You can check this by looking at Tools > Change Colors.
>
> Good luck,
> Theresa
>
>
> On Jun 26, 2012, at 2:34 AM, Angeline Lim wrote:
>
>> I thought it had to do with the image acquisition too -- But
>> I use the Volocity system to acquire the images (yes they are  
>> fluorescence, DAPI, eGFP and Alexafluo 594), i then did a max.  
>> projection with all 3 channels, and exported that as a tiff file.
>> open the tiff file in imageJ, did an RGB split -- that is when the  
>> blue shows up in the green...
>> On Volocity, when i looked at each channels, i don't see this. So  
>> it appears to be something that is software/post processing  
>> related, not in the raw data.
>
> ------------------------------------
> Theresa C. Swayne, Ph.D.
> Manager, Confocal and Specialized Microscopy Shared Resource
> Herbert Irving Comprehensive Cancer Center, Columbia University
> 1130 Saint Nicholas Ave, 222A
> New York, NY 10032
>
> 212-851-4613
>
> [hidden email]
> http://hiccc.columbia.edu/research/sharedresources/confocal
>

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