Ratiometric Z projections??
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Greetings ImageJ Gods,
I have a rather simple question (I think). I am doing ratiometric intracellular calcium imaging through a Z stack. I can not find much on the actual method of computing the various types of projections. If I am making an ave z projection of my ratiometric stack how should I be interpreting the resultant image? I have a feeling I may be misinterpreting my data. I would appreciate any and all feedback or suggestions! Thank you all! Brandon |
Re: Ratiometric Z projections??
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Brandon,
The average z-projection is computing the average digital value of your stack at each pixel location in your region of interest. For example, say you have four images in a stack where the grey values at pixel location (0,0) are 128, 135, 115, and 145. Then, the average z-projection will yield the value 131 (or 130.75 if working with 32-bit float images) at pixel (0,0). Hope that helps, Aaron. On Wed, Mar 27, 2013 at 1:10 PM, biomicrogen <[hidden email]> wrote: > Greetings ImageJ Gods, > > I have a rather simple question (I think). I am doing ratiometric > intracellular calcium imaging through a Z stack. I can not find much on > the > actual method of computing the various types of projections. If I am > making > an ave z projection of my ratiometric stack how should I be interpreting > the > resultant image? I have a feeling I may be misinterpreting my data. > > I would appreciate any and all feedback or suggestions! > > Thank you all! > > Brandon > > > > -- > View this message in context: > http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460.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 |
Re: Ratiometric Z projections??
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Hi Brandon,
in ratiometry as I understand it you should have two stacks recorded in channels of different wavelength. The ratiometric information is in the ratio of the intensities of those channels in each point. Why you are dealing with stacks you didn't explain. I assume they are Z-stacks you recorded so that you are sure the organelle you are watching is included. The ratio of the channels should be valid as long as you use the same projection method on the stacks of both channels. I would rather look at the max projection though. That way the organelle could have its brightest image in different slices of both channels and the max projection would give you the intensity of the slice where the organelle is in focus best regardless if the z-pos of that slice differs between channel 1 and 2. Hth Christian --- Christian Goosmann Mikroskopie Max-Planck-Institut für Infektionsbiologie Campus Charité Mitte Charitéplatz 1 10117 Berlin Tel.: +49 30 28460 388 Aaron Hendrickson wrote: > Brandon, > > The average z-projection is computing the average digital value of your > stack at each pixel location in your region of interest. For example, say > you have four images in a stack where the grey values at pixel location > (0,0) are 128, 135, 115, and 145. Then, the average z-projection will > yield the value 131 (or 130.75 if working with 32-bit float images) at > pixel (0,0). > > Hope that helps, > Aaron. > > On Wed, Mar 27, 2013 at 1:10 PM, biomicrogen <[hidden email]> wrote: > >> Greetings ImageJ Gods, >> >> I have a rather simple question (I think). I am doing ratiometric >> intracellular calcium imaging through a Z stack. I can not find much on >> the >> actual method of computing the various types of projections. If I am >> making >> an ave z projection of my ratiometric stack how should I be interpreting >> the >> resultant image? I have a feeling I may be misinterpreting my data. >> >> I would appreciate any and all feedback or suggestions! >> >> Thank you all! >> >> Brandon >> >> >> >> -- >> View this message in context: >> http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460.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 -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Thank you for your replies. I am doing ratiometric calcium imaging in a prokaryotic system. These cells are quite large (relative to other prokaryotes, and they are multicellular) and I am interested in the distribution of calcium ions. Because of the three dimensionality of these organisms I need to take z-stacks. If I create a ratiometric stack using the 2 different wavelength stacks then create a max z projection of the ratio stack, will I be artificially raising the ratios I am looking at?
Brandon S. Guida Mc.S., M.S.
Microbiology, A&P, and Biotechnology Instructor Ph.D Candidate, ASU School of Life Science Vice President, School of Life Sciences Graduate Student Community "It is not enough to just believe what you see, you must understand what you see." - Leonardo Da Vinci "A human being should be able to change a diaper, plan an invasion, butcher a hog, conn a ship, design a building, write a sonnet, balance accounts, build a wall, set a bone, comfort the dying, take orders, give orders, cooperate, act alone, solve equations, analyze a new problem, pitch manure, program a computer, cook a tasty meal, fight efficiently, die gallantly. Specialization is for insects." - Robert Heinlein. -----Original Message-----
From: Christian Goosmann-2 [via ImageJ] <[hidden email]> To: biomicrogen <[hidden email]> Sent: Thu, Mar 28, 2013 6:17 am Subject: Re: Ratiometric Z projections??
