*****
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 <[log in to unmask]>
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 <[log in to unmask]
> > 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: [log in to unmask]
>>> 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
>
>
|
