Hi! I'm Greg
After admiring all of your images through college and into grad school, it seems I owe it to the community to post some photos of my own.
This is an echinopluteus larva of Dendraster excentricus, about 400 um long
Confocal laser scanning microscopy is a really neat technology that has become pretty popular. It creates three dimensional images by layering optical slices, much like the optical stacks many of you do here (which I hope to learn.) The only difference is the use of fluorescent stains. In this case, phalloidin (red) tailored for nuclei, and hoechst (blue) for actin, ie; muscle. Ultimately, though the colors are digitized and can be altered using the image processing program.
Nice to meet you all. Thanks for all of your inspiring images over the years.
Greg
[img]http://www.photomacrography.net/forum/u ... JPEG_2.jpg[/img]
sand dollar larva through confocal
Moderators: rjlittlefield, ChrisR, Chris S., Pau
-
Greg Gavelis
- Posts: 11
- Joined: Wed Nov 09, 2011 4:04 pm
- Location: Vancouver, British Columbia
- Contact:
-
rjlittlefield
- Site Admin
- Posts: 23603
- Joined: Tue Aug 01, 2006 8:34 am
- Location: Richland, Washington State, USA
- Contact:
Greg, welcome aboard! An excellent image, and definitely out of the ordinary for this forum.
The reason your image didn't appear inline in your own post is that the post is tagged with "Disable BBCode in this post". Your default for this setting is controlled by a checkbox in your profile, and the setting can be changed for an individual post with the checkboxes just below the text entry box. There's also a Preview button that you can use to check that the post is going to look right, before you finally Submit it.
--Rik
The reason your image didn't appear inline in your own post is that the post is tagged with "Disable BBCode in this post". Your default for this setting is controlled by a checkbox in your profile, and the setting can be changed for an individual post with the checkboxes just below the text entry box. There's also a Preview button that you can use to check that the post is going to look right, before you finally Submit it.
--Rik
-
Cactusdave
- Posts: 1631
- Joined: Tue Jun 09, 2009 12:40 pm
- Location: Bromley, Kent, UK
-
Franz Neidl
- Posts: 747
- Joined: Wed Jan 14, 2009 11:59 am
- Location: Italy
-
Tardigrade37
- Posts: 134
- Joined: Tue Mar 04, 2008 7:38 pm
-
Cactusdave
- Posts: 1631
- Joined: Tue Jun 09, 2009 12:40 pm
- Location: Bromley, Kent, UK
Phalloidin specifically binds to actin.
Quote:
Phalloidin is a bicyclic peptide that belongs to a family of toxins isolated from the deadly Amanita phalloides “death cap” mushroom and is commonly used in imaging applications to selectively label F-actin in fixed cells, permeabilized cells, and cell-free experiments. Labeled phalloidin conjugates have similar affinity for both large and small filaments and bind in a stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and nonmuscle cells; they reportedly do not bind to monomeric G-actin, unlike some antibodies against actin.
http://www.invitrogen.com/site/us/en/ho ... Actin.html
The colour you actually observe in fluorescence microscopy using phalloidin as a probe for actin will depend upon the fluorophore (fluorescent chemical) linked to it, usually in practice a red or yellowish green fluorescing fluorophore is used and red contrasts better with blue stained nuclei.
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA.
Quote:
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by the Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra.
http://en.wikipedia.org/wiki/Hoechst_stain
As the original poster notes, I guess you can now fiddle with the final colours of the digital image on the computer if you want. Not possible in my pre-digital era of using the technique and I'm not quite sure why you would want to.
Quote:
Phalloidin is a bicyclic peptide that belongs to a family of toxins isolated from the deadly Amanita phalloides “death cap” mushroom and is commonly used in imaging applications to selectively label F-actin in fixed cells, permeabilized cells, and cell-free experiments. Labeled phalloidin conjugates have similar affinity for both large and small filaments and bind in a stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and nonmuscle cells; they reportedly do not bind to monomeric G-actin, unlike some antibodies against actin.
http://www.invitrogen.com/site/us/en/ho ... Actin.html
The colour you actually observe in fluorescence microscopy using phalloidin as a probe for actin will depend upon the fluorophore (fluorescent chemical) linked to it, usually in practice a red or yellowish green fluorescing fluorophore is used and red contrasts better with blue stained nuclei.
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA.
Quote:
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by the Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra.
http://en.wikipedia.org/wiki/Hoechst_stain
As the original poster notes, I guess you can now fiddle with the final colours of the digital image on the computer if you want. Not possible in my pre-digital era of using the technique and I'm not quite sure why you would want to.
Leitz Ortholux 1, Zeiss standard, Nikon Diaphot inverted, Canon photographic gear
-
Greg Gavelis
- Posts: 11
- Joined: Wed Nov 09, 2011 4:04 pm
- Location: Vancouver, British Columbia
- Contact:
Thanks for your your help, Rik. And Tardigrade, you are right that I had the stains switched--oh the errs of late night posting.
It is true that phalloidin and hoechst both represent the binding components molecular tracers, which have two components. Phalloidin, in this case, is coupled to Alexa 440 stain for fluorescence and Hoechst is coupled to DAPI stain. Each fluoresces at at a signature wavelength, and the typical confocal microscope has 3 lasers to acomodate three overlaid "channels" of color, which are then merged into one integrated image.
That is a great question, Franz. The answer is that this image was "just for fun." At the time I was studying the early development of nemertean larvae, and since I had some stain left over, I decided to image this slightly more photogenic specimen.
It is true that phalloidin and hoechst both represent the binding components molecular tracers, which have two components. Phalloidin, in this case, is coupled to Alexa 440 stain for fluorescence and Hoechst is coupled to DAPI stain. Each fluoresces at at a signature wavelength, and the typical confocal microscope has 3 lasers to acomodate three overlaid "channels" of color, which are then merged into one integrated image.
That is a great question, Franz. The answer is that this image was "just for fun." At the time I was studying the early development of nemertean larvae, and since I had some stain left over, I decided to image this slightly more photogenic specimen.