Imaging luminescence on an epifluorescence microscope - waste of time?

[Mel Symeonides]

*****
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
[hidden email]
Phone: [phone]

[Michael Giacomelli]

*****
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 Menelaos,

The first thing I would do is remove the filter cube.  While FITC will line
up with the maximum of your luciferase, you are still losing a lot of light
at the edges as the emission of luciferase is fairly broad relative to most
FITC filters.

Assuming your system is completely light tight (as in, _absolutely_ no
light is getting in, if not, make it so) you can also try integrating
longer.  I had one of the older TEC cooled CoolSnaps, and they have very
low noise even at long integration times.

Mike

On Sun, Dec 14, 2014 at 1:06 PM, Menelaos Symeonides <[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.
> *****
>
> 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
> [hidden email]
> Phone: [phone]
>

[James Pawley]

*****
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
>[hidden email]
>Phone: [phone]

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 [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Michael Giacomelli]

*****
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 <[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.
> *****
>
>  *****
>> 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
>> [hidden email]
>> Phone: [phone]
>>
>
> 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 [phone], email <[hidden email]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> 1-604-989-6146
>

[Zdenek Svindrych]

*****
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 Menelaos,
also make sure that the DeltaVision scope does not block the emission path
when you block the excitation (simply shine onto your sample with a
flashlight and you should see it on the camera). Longer integration
(minutes) should not be a problem with a cooled CCD.

Best, zdenek



---------- Původní zpráva ----------
Od: Menelaos Symeonides <[hidden email]>
Komu: [hidden email]
Datum: 14. 12. 2014 13:02:31
Předmět: Imaging luminescence on an epifluorescence microscope - waste of
time?

"*****
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
[hidden email]
Phone: [phone]"

[George McNamara]

*****
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 Menlaos and listserv,

Read carefully:

Sorre 2014 Encoding of Temporal Signals by the TGF-? Pathway and
Implications for Embryonic Patterning. Devel Cell 30: 334--34
http://www.sciencedirect.com/science/article/pii/S1534580714003426
especially the supplemental file(s).
most of the paper is about TGF-beta signaling - very weak = hard.
Their positive control CAG promoter --> Promega NanoLuc + Furimazine
(coelenterazine analog co-optimized with Nano-Luc) is what you want.

My suggestions:
* take Jim's suggestions below ... maximum binning (if CCD or EMCCD).
* Write Sorre et al for their CAGp--> NanoLuc (you may need to purchase
a Promega product to be legal).
* long term (10+ minutes, maybe 5+ minutes) coelenterazine or furimazine
may be toxic (do what Sorre et al did).
* phenol red is bad.
* serum is bad (low concentration may be ok ... NanoLuc works in live
mice, so can't be too bad).
* coelenterazine's can be subject to effluc through 'MDR' pumps (see
http://www.ncbi.nlm.nih.gov/pubmed/18713866 and more usefully
http://www.ncbi.nlm.nih.gov/pubmed/24798747 ... some of these
transporters can move molecules in either direction).

Plan B: read (carefully) Saito 2013 Luminescent proteins for high-speed
single-cell and whole-body imaging, Nat Commun, and if impressed, write
Take Nagai for his Nano-Lantern,
http://www.ncbi.nlm.nih.gov/pubmed/23232392
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535334/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535334/#S1

My advice: make some popcorn and download Saito's video 1 (not a
microscope experiment)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535334/bin/ncomms2248-s2.mov

