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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 <[log in to unmask]> on behalf of George McNamara <[log in to unmask]>
Sent: 08 April 2015 03:36
To: [log in to unmask]
Subject: Re: camera for FISH

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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<[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
>>>>
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>>>
>>>   --
>>>
>>>
>>>
>>> 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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