Hi Brandon,
in ratiometry as I understand it you should have two stacks recorded in channels of different wavelength. The ratiometric information is in the ratio of the intensities of those channels in each point. Why you are dealing with stacks you didn't explain. I assume they are Z-stacks you recorded so that you are sure the organelle you are watching is included. The ratio of the channels should be valid as long as you use the same projection method on the stacks of both channels. I would rather look at the max projection though. That way the organelle could have its brightest image in different slices of both channels and the max projection would give you the intensity of the slice where the organelle is in focus best regardless if the z-pos of that slice differs between channel 1 and 2. Hth Christian --- Christian Goosmann Mikroskopie Max-Planck-Institut für Infektionsbiologie Campus Charité Mitte Charitéplatz 1 10117 Berlin Tel.: +49 30 28460 388 Aaron Hendrickson wrote: > Brandon, > > The average z-projection is computing the average digital value of your > stack at each pixel location in your region of interest. For example, say > you have four images in a stack where the grey values at pixel location > (0,0) are 128, 135, 115, and 145. Then, the average z-projection will > yield the value 131 (or 130.75 if working with 32-bit float images) at > pixel (0,0). > > Hope that helps, > Aaron. > > On Wed, Mar 27, 2013 at 1:10 PM, biomicrogen <[hidden email]> wrote: > >> Greetings ImageJ Gods, >> >> I have a rather simple question (I think). I am doing ratiometric >> intracellular calcium imaging through a Z stack. I can not find much on >> the >> actual method of computing the various types of projections. If I am >> making >> an ave z projection of my ratiometric stack how should I be interpreting >> the >> resultant image? I have a feeling I may be misinterpreting my data. >> >> I would appreciate any and all feedback or suggestions! >> >> Thank you all! >> >> Brandon >> >> >> >> -- >> View this message in context: >> http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460.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 -- 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.n6.nabble.com/Ratiometric-Z-projections-tp5002460p5002472.html
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Re: Ratiometric Z projections??
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if you already have a ratiometric stack, I would not do a max projection
but either use Plugin>stack>MeasureStack (with the correct measurements in Analyse>setMeasurements...) or the average projection, giving you the average ratio for each xy position on the entire z serie. But be careful with background pixel (set to NaN?) Jean-Philippe Grossier 2013/3/28 biomicrogen <[hidden email]> > Thank you for your replies. I am doing ratiometric calcium imaging in a > prokaryotic system. These cells are quite large (relative to other > prokaryotes, and they are multicellular) and I am interested in the > distribution of calcium ions. Because of the three dimensionality of these > organisms I need to take z-stacks. If I create a ratiometric stack using > the 2 different wavelength stacks then create a max z projection of the > ratio stack, will I be artificially raising the ratios I am looking at? > > > > > Brandon S. Guida Mc.S., M.S. > Microbiology, A&P, and Biotechnology Instructor > Ph.D Candidate, ASU School of Life Science > Vice President, School of Life Sciences Graduate Student Community > > "It is not enough to just believe what you see, you must understand what > you see." - Leonardo Da Vinci > > "A human being should be able to change a diaper, plan an invasion, > butcher a hog, conn a ship, design a building, write a sonnet, balance > accounts, build a wall, set a bone, comfort the dying, take orders, give > orders, cooperate, act alone, solve equations, analyze a new problem, pitch > manure, program a computer, cook a tasty meal, fight efficiently, die > gallantly. Specialization is for insects." - Robert Heinlein. > > > > > > -----Original Message----- > From: Christian Goosmann-2 [via ImageJ] < > [hidden email]> > To: biomicrogen <[hidden email]> > Sent: Thu, Mar 28, 2013 6:17 am > Subject: Re: Ratiometric Z projections?? > > > Hi Brandon, > in ratiometry as I understand it you should have two stacks recorded in > channels of different wavelength. The ratiometric information is in the > ratio of the intensities of those channels in each point. Why you are > dealing with stacks you didn't explain. I assume they are Z-stacks you > recorded so that you are sure the organelle you are watching is > included. The ratio of the channels should be valid as long as you use > the same projection method on the stacks of both channels. I would > rather look at the max projection though. That way the organelle could > have its brightest image in different slices of both channels and the > max projection would give you the intensity of the slice where the > organelle is in focus best regardless if the z-pos of that slice differs > between channel 1 and 2. > Hth > Christian > > --- > Christian Goosmann > Mikroskopie > Max-Planck-Institut für Infektionsbiologie > Campus Charité Mitte > Charitéplatz 1 > 10117 Berlin > Tel.: +49 30 28460 388 > > Aaron Hendrickson wrote: > > > Brandon, > > > > The average z-projection is computing the average digital value of your > > stack at each pixel location in your region of interest. For example, say > > you have four images in a stack where the grey values at pixel location > > (0,0) are 128, 135, 115, and 145. Then, the average z-projection will > > yield the value 131 (or 130.75 if working with 32-bit float images) at > > pixel (0,0). > > > > Hope that helps, > > Aaron. > > > > On Wed, Mar 27, 2013 at 1:10 PM, biomicrogen <[hidden email]> wrote: > > > >> Greetings ImageJ Gods, > >> > >> I have a rather simple question (I think). I am doing ratiometric > >> intracellular calcium imaging through a Z stack. I can not find much on > >> the > >> actual method of computing the various types of projections. If I am > >> making > >> an ave z projection of my ratiometric stack how should I be interpreting > >> the > >> resultant image? I have a feeling I may be misinterpreting my data. > >> > >> I would appreciate any and all feedback or suggestions! > >> > >> Thank you all! > >> > >> Brandon > >> > >> > >> > >> -- > >> View this message in context: > >> > http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460.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 > > -- > 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.n6.nabble.com/Ratiometric-Z-projections-tp5002460p5002472.html > > To unsubscribe from Ratiometric Z > projections??, click here. > NAML > > > > > > -- > View this message in context: > http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460p5002474.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 |
Re: Ratiometric Z projections??
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In reply to this post by biomicrogen
Hi,
The maximum intensity projection is inappropriate for quantitation. It will skew your results to the brightest intensities. Ratio your dark frame corrected stacks with Process>Image Calculator> Divide> Stack 1 by Stack 2 to get a stack of ratio images. The ratio stack can be measured with the Plug in mentioned by Jean-Phillipe, or it can be converted to an average projection or a summed intensity projection of the ratio stack, then measured. Alternatively, create summed or averaged projections of the 2 source stacks and divide them with the Image Calculator to obtain a single image result. Regards, Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Cellular Morphology Core Center on Human Development and Disability Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] [hidden email] On Mar 28, 2013, at 7:42 AM, biomicrogen <[hidden email]> wrote: > Thank you for your replies. I am doing ratiometric calcium imaging in a prokaryotic system. These cells are quite large (relative to other prokaryotes, and they are multicellular) and I am interested in the distribution of calcium ions. Because of the three dimensionality of these organisms I need to take z-stacks. If I create a ratiometric stack using the 2 different wavelength stacks then create a max z projection of the ratio stack, will I be artificially raising the ratios I am looking at? > > > > > Brandon S. Guida Mc.S., M.S. > Microbiology, A&P, and Biotechnology Instructor > Ph.D Candidate, ASU School of Life Science > Vice President, School of Life Sciences Graduate Student Community > > "It is not enough to just believe what you see, you must understand what you see." - Leonardo Da Vinci > > "A human being should be able to change a diaper, plan an invasion, butcher a hog, conn a ship, design a building, write a sonnet, balance accounts, build a wall, set a bone, comfort the dying, take orders, give orders, cooperate, act alone, solve equations, analyze a new problem, pitch manure, program a computer, cook a tasty meal, fight efficiently, die gallantly. Specialization is for insects." - Robert Heinlein. > > > > > > -----Original Message----- > From: Christian Goosmann-2 [via ImageJ] <[hidden email]> > To: biomicrogen <[hidden email]> > Sent: Thu, Mar 28, 2013 6:17 am > Subject: Re: Ratiometric Z projections?? > > > Hi Brandon, > in ratiometry as I understand it you should have two stacks recorded in > channels of different wavelength. The ratiometric information is in the > ratio of the intensities of those channels in each point. Why you are > dealing with stacks you didn't explain. I assume they are Z-stacks you > recorded so that you are sure the organelle you are watching is > included. The ratio of the channels should be valid as long as you use > the same projection method on the stacks of both channels. I would > rather look at the max projection though. That way the organelle could > have its brightest image in different slices of both channels and the > max projection would give you the intensity of the slice where the > organelle is in focus best regardless if the z-pos of that slice differs > between channel 1 and 2. > Hth > Christian > > --- > Christian Goosmann > Mikroskopie > Max-Planck-Institut für Infektionsbiologie > Campus Charité Mitte > Charitéplatz 1 > 10117 Berlin > Tel.: +49 30 28460 388 > > Aaron Hendrickson wrote: > >> Brandon, >> >> The average z-projection is computing the average digital value of your >> stack at each pixel location in your region of interest. For example, say >> you have four images in a stack where the grey values at pixel location >> (0,0) are 128, 135, 115, and 145. Then, the average z-projection will >> yield the value 131 (or 130.75 if working with 32-bit float images) at >> pixel (0,0). >> >> Hope that helps, >> Aaron. >> >> On Wed, Mar 27, 2013 at 1:10 PM, biomicrogen <[hidden email]> wrote: >> >>> Greetings ImageJ Gods, >>> >>> I have a rather simple question (I think). I am doing ratiometric >>> intracellular calcium imaging through a Z stack. I can not find much on >>> the >>> actual method of computing the various types of projections. If I am >>> making >>> an ave z projection of my ratiometric stack how should I be interpreting >>> the >>> resultant image? I have a feeling I may be misinterpreting my data. >>> >>> I would appreciate any and all feedback or suggestions! >>> >>> Thank you all! >>> >>> Brandon >>> >>> >>> >>> -- >>> View this message in context: >>> http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460.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 > > -- > 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.n6.nabble.com/Ratiometric-Z-projections-tp5002460p5002472.html > > To unsubscribe from Ratiometric Z projections??, click here. > NAML > > > > > > -- > View this message in context: http://imagej.1557.n6.nabble.com/Ratiometric-Z-projections-tp5002460p5002474.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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