best wishes and may all your photons be detectable,

George
p.s. localizng FPs or Nano-Lantern, or NanoLuc: more FPs (or
Nano-Lantern or Nano-Luc) in smaller volume is better (and enables
multiplexing other stuff) ...
see http://works.bepress.com/gmcnamara/42 and
http://works.bepress.com/gmcnamara/63 for ideas and references --- I am
getting back to writing up "T-Bow" (Rainbow T-cells and tumor cells) for
the current NIH Single Cell Analysis Program InnoCentive Challenge
proposal as soon as I hit the Send button (since the deadline is Monday,
https://www.innocentive.com/ar/challenge/9933618 - everyone is welcome
to write NIH and recommend our proposal be approved for Phase 1) ...
look for the proposal on my site as number"65" in a couple of days), and
Tanembaum et al 2014, http://www.ncbi.nlm.nih.gov/pubmed/25307933 for
related published work.
Tanenbaum et al used dCas9-tag42 or dCas9-tag10, sdAb-EGFP with
endogenous genomic sites (EGFP is so 1996), I am proposing synthetic
tandem repeats (100 repeats per transposon), TALE-tag10, and
'binder'-nls-FP-FP with state of the art FP's (mNeonGreen is 3x brighter
than EGFP, bfloGFPa1 is almost 4x brighter than EGFP), so if funded for
implementation, ours would be, at saturation (hopefully not), per
synthetic tandem repeat array: 100 * 10 * 2 = 2000 state of the art FP
molecules in a diffraction limited spot (~200x200x600 nm if high NA
lens, something like ~400x400x1200 nm if 0.7NA). For comparison, see
Figure 4A (512 EGFP equivalents but smoggy background from
overerxpression) of:

*Robinett* CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont
AS 1996 In vivo localization of DNA sequences and visualization of
large-scale chromatin organization using lac operator/repressor
recognition. <http://www.ncbi.nlm.nih.gov/pubmed/8991083> J Cell Biol
135: 1685-700. PMID: 8991083.
http://jcb.rupress.org/content/135/6/1685.long   (open access).


On 12/14/2014 1:25 PM, James Pawley 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
>> [hidden email]
>> Phone: [phone]
>
> 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

--



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

[James Pawley]

*****
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,

As I understand it, these specimens are in water.
Any light leaving the specimen at an angle
corresponding to more than NA1.25 will be either
mostly or totally internally reflected at the
water/coverslip interface. More to the point,
when using an NA 1.42 oil objective, for light
emerging more than a micron or two inside the wet
specimen there will be a massive SA.

I don't know about Olympus but Zeiss makes a 10x
NA.45 Plan-APO (and a 10x 0.5, Fluar). At .45,
the SA is relatively minor for the first 10 µm.
Assuming that the actual light-gathering of the
1.42 will be closer to 1.2 for a water specimen,
the mag of the 10x is 6x smaller and the NA only
3x smaller so you come out about 4x more light to
a fixed-size pixel at the camera. To this you can
add the extra "now in-focus" light from the 3x
greater depth-of-focus (supposing that the
specimen emits light throughout this z-range).
12x is quite a difference.

Of course, we don't really know what "structure" Menelaos is hoping to see.

Each Luciferase emission will at most register as
one amplified photoelectron in an pixel of the
EM-CCD (If there is a lot of SA, this
photoelectron will be in the wrong pixel but we
won't know this if there is no fine structure to
the luciferase distribution.).

However, you can't make an image from one
detected photoelectron. There has to be some
contrast: some places from which more photons
emerge and other places where they are not found.

Are these dark areas large or small? Is he merely
wanting to see which cells light up (i.e., a bob
about 10µm in diameter, i.e., say 4x4µm pixels
referred to the specimen)?

In any case, at these signal levels, resolution
is limited far more by Poisson Noise  (probably
to a few µm) than by the Abbe equation. The only
way that higher NA will give improved spatial
resolution is if it allows you to count more
photons. As noted above, the 1.42 (really 1.2)
can acquire more photons but aberrations will
prevent them from being detected in the right
place. This may be OK if you only want to detect
blobs of cells but you may have to bin your CCD
more than 2x2 to see it, especially when the
noise floor of the Sony chip is usually about
+/-8 photoelectrons.

Otherwise, work on the stray light and see if you
can borrow an EM-CCD and a low-mag, high-NA lens
from Olympus.

