lif-files histogram
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Hello,
i have a bunch of -lif files and try to look at the histograms. When i look at the histograms directly, they seems to be binned. Only values ending on 0 or 5 show any values. However when i save individual stacks as .jpg-image and reopen it, the histogram is no longer binned, but continuous. I use 8-bit images and the bio-format importer in ImageJ to open the images. Does anyone know what the problem could be? Thanks, Stefan |
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Hi Stefan,
JPEG is a lossy compression, it modifies the image data. http://fiji.sc/IP_Principles#Why_.28lossy.29_JPEGs_should_not_be_used_in_imaging Michael ________________________________________________________________ On May 15, 2015, at 10:53, stefan_dillinger wrote: > Hello, > > i have a bunch of -lif files and try to look at the histograms. When i look > at the histograms directly, they seems to be binned. Only values ending on 0 > or 5 show any values. However when i save individual stacks as .jpg-image > and reopen it, the histogram is no longer binned, but continuous. I use > 8-bit images and the bio-format importer in ImageJ to open the images. > > > Does anyone know what the problem could be? > > > Thanks, > > Stefan -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Hi Michael,
thanks for the answer. Yes i know that jpg is a compression and i don't want to use that. The problem is that the histogram in the life 8-bit image shows this binning and i am not sure why this is the case, it should be continuous, right? Stefan |
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On May 15, 2015, at 10:53, stefan_dillinger wrote:
> i have a bunch of -lif files and try to look at the histograms. When i look > at the histograms directly, they seems to be binned. Only values ending on 0 > or 5 show any values. On May 15, 2015, at 12:07, stefan_dillinger wrote: > The problem is that the histogram in the life 8-bit image shows > this binning and i am not sure why this is the case, it should be > continuous, right? Hi Stefan, it depends on how your data were generated. E.g. if you have a sensor with 12 bits resolution, the acquisition software might multiply the raw data with some value to better use the full 16-bit range of the data and also to reduce the digitization noise upon further operations such as background subtractions and flat field correction. For 8-bit data, this is more unusual. Some integrating cameras (Mintron, WaTeC) with analog video output seems to use only 5 or 6 bits, not 8-bit values when used with exposure times longer than the TV frame rate. If the analog gain is not exactly 1, this may result in steps of roughly 5. If you post which instrument you have used, maybe someone knows more about it. Michael -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Thanks for the answeres so far! I am using the Leica SP8 microscope and i'm using 8-bit images. The Problem is, that i cannot get an continous histogram in ImageJ and i am worried about losing some information. maybe it might be due to the reverse engineering of the .lif file protocol...? Furthermore, if i convert the images from RGB to 8-bit and back, it changes every time... Thanks, Stefan
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is the problem that your image is in an RGB format but is really a greyscale image that uses a false colour scale ?
So the RGB image looks correct on the screen but a histogram taken from one of the 3 layers seems weird. test it by saving a single image from the microscope as greyscale and as an RGB image and opening both in imageJ and looking at the histograms ________________________________________ From: ImageJ Interest Group [[hidden email]] on behalf of stefan_dillinger [[hidden email]] Sent: 19 May 2015 14:33 To: [hidden email] Subject: Re: lif-files histogram Thanks for the answeres so far! I am using the Leica SP8 microscope and i'm using 8-bit images. The Problem is, that i cannot get an continous histogram in ImageJ and i am worried about losing some information. maybe it might be due to the reverse engineering of the .lif file protocol...? Furthermore, if i convert the images from RGB to 8-bit and back, it changes every time... Thanks, Stefan >>> "Michael Schmid-3 [via ImageJ]" <[hidden email]> 5/15/2015 3:23 PM >>> On May 15, 2015, at 10:53, stefan_dillinger wrote: > i have a bunch of -lif files and try to look at the histograms. When i look > at the histograms directly, they seems to be binned. Only values ending on 0 > or 5 show any values. On May 15, 2015, at 12:07, stefan_dillinger wrote: > The problem is that the histogram in the life 8-bit image shows > this binning and i am not sure why this is the case, it should be > continuous, right? Hi Stefan, it depends on how your data were generated. E.g. if you have a sensor with 