Agarose
Locked
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello all !
recently I have been trying to image the fluorescent beads inserted to the agarose gel.I make .1% agarose gel and while it's cooling but not still solidified I pour the solution for the beads into the gel and then I use vortex to mix
them.for the last sample,I have tried 3ul of beads solution to the 1.5 ml of gel and mixed them but from none of the samples I could capture beads with our two-photon microscopy.I appreciate if you could give me any kind of information that can help.
Thanks
Sarah
|
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Sarah - Can you see the beads on your slide by phase contrast or DIC imaging so that you are sure they are there? Tamara On Wed, 23 Jan 2008 15:19:20 -0500 Sarah Kefayati <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all ! > > recently I have been trying to image the fluorescent >beads inserted to the > agarose gel.I make .1% agarose gel and while it's >cooling but not still > solidified I pour the solution for the beads into the >gel and then I use > vortex to mix them.for the last sample,I have tried 3ul >of beads solution to > the 1.5 ml of gel and mixed them but from none of the >samples I could > capture beads with our two-photon microscopy.I >appreciate if you could give > me any kind of information that can help. > > Thanks > Sarah *************************** Tamara Howard Cell Biology & Physiology UNM-HSC Albuquerque, NM *************************** |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
That's very good idea but our microscope is not equipped by DIC unfortunately.
On Jan 23, 2008 3:21 PM, Tamara A Howard <[hidden email]> wrote: Search the CONFOCAL archive at |
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello, I used agarose (but I do not remember the concentration) for the same purpose. My first recommendation: verify the working distance of your objective. The agarose can be very thick! My method: 1- Vortex diluted beads 2- dry beads on a coverslip 3- warm agarose (no more than needed) 4- put 1-2 drops on agarose on the coverslip 5- put microscope slide on coverslip (making sure it does not crush the beads) 6- allow to cool thoroughly 7- check with air objective for fluorescence Notes: I did this with Molecular Probes' Constellation mix and the agarose remixed the beads over its thickness. I did this using Biofolie dish for my water immersion, no coverslip objective. I hope this helps. Sophie ____________________________________________________ Sophie M. K. Brunet, Ph. D. Research Officer Optical Spectroscopy, Laser Systems and Applications Chemistry 112 sessional lecturer [hidden email] 306-966-1719 (office) 306-966-1702 (fax) ____________________________________________________ Saskatchewan Structural Sciences Centre University of Saskatchewan Thorvaldson Bldg. 110 Science Place Saskatoon, Sk S7N 5C9 ____________________________________________________ Quoting Sarah Kefayati <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all ! > > recently I have been trying to image the fluorescent beads inserted to the > agarose gel.I make .1% agarose gel and while it's cooling but not still > solidified I pour the solution for the beads into the gel and then I use > vortex to mix them.for the last sample,I have tried 3ul of beads solution to > the 1.5 ml of gel and mixed them but from none of the samples I could > capture beads with our two-photon microscopy.I appreciate if you could give > me any kind of information that can help. > > Thanks > Sarah > |
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Here is what I have learned making similar samples.
Bigger beads are much brighter and easier to find at depth than something small such as what you would use for a PSF measurement. It is difficult to mix the beads very evenly through the viscous agarose. You usually get streaks of beads which can be hard to find. Kind of like a marble cake. Also it is helpful to squirt some beads onto the surface of the agarose so you know where the top is and can measure the depth. Using my 2-photon microscope I have been able to see two millimeters (the working distance of my objective) into 1% agarose with out noticing a change in intensity of the beads. Paul -- Paul Herzmark Specialist [hidden email] Department of Molecular and Cell Biology 479 Life Science Addition University of California, Berkeley Berkeley, CA 94720-3200 (510) 643-9603 On Jan 23, 2008 12:26 PM, Sarah Kefayati <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal That's very good idea but our microscope is not equipped by DIC unfortunately. |
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Sarah We have developed a technique we coined "Fluorescent Microangiography", where we perfuse an anatomical area of an animal with 0.2um fluorescent beads in 1% low melting point agarose. These are polystyrene beads from Molecular Probes and they are almost too bright, and they never bleach. The local vasculature is outlined really beautifully. I did some calibration by filling microcapillaries with this solution just to check intensities, depth attenuation, etc. Worked very well. Dutly, A.E. et al., Laboratory Investigation, 2006 86:409-416. - a technique paper Let me know if you need additional details. Judy Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 [hidden email] >>> Sarah Kefayati <[hidden email]> 01/23/08 3:19 PM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all ! recently I have been trying to image the fluorescent beads inserted to the agarose gel.I make .1% agarose gel and while it's cooling but not still solidified I pour the solution for the beads into the gel and then I use vortex to mix them.for the last sample,I have tried 3ul of beads solution to the 1.5 ml of gel and mixed them but from none of the samples I could capture beads with our two-photon microscopy.I appreciate if you could give me any kind of information that can help. Thanks Sarah |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Agarose may not be the easiest gelling agent to use in these contexts, although obviously from Judy's work , it can be used very productively. Biostatus sells CyGel, which is liquid at low temperatures and changes from sol to gel (or certainly thickens to a useful extent) at moderate temperatures (from 15 to 50 degrees centigrade). It has the advantage that cells can be transfixed without the danger of cooking them, and beads can also be held still without stripping them of dye - I have no commercial interest in Biostatus (indeed I read their msds sheet to see what they make the gel from so I could make my own I was so impressed by the product's usefulness). We use it to instantly immobilise suspension cells for use on our inverted microscope without the need to spin them down then seal coverslips - probably missing many applications. It would seem that Judy's work may be made easier using them rather than agarose, along with many drug-delivery applications (a flick through patent sites shows that similar or identical copolymers are being exploited widely). Cheers Ray ----- Original Message ----- From: "Judy Trogadis" <[hidden email]> To: <[hidden email]> Sent: Wednesday, January 23, 2008 8:52 PM Subject: Re: Agarose Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Sarah We have developed a technique we coined "Fluorescent Microangiography", where we perfuse an anatomical area of an animal with 0.2um fluorescent beads in 1% low melting point agarose. These are polystyrene beads from Molecular Probes and they are almost too bright, and they never bleach. The local vasculature is outlined really beautifully. I did some calibration by filling microcapillaries with this solution just to check intensities, depth attenuation, etc. Worked very well. Dutly, A.E. et al., Laboratory Investigation, 2006 86:409-416. - a technique paper Let me know if you need additional details. Judy Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 [hidden email] >>> Sarah Kefayati <[hidden email]> 01/23/08 3:19 PM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all ! recently I have been trying to image the fluorescent beads inserted to the agarose gel.I make .1% agarose gel and while it's cooling but not still solidified I pour the solution for the beads into the gel and then I use vortex to mix them.for the last sample,I have tried 3ul of beads solution to the 1.5 ml of gel and mixed them but from none of the samples I could capture beads with our two-photon microscopy.I appreciate if you could give me any kind of information that can help. Thanks Sarah |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |