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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. ***** Dear all, just a quick survey....if I need to use a widefield microscope dedicated to acquire DNA e RNA FISH slides on cell with 100X obj (all of these experiments are either with home-made probes (not very strong signal) and small cell (lymphocytes))...... which source light (LED, Xenon..), but mainly which type of camera would you suggest? or even better which are the camera characteristics I cannot underrate? Thanks in advance for all your always useful replies. Valeria Valeria Berno, PhD Imaging Facility ______ Istituto Nazionale di Genetica Molecolare Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" Via F.Sforza 35 - 20122 - Milano (MI) - Italy Tel Off.: +39 02.00660 219 Tel Lab.:+39 02.00660 338 E-Mail: [hidden email] Web site: http://www.ingm.org SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la RICERCA SANITARIA. Grazie 1000...al 5 per mille! Please consider the environment before printing this mail note DISCLAIMER : Questa e-mail potrebbe contenere informazioni privilegiate o confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è pregato di contattare il mittente rispondendo a questa stessa dopodichè è pregato di cancellarla immediatamente. This e-mail may contain confidential or privileged information. If you think you have received this e-mail in error, please advise the sender by reply e-mail and then delete this e-mail immediately. |
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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. ***** Hi Valeria, I have done the exact experiments you are talking about. Homemade FISH probes by Nick translation of BACs to be used on human and mouse lymphocytes. I was able to get very nice images on our wide field microscope. We use this light source ( http://www.prior-us.com/Products/Illumination/Lumen-200-Pro.aspx) and this camera (http://www.prior-us.com/Products/Illumination/Lumen-200-Pro.aspx). Regards, Daniel Neems PhD Candidate, Kosak Laboratory Feinberg School of Medicine, Northwestern University Chicago, IL On Tue, Apr 7, 2015 at 8:27 AM, Valeria Berno <[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. > ***** > > Dear all, > > just a quick survey....if I need to use a widefield microscope dedicated > to acquire DNA e RNA FISH slides on cell with 100X obj (all of these > experiments are either with home-made probes (not very strong signal) and > small cell (lymphocytes))...... > > which source light (LED, Xenon..), but mainly which type of camera would > you suggest? or even better which are the camera characteristics I cannot > underrate? > > Thanks in advance for all your always useful replies. > > Valeria > > > Valeria Berno, PhD > Imaging Facility > ______ > > Istituto Nazionale di Genetica Molecolare > Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" > Via F.Sforza 35 - 20122 - Milano (MI) - Italy > Tel Off.: +39 02.00660 219 > Tel Lab.:+39 02.00660 338 > E-Mail: [hidden email] > Web site: http://www.ingm.org > > SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE > INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire > il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, > 730 e UNICO nella sezione relativa al finanziamento per la RICERCA > SANITARIA. Grazie 1000...al 5 per mille! > Please consider the environment before printing this mail note > > DISCLAIMER : > Questa e-mail potrebbe contenere informazioni privilegiate o > confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è > pregato di contattare il mittente rispondendo a questa stessa dopodichè è > pregato di cancellarla immediatamente. > > This e-mail may contain confidential or privileged information. If you > think you have received this e-mail in error, please advise the sender by > reply e-mail and then delete this e-mail immediately. |
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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. ***** Hi Valeria, I recommend a 4 megapixel or 5.5 megapixel scientific CMOS camera, for examples from Hamamatsu (FLASH4.0), PCO or Andor. these have the same pixel size (~6.8x6.8um) but 3x more pixels than a typical CCD camera (ex ORCA-ER) so 3x bigger field of view, and sCMOS have much faster focusing. I've been using Bruce & Butte GPU deconvolution for (almost) instant gratification deconvolution http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 See http://works.bepress.com/gmcnamara/37/ and other FISH data posted at http://works.bepress.com/gmcnamara for more examples. I am using the late 2013 NVidia TITAN card. The newest TITAN X should be somewhat faster. The free B&B Deconvolution is no longer available - see http://www.microvolution.com/ for the commercial version (disclosure: I was involved in acquiring the image on their home page). See http://stellarisfish.smugmug.com/ for the single RNA molecule FISH vendor galleries (Biosearch Tech). If the probe set you want is not part of BTI's catalogue line, takes about a week to make and ship 'custom' set. With my microscope (Lumencor SOLA in single LED mode, Leica DMI6000, 6 filter cubes) I recently found Quasar 705 is somewhat brighter than Quasar 670 for the same probe set. Quasar 570 and CAL Fluor Red 610 work fine. I avoid ordering probe sets in the green since the pancreatic cancer cells I FISH have high green fluorescent autofluorescence. Enjoy, George On 4/7/2015 8:27 AM, Valeria Berno 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. > ***** > > Dear all, > > just a quick survey....if I need to use a widefield microscope dedicated to acquire DNA e RNA FISH slides on cell with 100X obj (all of these experiments are either with home-made probes (not very strong signal) and small cell (lymphocytes))...... > > which source light (LED, Xenon..), but mainly which type of camera would you suggest? or even better which are the camera characteristics I cannot underrate? > > Thanks in advance for all your always useful replies. > > Valeria > > > Valeria Berno, PhD > Imaging Facility > ______ > > Istituto Nazionale di Genetica Molecolare > Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" > Via F.Sforza 35 - 20122 - Milano (MI) - Italy > Tel Off.: +39 02.00660 219 > Tel Lab.:+39 02.00660 338 > E-Mail: [hidden email] > Web site: http://www.ingm.org > > SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la RICERCA SANITARIA. Grazie 1000...al 5 per mille! > Please consider the environment before printing this mail note > > DISCLAIMER : > Questa e-mail potrebbe contenere informazioni privilegiate o confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è pregato di contattare il mittente rispondendo a questa stessa dopodichè è pregato di cancellarla immediatamente. > > This e-mail may contain confidential or privileged information. If you think you have received this e-mail in error, please advise the sender by reply e-mail and then delete this e-mail immediately. > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 |
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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. ***** Hey George, how dim would you say is dim in this case? How do you know when you need an amplified solution like EMCCD vs a good sCMOS? Craig On Tue, Apr 7, 2015 at 4:56 PM, George McNamara <[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. > ***** > > Hi Valeria, > > I recommend a 4 megapixel or 5.5 megapixel scientific CMOS camera, for > examples from Hamamatsu (FLASH4.0), PCO or Andor. these have the same pixel > size (~6.8x6.8um) but 3x more pixels than a typical CCD camera (ex ORCA-ER) > so 3x bigger field of view, and sCMOS have much faster focusing. > > I've been using Bruce & Butte GPU deconvolution for (almost) instant > gratification deconvolution > http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 > > See > http://works.bepress.com/gmcnamara/37/ > and other FISH data posted at http://works.bepress.com/gmcnamara > for more examples. I am using the late 2013 NVidia TITAN card. The newest > TITAN X should be somewhat faster. > The free B&B Deconvolution is no longer available - see > http://www.microvolution.com/ for the commercial version (disclosure: I > was involved in acquiring the image on their home page). > > See http://stellarisfish.smugmug.com/ for the single RNA > molecule FISH vendor galleries (Biosearch Tech). If the probe set you want > is not part of BTI's catalogue line, takes about a week to make and ship > 'custom' set. With my microscope (Lumencor SOLA in single LED mode, Leica > DMI6000, 6 filter cubes) I recently found Quasar 705 is somewhat brighter > than Quasar 670 for the same probe set. Quasar 570 and CAL Fluor Red 610 > work fine. I avoid ordering probe sets in the green since the pancreatic > cancer cells I FISH have high green fluorescent autofluorescence. > > Enjoy, > George > > > > > > On 4/7/2015 8:27 AM, Valeria Berno 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. >> ***** >> >> Dear all, >> >> just a quick survey....if I need to use a widefield microscope dedicated >> to acquire DNA e RNA FISH slides on cell with 100X obj (all of these >> experiments are either with home-made probes (not very strong signal) and >> small cell (lymphocytes))...... >> >> which source light (LED, Xenon..), but mainly which type of camera would >> you suggest? or even better which are the camera characteristics I cannot >> underrate? >> >> Thanks in advance for all your always useful replies. >> >> Valeria >> >> >> Valeria Berno, PhD >> Imaging Facility >> ______ >> >> Istituto Nazionale di Genetica Molecolare >> Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" >> Via F.Sforza 35 - 20122 - Milano (MI) - Italy >> Tel Off.: +39 02.00660 219 >> Tel Lab.:+39 02.00660 338 >> E-Mail: [hidden email] >> Web site: http://www.ingm.org >> >> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE >> INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire >> il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, >> 730 e UNICO nella sezione relativa al finanziamento per la RICERCA >> SANITARIA. Grazie 1000...al 5 per mille! >> Please consider the environment before printing this mail note >> >> DISCLAIMER : >> Questa e-mail potrebbe contenere informazioni privilegiate o >> confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è >> pregato di contattare il mittente rispondendo a questa stessa dopodichè è >> pregato di cancellarla immediatamente. >> >> This e-mail may contain confidential or privileged information. If you >> think you have received this e-mail in error, please advise the sender by >> reply e-mail and then delete this e-mail immediately. >> >> > > > -- > > > > George McNamara, Ph.D. > Single Cells Analyst > L.J.N. Cooper Lab > University of Texas M.D. Anderson Cancer Center > Houston, TX 77054 > Tattletales http://works.bepress.com/gmcnamara/42 > |
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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. ***** Hi Craig, I don't have an EMCCD handy to do this - see no reason to need EM anyway. Depends on the target and probe set. The Levesque et al 2013 SNV FISH paper, from Arjun Raj's lab used a CCD camera, even though the SNV site is detected with a single oligo with one fluorophore and single base different oligo with a different color fluorophore (the rest of the RNA is detected with up to 47 oligos, 20 bases each). http://www.ncbi.nlm.nih.gov/pubmed/23913259 http://www.nature.com/nmeth/journal/v10/n9/full/nmeth.2589.html see the key figure http://www.nature.com/nmeth/journal/v10/n9/fig_tab/nmeth.2589_F1.html With 20x/0.7NA and Cy3 filter set, I can see GAPDH Quasar 570 (Cy3 equivalent) by eye (room dark). I image on my lab's Hamamatsu FLASH4.0 sCMOS camera with leica plan apo 63x/1.3NA oil lens, 500 ms or 1000 ms for GAPDH, sometimes to 4000 ms for Quasar 670 or Quasar 705 for a single exon with under 20 oligos (each oligo has one fluorophore ... most probe sets have 48 oligos). These Biosearch Tech 'Stellaris' single molecule RNA FISH probe sets also work in flow cytometry, http://www.ncbi.nlm.nih.gov/pubmed/25505292 (not the only paper, just easiest for me to cite). George On 4/7/2015 7:20 PM, Craig Brideau wrote: > Hey George, how dim would you say is dim in this case? How do you know > when you need an amplified solution like EMCCD vs a good sCMOS? > > Craig > > On Tue, Apr 7, 2015 at 4:56 PM, George McNamara > <[hidden email] <mailto:[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. > ***** > > Hi Valeria, > > I recommend a 4 megapixel or 5.5 megapixel scientific CMOS camera, > for examples from Hamamatsu (FLASH4.0), PCO or Andor. these have > the same pixel size (~6.8x6.8um) but 3x more pixels than a typical > CCD camera (ex ORCA-ER) so 3x bigger field of view, and sCMOS have > much faster focusing. > > I've been using Bruce & Butte GPU deconvolution for (almost) > instant gratification deconvolution > http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 > <http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375> > > See > http://works.bepress.com/gmcnamara/37/ > and other FISH data posted at http://works.bepress.com/gmcnamara > for more examples. I am using the late 2013 NVidia TITAN card. The > newest TITAN X should be somewhat faster. > The free B&B Deconvolution is no longer available - see > http://www.microvolution.com/ for the commercial version > (disclosure: I was involved in acquiring the image on their home > page). > > See http://stellarisfish.smugmug.com/ for the single RNA > molecule FISH vendor galleries (Biosearch Tech). If the probe set > you want is not part of BTI's catalogue line, takes about a week > to make and ship 'custom' set. With my microscope (Lumencor SOLA > in single LED mode, Leica DMI6000, 6 filter cubes) I recently > found Quasar 705 is somewhat brighter than Quasar 670 for the same > probe set. Quasar 570 and CAL Fluor Red 610 work fine. I avoid > ordering probe sets in the green since the pancreatic cancer cells > I FISH have high green fluorescent autofluorescence. > > Enjoy, > George > > > > > > On 4/7/2015 8:27 AM, Valeria Berno 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. > ***** > > Dear all, > > just a quick survey....if I need to use a widefield microscope > dedicated to acquire DNA e RNA FISH slides on cell with 100X > obj (all of these experiments are either with home-made probes > (not very strong signal) and small cell (lymphocytes))...... > > which source light (LED, Xenon..), but mainly which type of > camera would you suggest? or even better which are the camera > characteristics I cannot underrate? > > Thanks in advance for all your always useful replies. > > Valeria > > > Valeria Berno, PhD > Imaging Facility > ______ > > Istituto Nazionale di Genetica Molecolare > Istutito Nazionale Genetica Molecolare "Romeo ed Enrica > Invernizzi" > Via F.Sforza 35 - 20122 - Milano (MI) - Italy > Tel Off.: +39 02.00660 219 > Tel Lab.:+39 02.00660 338 > E-Mail: [hidden email] <mailto:[hidden email]> > Web site: http://www.ingm.org > > SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla > FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di > Milano. Devi solo inserire il codice fiscale della Fondazione > - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella > sezione relativa al finanziamento per la RICERCA SANITARIA. > Grazie 1000...al 5 per mille! > Please consider the environment before printing this mail note > > DISCLAIMER : > Questa e-mail potrebbe contenere informazioni privilegiate o > confidenziali. Se pensa di aver ricevuto questa e-mail per > errore, è pregato di contattare il mittente rispondendo a > questa stessa dopodichè è pregato di cancellarla immediatamente. > > This e-mail may contain confidential or privileged > information. If you think you have received this e-mail in > error, please advise the sender by reply e-mail and then > delete this e-mail immediately. > > > > -- > > > > George McNamara, Ph.D. > Single Cells Analyst > L.J.N. Cooper Lab > University of Texas M.D. Anderson Cancer Center > Houston, TX 77054 > Tattletales http://works.bepress.com/gmcnamara/42 > > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 |
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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! So it comes down to 'do a lit review to see what other people used' and your own experience. Craig On Tue, Apr 7, 2015 at 6:45 PM, George McNamara <[hidden email]> wrote: > Hi Craig, > > I don't have an EMCCD handy to do this - see no reason to need EM anyway. > Depends on the target and probe set. The Levesque et al 2013 SNV FISH > paper, from Arjun Raj's lab used a CCD camera, even though the SNV site is > detected with a single oligo with one fluorophore and single base different > oligo with a different color fluorophore (the rest of the RNA is detected > with up to 47 oligos, 20 bases each). > http://www.ncbi.nlm.nih.gov/pubmed/23913259 > http://www.nature.com/nmeth/journal/v10/n9/full/nmeth.2589.html > see the key figure > http://www.nature.com/nmeth/journal/v10/n9/fig_tab/nmeth.2589_F1.html > > With 20x/0.7NA and Cy3 filter set, I can see GAPDH Quasar 570 (Cy3 > equivalent) by eye (room dark). I image on my lab's Hamamatsu FLASH4.0 > sCMOS camera with leica plan apo 63x/1.3NA oil lens, 500 ms or 1000 ms for > GAPDH, sometimes to 4000 ms for Quasar 670 or Quasar 705 for a single exon > with under 20 oligos (each oligo has one fluorophore ... most probe sets > have 48 oligos). > > These Biosearch Tech 'Stellaris' single molecule RNA FISH probe sets also > work in flow cytometry, > http://www.ncbi.nlm.nih.gov/pubmed/25505292 > (not the only paper, just easiest for me to cite). > > George > > > On 4/7/2015 7:20 PM, Craig Brideau wrote: > > Hey George, how dim would you say is dim in this case? How do you know > when you need an amplified solution like EMCCD vs a good sCMOS? > > Craig > > On Tue, Apr 7, 2015 at 4:56 PM, George McNamara <[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. >> ***** >> >> Hi Valeria, >> >> I recommend a 4 megapixel or 5.5 megapixel scientific CMOS camera, for >> examples from Hamamatsu (FLASH4.0), PCO or Andor. these have the same pixel >> size (~6.8x6.8um) but 3x more pixels than a typical CCD camera (ex ORCA-ER) >> so 3x bigger field of view, and sCMOS have much faster focusing. >> >> I've been using Bruce & Butte GPU deconvolution for (almost) instant >> gratification deconvolution >> http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 >> >> See >> http://works.bepress.com/gmcnamara/37/ >> and other FISH data posted at http://works.bepress.com/gmcnamara >> for more examples. I am using the late 2013 NVidia TITAN card. The newest >> TITAN X should be somewhat faster. >> The free B&B Deconvolution is no longer available - see >> http://www.microvolution.com/ for the commercial version (disclosure: I >> was involved in acquiring the image on their home page). >> >> See http://stellarisfish.smugmug.com/ for the single RNA >> molecule FISH vendor galleries (Biosearch Tech). If the probe set you want >> is not part of BTI's catalogue line, takes about a week to make and ship >> 'custom' set. With my microscope (Lumencor SOLA in single LED mode, Leica >> DMI6000, 6 filter cubes) I recently found Quasar 705 is somewhat brighter >> than Quasar 670 for the same probe set. Quasar 570 and CAL Fluor Red 610 >> work fine. I avoid ordering probe sets in the green since the pancreatic >> cancer cells I FISH have high green fluorescent autofluorescence. >> >> Enjoy, >> George >> >> >> >> >> >> On 4/7/2015 8:27 AM, Valeria Berno 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. >>> ***** >>> >>> Dear all, >>> >>> just a quick survey....if I need to use a widefield microscope dedicated >>> to acquire DNA e RNA FISH slides on cell with 100X obj (all of these >>> experiments are either with home-made probes (not very strong signal) and >>> small cell (lymphocytes))...... >>> >>> which source light (LED, Xenon..), but mainly which type of camera would >>> you suggest? or even better which are the camera characteristics I cannot >>> underrate? >>> >>> Thanks in advance for all your always useful replies. >>> >>> Valeria >>> >>> >>> Valeria Berno, PhD >>> Imaging Facility >>> ______ >>> >>> Istituto Nazionale di Genetica Molecolare >>> Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" >>> Via F.Sforza 35 - 20122 - Milano (MI) - Italy >>> Tel Off.: +39 02.00660 219 >>> Tel Lab.:+39 02.00660 338 >>> E-Mail: [hidden email] >>> Web site: http://www.ingm.org >>> >>> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla >>> FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo >>> inserire il codice fiscale della Fondazione - 04175700964 - in tutti i >>> modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la >>> RICERCA SANITARIA. Grazie 1000...al 5 per mille! >>> Please consider the environment before printing this mail note >>> >>> DISCLAIMER : >>> Questa e-mail potrebbe contenere informazioni privilegiate o >>> confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è >>> pregato di contattare il mittente rispondendo a questa stessa dopodichè è >>> pregato di cancellarla immediatamente. >>> >>> This e-mail may contain confidential or privileged information. If you >>> think you have received this e-mail in error, please advise the sender by >>> reply e-mail and then delete this e-mail immediately. >>> >>> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales http://works.bepress.com/gmcnamara/42 >> > > > > -- > > > > George McNamara, Ph.D. > Single Cells Analyst > L.J.N. Cooper Lab > University of Texas M.D. Anderson Cancer Center > Houston, TX 77054 > Tattletales http://works.bepress.com/gmcnamara/42 > > |
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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. ***** Hi Craig, I had the Leica DMI6000 microscope and FLASH4.0 sCMOS camera before I started doing (or even thinking about) single molecule RNA FISHing. I knew what the vendor typically used (Nikon scope, oil immersion lens, Photometrics CoolSNAP) for their web galleries ( http://stellarisfish.smugmug.com/ ), so was confident our lab microscope would work. I was also confident my lab's scope field of view and throughput would be much higher than going upstairs to the OMX with 2 EMCCDs (and tiny field of view, and I've only been able to get it to give good images in deconvolution mode, not 3D-SIM). George p.s. check out the Stellaris RNA FISH full length promotional video on the left side of http://stellarisfish.smugmug.com about as good as this recent web video http://chronicle.com/blogs/ticker/watch-a-professors-elaborate-april-fools-joke-slay-his-lecture-class/96633 On 4/7/2015 8:49 PM, Craig Brideau 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! So it comes down to 'do a lit review to see what other people used' > and your own experience. > > Craig > > On Tue, Apr 7, 2015 at 6:45 PM, George McNamara<[hidden email]> > wrote: > > >> Hi Craig, >> >> I don't have an EMCCD handy to do this - see no reason to need EM anyway. >> Depends on the target and probe set. The Levesque et al 2013 SNV FISH >> paper, from Arjun Raj's lab used a CCD camera, even though the SNV site is >> detected with a single oligo with one fluorophore and single base different >> oligo with a different color fluorophore (the rest of the RNA is detected >> with up to 47 oligos, 20 bases each). >> http://www.ncbi.nlm.nih.gov/pubmed/23913259 >> http://www.nature.com/nmeth/journal/v10/n9/full/nmeth.2589.html >> see the key figure >> http://www.nature.com/nmeth/journal/v10/n9/fig_tab/nmeth.2589_F1.html >> >> With 20x/0.7NA and Cy3 filter set, I can see GAPDH Quasar 570 (Cy3 >> equivalent) by eye (room dark). I image on my lab's Hamamatsu FLASH4.0 >> sCMOS camera with leica plan apo 63x/1.3NA oil lens, 500 ms or 1000 ms for >> GAPDH, sometimes to 4000 ms for Quasar 670 or Quasar 705 for a single exon >> with under 20 oligos (each oligo has one fluorophore ... most probe sets >> have 48 oligos). >> >> These Biosearch Tech 'Stellaris' single molecule RNA FISH probe sets also >> work in flow cytometry, >> http://www.ncbi.nlm.nih.gov/pubmed/25505292 >> (not the only paper, just easiest for me to cite). >> >> George >> >> >> On 4/7/2015 7:20 PM, Craig Brideau wrote: >> >> Hey George, how dim would you say is dim in this case? How do you know >> when you need an amplified solution like EMCCD vs a good sCMOS? >> >> Craig >> >> On Tue, Apr 7, 2015 at 4:56 PM, George McNamara<[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. >>> ***** >>> >>> Hi Valeria, >>> >>> I recommend a 4 megapixel or 5.5 megapixel scientific CMOS camera, for >>> examples from Hamamatsu (FLASH4.0), PCO or Andor. these have the same pixel >>> size (~6.8x6.8um) but 3x more pixels than a typical CCD camera (ex ORCA-ER) >>> so 3x bigger field of view, and sCMOS have much faster focusing. >>> >>> I've been using Bruce& Butte GPU deconvolution for (almost) instant >>> gratification deconvolution >>> http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 >>> >>> See >>> http://works.bepress.com/gmcnamara/37/ >>> and other FISH data posted at http://works.bepress.com/gmcnamara >>> for more examples. I am using the late 2013 NVidia TITAN card. The newest >>> TITAN X should be somewhat faster. >>> The free B&B Deconvolution is no longer available - see >>> http://www.microvolution.com/ for the commercial version (disclosure: I >>> was involved in acquiring the image on their home page). >>> >>> See http://stellarisfish.smugmug.com/ for the single RNA >>> molecule FISH vendor galleries (Biosearch Tech). If the probe set you want >>> is not part of BTI's catalogue line, takes about a week to make and ship >>> 'custom' set. With my microscope (Lumencor SOLA in single LED mode, Leica >>> DMI6000, 6 filter cubes) I recently found Quasar 705 is somewhat brighter >>> than Quasar 670 for the same probe set. Quasar 570 and CAL Fluor Red 610 >>> work fine. I avoid ordering probe sets in the green since the pancreatic >>> cancer cells I FISH have high green fluorescent autofluorescence. >>> >>> Enjoy, >>> George >>> >>> >>> >>> >>> >>> On 4/7/2015 8:27 AM, Valeria Berno 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. >>>> ***** >>>> >>>> Dear all, >>>> >>>> just a quick survey....if I need to use a widefield microscope dedicated >>>> to acquire DNA e RNA FISH slides on cell with 100X obj (all of these >>>> experiments are either with home-made probes (not very strong signal) and >>>> small cell (lymphocytes))...... >>>> >>>> which source light (LED, Xenon..), but mainly which type of camera would >>>> you suggest? or even better which are the camera characteristics I cannot >>>> underrate? >>>> >>>> Thanks in advance for all your always useful replies. >>>> >>>> Valeria >>>> >>>> >>>> Valeria Berno, PhD >>>> Imaging Facility >>>> ______ >>>> >>>> Istituto Nazionale di Genetica Molecolare >>>> Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" >>>> Via F.Sforza 35 - 20122 - Milano (MI) - Italy >>>> Tel Off.: +39 02.00660 219 >>>> Tel Lab.:+39 02.00660 338 >>>> E-Mail: [hidden email] >>>> Web site: http://www.ingm.org >>>> >>>> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla >>>> FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo >>>> inserire il codice fiscale della Fondazione - 04175700964 - in tutti i >>>> modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la >>>> RICERCA SANITARIA. Grazie 1000...al 5 per mille! >>>> Please consider the environment before printing this mail note >>>> >>>> DISCLAIMER : >>>> Questa e-mail potrebbe contenere informazioni privilegiate o >>>> confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è >>>> pregato di contattare il mittente rispondendo a questa stessa dopodichè è >>>> pregato di cancellarla immediatamente. >>>> >>>> This e-mail may contain confidential or privileged information. If you >>>> think you have received this e-mail in error, please advise the sender by >>>> reply e-mail and then delete this e-mail immediately. >>>> >>>> >>>> >>> >>> -- >>> >>> >>> >>> George McNamara, Ph.D. >>> Single Cells Analyst >>> L.J.N. Cooper Lab >>> University of Texas M.D. Anderson Cancer Center >>> Houston, TX 77054 >>> Tattletales http://works.bepress.com/gmcnamara/42 >>> >>> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales http://works.bepress.com/gmcnamara/42 >> >> >> > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 |
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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. ***** Hi Valeria We do a lot of FISH at the IGMM in Edinburgh and use various probes which are commercial or homemade with weaker signals. Although CCD may be a bit old school we find the Coolsnap HQ2 to suit our needs very well as we do multi-colour FISH where we need to compare expression across 2 different fluorophores. Some of the newer sCMOS and EMCCD cameras don't have the nice flat Quantum efficiency that the HQ2 affords across the visible spectrum. It has smaller Pixels than EMCCD which we need for resolution and none of the fixed pattern noise which needs to be corrected (even on a Flash4). So for multicolour FISH it does well. The jury is still out as far as I am concerned with SIM and FISH. You need a good signal or SIM won't work so well and there are artefacts in image reconstruction which need sorting out carefully - particularly with weaker signals. We're considering 2D TIRF SIM for the improved resolution. Hope this is of some use, there is a very recent paper in Genes and Development about how we have done our FISH imaging which may be of use. Ann Dr Ann Wheeler Head of Advanced Imaging Resource Institute of Genetics and Molecular Medicine + ESRIC University of Edinburgh EH4 2XU UK ________________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of George McNamara <[hidden email]> Sent: 08 April 2015 03:36 To: [hidden email] Subject: Re: camera for FISH ***** 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. ***** Hi Craig, I had the Leica DMI6000 microscope and FLASH4.0 sCMOS camera before I started doing (or even thinking about) single molecule RNA FISHing. I knew what the vendor typically used (Nikon scope, oil immersion lens, Photometrics CoolSNAP) for their web galleries ( http://stellarisfish.smugmug.com/ ), so was confident our lab microscope would work. I was also confident my lab's scope field of view and throughput would be much higher than going upstairs to the OMX with 2 EMCCDs (and tiny field of view, and I've only been able to get it to give good images in deconvolution mode, not 3D-SIM). George p.s. check out the Stellaris RNA FISH full length promotional video on the left side of http://stellarisfish.smugmug.com about as good as this recent web video http://chronicle.com/blogs/ticker/watch-a-professors-elaborate-april-fools-joke-slay-his-lecture-class/96633 On 4/7/2015 8:49 PM, Craig Brideau 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! So it comes down to 'do a lit review to see what other people used' > and your own experience. > > Craig > > On Tue, Apr 7, 2015 at 6:45 PM, George McNamara<[hidden email]> > wrote: > > >> Hi Craig, >> >> I don't have an EMCCD handy to do this - see no reason to need EM anyway. >> Depends on the target and probe set. The Levesque et al 2013 SNV FISH >> paper, from Arjun Raj's lab used a CCD camera, even though the SNV site is >> detected with a single oligo with one fluorophore and single base different >> oligo with a different color fluorophore (the rest of the RNA is detected >> with up to 47 oligos, 20 bases each). >> http://www.ncbi.nlm.nih.gov/pubmed/23913259 >> http://www.nature.com/nmeth/journal/v10/n9/full/nmeth.2589.html >> see the key figure >> http://www.nature.com/nmeth/journal/v10/n9/fig_tab/nmeth.2589_F1.html >> >> With 20x/0.7NA and Cy3 filter set, I can see GAPDH Quasar 570 (Cy3 >> equivalent) by eye (room dark). I image on my lab's Hamamatsu FLASH4.0 >> sCMOS camera with leica plan apo 63x/1.3NA oil lens, 500 ms or 1000 ms for >> GAPDH, sometimes to 4000 ms for Quasar 670 or Quasar 705 for a single exon >> with under 20 oligos (each oligo has one