12 bits resolution, the acquisition software might multiply the raw data with some value to better use the full 16-bit range of the data and also to reduce the digitization noise upon further operations such as background subtractions and flat field correction. For 8-bit data, this is more unusual. Some integrating cameras (Mintron, WaTeC) with analog video output seems to use only 5 or 6 bits, not 8-bit values when used with exposure times longer than the TV frame rate. If the analog gain is not exactly 1, this may result in steps of roughly 5. If you post which instrument you have used, maybe someone knows more about it. Michael -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html If you reply to this email, your message will be added to the discussion below: http://imagej.1557.x6.nabble.com/lif-files-histogram-tp5012839p5012844.html To unsubscribe from lif-files histogram, click here ( NAML ( http://imagej.1557.x6.nabble.com/template/NamlServlet.jtp?macro=macro_viewer&id=instant_html%21nabble%3Aemail.naml&base=nabble.naml.namespaces.BasicNamespace-nabble.view.web.template.NabbleNamespace-nabble.view.web.template.NodeNamespace&breadcrumbs=notify_subscribers%21nabble%3Aemail.naml-instant_emails%21nabble%3Aemail.naml-send_instant_email%21nabble%3Aemail.naml ) -- View this message in context: http://imagej.1557.x6.nabble.com/lif-files-histogram-tp5012839p5012865.html Sent from the ImageJ mailing list archive at Nabble.com. -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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In reply to this post by stefan_dillinger
Hi Stefan,
are you using the microscope in confocal mode or imaging? According to the datasheet, the detectors for confocal mode should have 12 or 16 bits resolution. So it's strange if you get an 8-bit file, not a 16-bit file! If you don't have any settings in the software that reduce the bit depth, maybe you can use the microscope's software to convert the original lif files to tiff and see what you get. If it does not match, it might be an issue for the Bioformats group (assuming you use Bioformats to read the .lif files). Michael ________________________________________________________________ On May 19, 2015, at 14:33, stefan_dillinger wrote: > Thanks for the answeres so far! > > I am using the Leica SP8 microscope and i'm using 8-bit images. The Problem is, that i cannot get an continous histogram in ImageJ and i am worried about losing some information. maybe it might be due to the reverse engineering of the .lif file protocol...? Furthermore, if i convert the images from RGB to 8-bit and back, it changes every time... > > Thanks, > > > Stefan > > > > > >>>> "Michael Schmid-3 [via ImageJ]" <[hidden email]> 5/15/2015 3:23 PM >>> > On May 15, 2015, at 10:53, stefan_dillinger wrote: > >> i have a bunch of -lif files and try to look at the histograms. When i look >> at the histograms directly, they seems to be binned. Only values ending on 0 >> or 5 show any values. > > On May 15, 2015, at 12:07, stefan_dillinger wrote: > >> The problem is that the histogram in the life 8-bit image shows >> this binning and i am not sure why this is the case, it should be >> continuous, right? > > Hi Stefan, > > it depends on how your data were generated. E.g. if you have a sensor with 12 bits resolution, the acquisition software might multiply the raw data with some value to better use the full 16-bit range of the data and also to reduce the digitization noise upon further operations such as background subtractions and flat field correction. > > For 8-bit data, this is more unusual. Some integrating cameras (Mintron, WaTeC) with analog video output seems to use only 5 or 6 bits, not 8-bit values when used with exposure times longer than the TV frame rate. If the analog gain is not exactly 1, this may result in steps of roughly 5. > > If you post which instrument you have used, maybe someone knows more about it. > > Michael > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > > > > If you reply to this email, your message will be added to the discussion below: > > http://imagej.1557.x6.nabble.com/lif-files-histogram-tp5012839p5012844.html > > To unsubscribe from lif-files histogram, click here ( > NAML ( http://imagej.1557.x6.nabble.com/template/NamlServlet.jtp?macro=macro_viewer&id=instant_html%21nabble%3Aemail.naml&base=nabble.naml.namespaces.BasicNamespace-nabble.view.web.template.NabbleNamespace-nabble.view.web.template.NodeNamespace&breadcrumbs=notify_subscribers%21nabble%3Aemail.naml-instant_emails%21nabble%3Aemail.naml-send_instant_email%21nabble%3Aemail.naml ) > > > > > -- > View this message in context: http://imagej.1557.x6.nabble.com/lif-files-histogram-tp5012839p5012865.html > Sent from the ImageJ mailing list archive at Nabble.com. > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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