DIC imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** This is not a confocal question but I was hoping that some one would have a suggestion. I am using a Leica SP5 to do some DIC imaging of live cells. The cells are adherent and in a Matek dish. After collecting the image I gat a nice image of the cells but a background of circular lines through out the image. I am using the 488 laser line as the illumination source. Is this refracted light from the coverslip? Thanks for any help! Mike |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Do you have the lid on or off of the dish? Craig Brideau On Thu, May 22, 2014 at 11:58 AM, Mike Tighe <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > This is not a confocal question but I was hoping that some one would have > a suggestion. I am using a Leica SP5 to do some DIC imaging of live cells. > The cells are adherent and in a Matek dish. After collecting the image I > gat a nice image of the cells but a background of circular lines through > out the image. I am using the 488 laser line as the illumination source. Is > this refracted light from the coverslip? > > > > Thanks for any help! > > Mike > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Lid was on. ________________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Craig Brideau <[hidden email]> Sent: Thursday, May 22, 2014 2:22 PM To: [hidden email] Subject: Re: DIC imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Do you have the lid on or off of the dish? Craig Brideau On Thu, May 22, 2014 at 11:58 AM, Mike Tighe <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > This is not a confocal question but I was hoping that some one would have > a suggestion. I am using a Leica SP5 to do some DIC imaging of live cells. > The cells are adherent and in a Matek dish. After collecting the image I > gat a nice image of the cells but a background of circular lines through > out the image. I am using the 488 laser line as the illumination source. Is > this refracted light from the coverslip? > > > > Thanks for any help! > > Mike > |
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In reply to this post by Craig Brideau
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I agree with Craig, DIC optics requires glass only in the light path. We replace the plastic lid with a large glass cover slip. Also, it is critical to adjust the condenser for Köhler illumination - even though it is a confocal the DIC light path is still wide field. Jim Denegre On 5/22/14, 2:22 PM, "Craig Brideau" <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >Post images on http://www.imgur.com and include the link in your posting. >***** > >Do you have the lid on or off of the dish? > >Craig Brideau > > >On Thu, May 22, 2014 at 11:58 AM, Mike Tighe ><[hidden email]>wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your >>posting. >> ***** >> >> This is not a confocal question but I was hoping that some one would >>have >> a suggestion. I am using a Leica SP5 to do some DIC imaging of live >>cells. >> The cells are adherent and in a Matek dish. After collecting the image I >> gat a nice image of the cells but a background of circular lines through >> out the image. I am using the 488 laser line as the illumination >>source. Is >> this refracted light from the coverslip? >> >> >> >> Thanks for any help! >> >> Mike >> The information in this email, including attachments, may be confidential and is intended solely for the addressee(s). If you believe you received this email by mistake, please notify the sender by return email as soon as possible. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Thanks! (Oops) ________________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of James Denegre <[hidden email]> Sent: Thursday, May 22, 2014 2:50 PM To: [hidden email] Subject: Re: DIC imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I agree with Craig, DIC optics requires glass only in the light path. We replace the plastic lid with a large glass cover slip. Also, it is critical to adjust the condenser for Köhler illumination - even though it is a confocal the DIC light path is still wide field. Jim Denegre On 5/22/14, 2:22 PM, "Craig Brideau" <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >Post images on http://www.imgur.com and include the link in your posting. >***** > >Do you have the lid on or off of the dish? > >Craig Brideau > > >On Thu, May 22, 2014 at 11:58 AM, Mike Tighe ><[hidden email]>wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your >>posting. >> ***** >> >> This is not a confocal question but I was hoping that some one would >>have >> a suggestion. I am using a Leica SP5 to do some DIC imaging of live >>cells. >> The cells are adherent and in a Matek dish. After collecting the image I >> gat a nice image of the cells but a background of circular lines through >> out the image. I am using the 488 laser line as the illumination >>source. Is >> this refracted light from the coverslip? >> >> >> >> Thanks for any help! >> >> Mike >> The information in this email, including attachments, may be confidential and is intended solely for the addressee(s). If you believe you received this email by mistake, please notify the sender by return email as soon as possible. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Ibidi makes a DIC compatible lid with a very flat window on the top for their dishes. The rings you saw were probably slight thickness variations in the plastic from the center to the edge of the lid. Craig Brideau On Thu, May 22, 2014 at 2:53 PM, Mike Tighe <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Thanks! (Oops) > ________________________________________ > From: Confocal Microscopy List <[hidden email]> on > behalf of James Denegre <[hidden email]> > Sent: Thursday, May 22, 2014 2:50 PM > To: [hidden email] > Subject: Re: DIC imaging > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I agree with Craig, DIC optics requires glass only in the light path. We > replace the plastic lid with a large glass cover slip. Also, it is > critical to adjust the condenser for Köhler illumination - even though it > is a confocal the DIC light path is still wide field. > > Jim Denegre > > > > On 5/22/14, 2:22 PM, "Craig Brideau" <[hidden email]> wrote: > > >***** > >To join, leave or search the confocal microscopy listserv, go to: > >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >Post images on http://www.imgur.com and include the link in your posting. > >***** > > > >Do you have the lid on or off of the dish? > > > >Craig Brideau > > > > > >On Thu, May 22, 2014 at 11:58 AM, Mike Tighe > ><[hidden email]>wrote: > > > >> ***** > >> To join, leave or search the confocal microscopy listserv, go to: > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >> Post images on http://www.imgur.com and include the link in your > >>posting. > >> ***** > >> > >> This is not a confocal question but I was hoping that some one would > >>have > >> a suggestion. I am using a Leica SP5 to do some DIC imaging of live > >>cells. > >> The cells are adherent and in a Matek dish. After collecting the image I > >> gat a nice image of the cells but a background of circular lines through > >> out the image. I am using the 488 laser line as the illumination > >>source. Is > >> this refracted light from the coverslip? > >> > >> > >> > >> Thanks for any help! > >> > >> Mike > >> > > > The information in this email, including attachments, may be confidential > and is intended solely for the addressee(s). If you believe you received > this email by mistake, please notify the sender by return email as soon as > possible. > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** The problem with DIC and plastic also stems from photoelasticity in plastic. The DIC phenomenon assumes that there will only be an optical path difference between the ordinary and extraordinary waves due to only the sample. The birefringence of plastic, due to photoelasticity, can also induce and optical path difference due to the two refractive indeces the to waves encounter in the plastic (hold a tissue culture plate between two crossed polarizers, or, if you don't have a polarizer, look at the light from an LCD computer monitor reflecting off of a tissue culture plate in a dark room). As such, the final image is the DIC effect due to the optical path difference in the specimen + the optical path difference due to the plastic dish. Using glass bottomed dishes, or other optical contrast methods including brightfield with the aperture diaphragm closed or oblique illumination, will give you a more uniform contrasting effect. -Ben Smith ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of Craig Brideau [[hidden email]] Sent: Thursday, May 22, 2014 4:36 PM To: [hidden email] Subject: Re: DIC imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Ibidi makes a DIC compatible lid with a very flat window on the top for their dishes. The rings you saw were probably slight thickness variations in the plastic from the center to the edge of the lid. Craig Brideau On Thu, May 22, 2014 at 2:53 PM, Mike Tighe <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Thanks! (Oops) > ________________________________________ > From: Confocal Microscopy List <[hidden email]> on > behalf of James Denegre <[hidden email]> > Sent: Thursday, May 22, 2014 2:50 PM > To: [hidden email] > Subject: Re: DIC imaging > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I agree with Craig, DIC optics requires glass only in the light path. We > replace the plastic lid with a large glass cover slip. Also, it is > critical to adjust the condenser for Köhler illumination - even though it > is a confocal the DIC light path is still wide field. > > Jim Denegre > > > > On 5/22/14, 2:22 PM, "Craig Brideau" <[hidden email]> wrote: > > >***** > >To join, leave or search the confocal microscopy listserv, go to: > >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >Post images on http://www.imgur.com and include the link in your posting. > >***** > > > >Do you have the lid on or off of the dish? > > > >Craig Brideau > > > > > >On Thu, May 22, 2014 at 11:58 AM, Mike Tighe > ><[hidden email]>wrote: > > > >> ***** > >> To join, leave or search the confocal microscopy listserv, go to: > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >> Post images on http://www.imgur.com and include the link in your > >>posting. > >> ***** > >> > >> This is not a confocal question but I was hoping that some one would > >>have > >> a suggestion. I am using a Leica SP5 to do some DIC imaging of live > >>cells. > >> The cells are adherent and in a Matek dish. After collecting the image I > >> gat a nice image of the cells but a background of circular lines through > >> out the image. I am using the 488 laser line as the illumination > >>source. Is > >> this refracted light from the coverslip? > >> > >> > >> > >> Thanks for any help! > >> > >> Mike > >> > > > The information in this email, including attachments, may be confidential > and is intended solely for the addressee(s). If you believe you received > this email by mistake, please notify the sender by return email as soon as > possible. > |