fluorophore ... most probe sets >> have 48 oligos). >> >> These Biosearch Tech 'Stellaris' single molecule RNA FISH probe sets also >> work in flow cytometry, >> http://www.ncbi.nlm.nih.gov/pubmed/25505292 >> (not the only paper, just easiest for me to cite). >> >> George >> >> >> On 4/7/2015 7:20 PM, Craig Brideau wrote: >> >> Hey George, how dim would you say is dim in this case? How do you know >> when you need an amplified solution like EMCCD vs a good sCMOS? >> >> Craig >> >> On Tue, Apr 7, 2015 at 4:56 PM, George McNamara<[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. >>> ***** >>> >>> Hi Valeria, >>> >>> I recommend a 4 megapixel or 5.5 megapixel scientific CMOS camera, for >>> examples from Hamamatsu (FLASH4.0), PCO or Andor. these have the same pixel >>> size (~6.8x6.8um) but 3x more pixels than a typical CCD camera (ex ORCA-ER) >>> so 3x bigger field of view, and sCMOS have much faster focusing. >>> >>> I've been using Bruce& Butte GPU deconvolution for (almost) instant >>> gratification deconvolution >>> http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 >>> >>> See >>> http://works.bepress.com/gmcnamara/37/ >>> and other FISH data posted at http://works.bepress.com/gmcnamara >>> for more examples. I am using the late 2013 NVidia TITAN card. The newest >>> TITAN X should be somewhat faster. >>> The free B&B Deconvolution is no longer available - see >>> http://www.microvolution.com/ for the commercial version (disclosure: I >>> was involved in acquiring the image on their home page). >>> >>> See http://stellarisfish.smugmug.com/ for the single RNA >>> molecule FISH vendor galleries (Biosearch Tech). If the probe set you want >>> is not part of BTI's catalogue line, takes about a week to make and ship >>> 'custom' set. With my microscope (Lumencor SOLA in single LED mode, Leica >>> DMI6000, 6 filter cubes) I recently found Quasar 705 is somewhat brighter >>> than Quasar 670 for the same probe set. Quasar 570 and CAL Fluor Red 610 >>> work fine. I avoid ordering probe sets in the green since the pancreatic >>> cancer cells I FISH have high green fluorescent autofluorescence. >>> >>> Enjoy, >>> George >>> >>> >>> >>> >>> >>> On 4/7/2015 8:27 AM, Valeria Berno 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. >>>> ***** >>>> >>>> Dear all, >>>> >>>> just a quick survey....if I need to use a widefield microscope dedicated >>>> to acquire DNA e RNA FISH slides on cell with 100X obj (all of these >>>> experiments are either with home-made probes (not very strong signal) and >>>> small cell (lymphocytes))...... >>>> >>>> which source light (LED, Xenon..), but mainly which type of camera would >>>> you suggest? or even better which are the camera characteristics I cannot >>>> underrate? >>>> >>>> Thanks in advance for all your always useful replies. >>>> >>>> Valeria >>>> >>>> >>>> Valeria Berno, PhD >>>> Imaging Facility >>>> ______ >>>> >>>> Istituto Nazionale di Genetica Molecolare >>>> Istutito Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi" >>>> Via F.Sforza 35 - 20122 - Milano (MI) - Italy >>>> Tel Off.: +39 02.00660 219 >>>> Tel Lab.:+39 02.00660 338 >>>> E-Mail: [hidden email] >>>> Web site: http://www.ingm.org >>>> >>>> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla >>>> FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo >>>> inserire il codice fiscale della Fondazione - 04175700964 - in tutti i >>>> modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la >>>> RICERCA SANITARIA. Grazie 1000...al 5 per mille! >>>> Please consider the environment before printing this mail note >>>> >>>> DISCLAIMER : >>>> Questa e-mail potrebbe contenere informazioni privilegiate o >>>> confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è >>>> pregato di contattare il mittente rispondendo a questa stessa dopodichè è >>>> pregato di cancellarla immediatamente. >>>> >>>> This e-mail may contain confidential or privileged information. If you >>>> think you have received this e-mail in error, please advise the sender by >>>> reply e-mail and then delete this e-mail immediately. >>>> >>>> >>>> >>> >>> -- >>> >>> >>> >>> George McNamara, Ph.D. >>> Single Cells Analyst >>> L.J.N. Cooper Lab >>> University of Texas M.D. Anderson Cancer Center >>> Houston, TX 77054 >>> Tattletales http://works.bepress.com/gmcnamara/42 >>> >>> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales http://works.bepress.com/gmcnamara/42 >> >> >> > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 -- The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. |
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