*****
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.
*****

That particular CCD does support binning, which enables electronic
reduction of magnification without sacrificing NA, so rather than trying a
lower magnification objective, I'd stay with the 1.42 NA and maybe try 2x
binning (which would give you the equivalent pixel size as a 30x objective)
if needed.

Regarding binning, keep in mind that it reduces read noise, but does not
reduce thermal noise.  For long integration times where thermal noise tends
to be a bigger factor than read noise, both binning and reducing the
magnification optically will only be marginally different than simply
downsampling the image in photoshop or matlab.

Really though the key is eliminating all sources of photon loss in the
system.  Use the highest NA you can, and make sure all excess filters are
removed.  Ideally you want just the sample, the objective, the tubes lens
and the camera.  Then reduce your noise as best you can, so all sources of
stray light.  Finally increase the integration time until you see
something.

Mike

On Sun, Dec 14, 2014 at 2:25 PM, James Pawley <[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.
> *****
>
>  *****
>> 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've obtained samples of some Renilla luciferase substrates from
>> NanoLight for the purpose of visualizing bioluminescence in living cells.
>> These substrates are formulated for various emission peaks, one being V-CTZ
>> which has a peak around 520 nm, which nicely fits in the FITC filter set.
>>
>> I tried transfecting some HEK293T cells with Rluc8 (highly active version
>> of the luciferase), then loading them up with the substrate and imaging
>> them live on our DeltaVision microscope. We have an Olympus IX70 base,
>> CoolSnapHQ CCD camera, and I used a 60X Olympus 1.42NA oil objective. My
>> strategy was to set up a long exposure (2 to 10 sec) with no excitation
>> light (Ex set to BLOCK), collecting emitted light in the FITC channel. I
>> started imaging pretty much immediately after I put the substrate on the
>> cells. I know that the luciferase signal peaks in the first minute and then
>> drops off, but should still be detectable for about an hour after that.
>>
>> I was unable to get any signal whatsoever. The exposure was coming up
>> completely negative, I could not even see any autofluorescence, just CCD
>> noise at around 100 (of 4095) units, which is the standard background
>> noise. I had co-transfected GFP in my cells, and parallel (very short -
>> 0.05 sec) exposures with 100% excitation light showed very strong GFP
>> signal in almost every cell, so I know that the camera was collecting light
>> in the FITC channel and that the cells were transfected.
>>
>> I also tried two other substrates from NanoLight, Prolume Purple and
>> Prolume Purple II, which emit close to the DAPI range, and again had
>> absolutely no signal. I know for certain that my Rluc8 works in these
>> particular cells as it produces very high luminescence using Promega's kit,
>> though that assay is designed for lysed cells and uses a plate reader, and
>> is not suitable for imaging living cells.
>>
>> The luciferase substrate was loaded at the upper end of NanoLight's
>> recommended concentration for live cells (20 uM). I can try to increase the
>> substrate concentration somewhat, though the stock solution (about 12.5 mM)
>> is dissolved in 10% ethanol and there will be a point beyond which I would
>> be adding enough ethanol to the culture medium to affect the luciferase
>> reaction. I also don't really expect that I will suddenly go from ZERO
>> signal to something substantial/publishable just by increasing the
>> concentration.
>>
>> My conclusion is that the luminescence is far too dim for my camera and
>> it seems that I will never be able to see it. Is there anything else I can
>> do to increase my ability to see this signal in living cells on my
>> microscope? NanoLight suggested that I open the aperture on the microscope
>> to maximum, but I don't know how to do that or even if that is possible on
>> my setup. I know that there are bioluminescence microscopes like the
>> Olympus LV-200, but there does not seem to be anything like that on campus.
>> There are plenty of other epi scopes, however, and I can look around to see
>> whether there are more sensitive CCDs I could take advantage of, if that is
>> the main limiting factor.
>>
>>
>> --
>> Menelaos Symeonides
>> University of Vermont
>> Cell & Molecular Biology Graduate Program
>> Department of Microbiology and Molecular Genetics
>> 318 Stafford Hall
>> 95 Carrigan Dr
>> Burlington, VT 05405
>> [log in to unmask]
>> Phone: 802-656-1161
>>
>
> Dear Menelaos,
>
> The signal will inevitably be very weak. By magnifying the image, you
> spread a fixed number of photons over more pixels and the signal level
> drops with (mag) squared. Try lower magnification: bigger pixels, referred
> to the specimen, will contain more light emitting molecules. Of course,
> lower mag usually means lower NA, as signal/pixel goes with (NA) squared so
> you need a balance Remember, you are actually sampling a volume at each
> pixel and, as long as your specimen has some depth, lower NA will provide
> more depth of field and extends the volume sampled by the pixel on the
> camera in the z direction.
>
> You probably also need a different camera, one with lower read noise (like
> a good EM-CCD), higher raw QE (Back-illuminated), and cooled to a low
> enough temp to allow even longer exposures (minutes).  If this is
> impossible, try binning the pixels in your present camera. Not as good but
> cheaper! Just to see how bad the problem is.
>
> Finally, make a major effort to reduce stray light in the room: No
> operating display screens during image collection, back velvet cap on the
> far side of the specimen from the objective etc.  You may even have to put
> black tape over all the "holes" in your microscope between the objective
> and the camera (to block light from the inevitable LED indicator lights).
> With no exciting light, (turned off, not just blocked with a slider) you
> can  probably do without the emission filter because the luciferase
> reaction should be the only light source left.
>
> Good luck
> --
>               ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
> Canada, V0N3A0,
> Phone 604-885-0840, email <[log in to unmask]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> 1-604-989-6146
>