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Benjamin is absolutely correct re: birefringence of the plastic. I suspect hwat youare seeing are called "Newton's rings", due to interference from the plastic in the lid. I wrote a detailed "lay-science" article about DIC in American Lab, 1988. You should be able to download it from "The Library" at www.MicroscopyEducation.com. If you have problems, email me and I'll send you a PDF. Best regards, Barbara Foster, President & Chief Consultant Microscopy/Microscopy Education* www.MicroscopyEducation.com *A subsidiary of The Microscopy & Imaging Place, Inc. 7101 Royal Glen Trail, Suite A McKinney, TX 75070 P: 972-924-5310 F: 214-592-0277 MME is currently scheduling courses for now and through Fall 2014. Call us today for a free training evaluation. At 09:15 AM 5/23/2014, you wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >Post images on http://www.imgur.com and include the link in your posting. >***** > >The problem with DIC and plastic also stems from >photoelasticity in plastic. The DIC phenomenon >assumes that there will only be an optical path >difference between the ordinary and >extraordinary waves due to only the sample. The >birefringence of plastic, due to >photoelasticity, can also induce and optical >path difference due to the two refractive >indeces the to waves encounter in the plastic >(hold a tissue culture plate between two crossed >polarizers, or, if you don't have a polarizer, >look at the light from an LCD computer monitor >reflecting off of a tissue culture plate in a >dark room). As such, the final image is the DIC >effect due to the optical path difference in the >specimen + the optical path difference due to >the plastic dish. Using glass bottomed dishes, >or other optical contrast methods including >brightfield with the aperture diaphragm closed >or oblique illumination, will give you a more >uniform contrasting effect. -Ben Smith >________________________________________ From: >Confocal Microscopy List >[[hidden email]] on behalf of >Craig Brideau [[hidden email]] Sent: >Thursday, May 22, 2014 4:36 PM To: >[hidden email] Subject: Re: >DIC imaging ***** To join, leave or search the >confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >Post images on http://www.imgur.com and include >the link in your posting. ***** Ibidi makes a >DIC compatible lid with a very flat window on >the top for their dishes. The rings you saw were >probably slight thickness variations in the >plastic from the center to the edge of the lid. >Craig Brideau On Thu, May 22, 2014 at 2:53 PM, >Mike Tighe <[hidden email]>wrote: > >***** > To join, leave or search the confocal >microscopy listserv, go to: > >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and >include the link in your posting. > ***** > > >Thanks! (Oops) > >________________________________________ > From: >Confocal Microscopy List ><[hidden email]> on > behalf >of James Denegre <[hidden email]> > Sent: >Thursday, May 22, 2014 2:50 PM > To: >[hidden email] > Subject: Re: >DIC imaging > > ***** > To join, leave or search >the confocal microscopy listserv, go to: > >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and >include the link in your posting. > ***** > > I >agree with Craig, DIC optics requires glass only >in the light path. We > replace the plastic lid >with a large glass cover slip. Also, it is > >critical to adjust the condenser for Köhler >illumination - even though it > is a confocal >the DIC light path is still wide field. > > Jim >Denegre > > > > On 5/22/14, 2:22 PM, "Craig >Brideau" <[hidden email]> >wrote: > > >***** > >To join, leave or search >the confocal microscopy listserv, go >to: > >http://lists.umn.edu/cgi-bin/wa?A0=confoca >lmicroscopy > >Post images on >http://www.imgur.com and include the link in >your posting. > >***** > > > >Do you have the >lid on or off of the dish? > > > >Craig >Brideau > > > > > >On Thu, May 22, 2014 at 11:58 >AM, Mike >Tighe > ><[hidden email]>wrote: > > > > >> ***** > >> To join, leave or search the >confocal microscopy listserv, go to: > >> >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >> Post images on http://www.imgur.com and >include the link in your > >>posting. > >> >***** > >> > >> This is not a confocal question >but I was hoping that some one >would > >>have > >> a suggestion. I am using a >Leica SP5 to do some DIC imaging of >live > >>cells. > >> The cells are adherent and >in a Matek dish. After collecting the image >I > >> gat a nice image of the cells but a >background of circular lines through > >> out >the image. I am using the 488 laser line as the >illumination > >>source. Is > >> this refracted >light from the coverslip? > >> > >> > >> > >> >Thanks for any help! > >> > >> Mike > >> > > > >The information in this email, including >attachments, may be confidential > and is >intended solely for the addressee(s). If you >believe you received > this email by mistake, >please notify the sender by return email as soon as > possible. > |
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** There's a decent article on the Nikon website on imaging of birefringent samples in general: http://www.microscopyu.com/articles/polarized/polarizedintro.html It's got some pretty neat diagrams and example specimens. Craig Brideau On Sat, May 24, 2014 at 12:43 PM, Barbara Foster <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Benjamin is absolutely correct re: birefringence of the plastic. I > suspect hwat youare seeing are called "Newton's rings", due to interference > from the plastic in the lid. > > I wrote a detailed "lay-science" article about DIC in American Lab, 1988. > You should be able to download it from "The Library" at > www.MicroscopyEducation.com. If you have problems, email me and I'll > send you a PDF. > > Best regards, > Barbara Foster, President & Chief Consultant > Microscopy/Microscopy Education* > www.MicroscopyEducation.com > > *A subsidiary of The Microscopy & Imaging Place, Inc. > 7101 Royal Glen Trail, Suite A > McKinney, TX 75070 > P: 972-924-5310 > F: 214-592-0277 > > MME is currently scheduling courses for now and through Fall 2014. Call us > today for a free training evaluation. > > > > At 09:15 AM 5/23/2014, you wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> The problem with DIC and plastic also stems from photoelasticity in >> plastic. The DIC phenomenon assumes that there will only be an optical >> path difference between the ordinary and extraordinary waves due to only >> the sample. The birefringence of plastic, due to photoelasticity, can also >> induce and optical path difference due to the two refractive indeces the to >> waves encounter in the plastic (hold a tissue culture plate between two >> crossed polarizers, or, if you don't have a polarizer, look at the light >> from an LCD computer monitor reflecting off of a tissue culture plate in a >> dark room). As such, the final image is the DIC effect due to the optical >> path difference in the specimen + the optical path difference due to the >> plastic dish. Using glass bottomed dishes, or other optical contrast >> methods including brightfield with the aperture diaphragm closed or oblique >> illumination, will give you a more uniform contrasting effect. -Ben Smith >> ________________________________________ From: Confocal Microscopy List [ >> [hidden email]] on behalf of Craig Brideau [ >> [hidden email]] Sent: Thursday, May 22, 2014 4:36 PM To: >> [hidden email] Subject: Re: DIC imaging ***** To join, >> leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on >> http://www.imgur.com and include the link in your posting. ***** Ibidi >> makes a DIC compatible lid with a very flat window on the top for their >> dishes. The rings you saw were probably slight thickness variations in the >> plastic from the center to the edge of the lid. Craig Brideau On Thu, May >> 22, 2014 at 2:53 PM, Mike Tighe <[hidden email]>wrote: > >> ***** > To join, leave or search the confocal microscopy listserv, go to: > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on >> http://www.imgur.com and include the link in your posting. > ***** > > >> Thanks! (Oops) > ________________________________________ > From: >> Confocal Microscopy List <[hidden email]> on > behalf >> of James Denegre <[hidden email]> > Sent: Thursday, May 22, 2014 >> 2:50 PM > To: [hidden email] > Subject: Re: DIC >> imaging > > ***** > To join, leave or search the confocal microscopy >> listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on >> http://www.imgur.com and include the link in your posting. > ***** > > I >> agree with Craig, DIC optics requires glass only in the light path. We > >> replace the plastic lid with a large glass cover slip. Also, it is > >> critical to adjust the condenser for Köhler illumination - even though it > >> is a confocal the DIC light path is still wide field. > > Jim Denegre > > > >> > On 5/22/14, 2:22 PM, "Craig Brideau" <[hidden email]> wrote: >> > > >***** > >To join, leave or search the confocal microscopy listserv, go >> to: > >http://lists.umn.edu/cgi-bin/wa?A0=confoca lmicroscopy > >Post >> images on http://www.imgur.com and include the link in your posting. > >> >***** > > > >Do you have the lid on or off of the dish? > > > >Craig >> Brideau > > > > > >On Thu, May 22, 2014 at 11:58 AM, Mike Tighe > >< >> [hidden email]>wrote: > > > >> ***** > >> To join, leave >> or search the confocal microscopy listserv, go to: > >> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >> Post images >> on http://www.imgur.com and include the link in your > >>posting. > >> >> ***** > >> > >> This is not a confocal question but I was hoping that some >> one would > >>have > >> a suggestion. I am using a Leica SP5 to do some DIC >> imaging of live > >>cells. > >> The cells are adherent and in a Matek dish. >> After collecting the image I > >> gat a nice image of the cells but a >> background of circular lines through > >> out the image. I am using the 488 >> laser line as the illumination > >>source. Is > >> this refracted light >> from the coverslip? > >> > >> > >> > >> Thanks for any help! > >> > >> Mike >> > >> > > > The information in this email, including attachments, may be >> confidential > and is intended solely for the addressee(s). If you believe >> you received > this email by mistake, please notify the sender by return >> email as soon as > possible. > >> > |
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