Cheers,

Jim Pawley

>*****
>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 <[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.
>>  *****
>>
>>   *****
>  >> 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
>>>  [hidden email]
>>>  Phone: [phone]
>>>
>>
>>  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 [phone], email <[hidden email]>
>>  NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
>>  1-604-989-6146
>>

--
               ****************************************
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Guy Cox-2]

*****
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.
*****

OK, I've been a bit reluctant to join this discussion since I've never worked with a luminescence system.  But I have a lot of experience in imaging super-dim signals.  First of all I think that human eyes have to get involved.  Your EMCCD or whatever may be better on paper but the human visual system has a huge amount of processing built in.  Are you really sure that the camera isn't shuttered off when you cut the excitation?  Many are, for obvious reasons (not detecting completely spurious signals).  (A month or two ago I'd have expected Deltavision to have jumped in on that but now, as I understand, they have been taken over by a company that has no interest in the product.)  Someone else pointed out this potential problem, but it still seems to me that this is the most likely explanation.  And, as other people have said, remove the filter cube - face it, it does nothing!  

So, first of all, get totally dark adapted and see if you can see the signal by eye.  My gut feeling is that unless you have got the experiment totally wrong you will.  

Then we come to the optics, and Jim has made several valid points there.  There must be absolutely no spherical aberration - one emitting molecule needs to end up on one camera pixel.  So index matching is crucial - and if you don't know how to use a correction collar buy a ticket to Sydney and I'll show you! :)  What Jim didn't mention is that several manufacturers now make low-mag high NA objectives, and they will give you a lot of light-gathering power.

Finally, people have been doing luciferin - luciferase imaging successfully long before EMCCD and SCMOS and the latest high-brightness probes.  So it can't be that hard.  If you are really seeing absolutely nothing at all I'm sure it's a fault in your system rather than inadequate hardware.

                                                           Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Monday, 15 December 2014 11:26 AM
To: [hidden email]
Subject: Re: Imaging luminescence on an epifluorescence microscope - waste of time?

*****
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,

As I understand it, these specimens are in water.
Any light leaving the specimen at an angle corresponding to more than NA1.25 will be either mostly or totally internally reflected at the water/coverslip interface. More to the point, when using an NA 1.42 oil objective, for light emerging more than a micron or two inside the wet specimen there will be a massive SA.

I don't know about Olympus but Zeiss makes a 10x
NA.45 Plan-APO (and a 10x 0.5, Fluar). At .45, the SA is relatively minor for the first 10 µm.
Assuming that the actual light-gathering of the
1.42 will be closer to 1.2 for a water specimen, the mag of the 10x is 6x smaller and the NA only 3x smaller so you come out about 4x more light to a fixed-size pixel at the camera. To this you can add the extra "now in-focus" light from the 3x greater depth-of-focus (supposing that the specimen emits light throughout this z-range).
12x is quite a difference.

Of course, we don't really know what "structure" Menelaos is hoping to see.

Each Luciferase emission will at most register as one amplified photoelectron in an pixel of the EM-CCD (If there is a lot of SA, this photoelectron will be in the wrong pixel but we won't know this if there is no fine structure to the luciferase distribution.).

However, you can't make an image from one detected photoelectron. There has to be some
contrast: some places from which more photons emerge and other places where they are not found.

Are these dark areas large or small? Is he merely wanting to see which cells light up (i.e., a bob about 10µm in diameter, i.e., say 4x4µm pixels referred to the specimen)?

In any case, at these signal levels, resolution is limited far more by Poisson Noise  (probably to a few µm) than by the Abbe equation. The only way that higher NA will give improved spatial resolution is if it allows you to count more photons. As noted above, the 1.42 (really 1.2) can acquire more photons but aberrations will prevent them from being detected in the right place. This may be OK if you only want to detect blobs of cells but you may have to bin your CCD more than 2x2 to see it, especially when the noise floor of the Sony chip is usually about
+/-8 photoelectrons.

Otherwise, work on the stray light and see if you can borrow an EM-CCD and a low-mag, high-NA lens from Olympus.

Cheers,

Jim Pawley

>*****
>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 <[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.
>>  *****
>>
>>   *****
>  >> 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
>>>  [hidden email]
>>>  Phone: [phone]
>>>
>>
>>  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 [phone], email
>> <[hidden email]>  NEW! NEW! AND DIFFERENT Cell (when I remember to
>> turn it on!)
>>  1-604-989-6146
>>

--
               ****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone [phone], email <[hidden email]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Jens]

*****
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 Menelaos,

You could also built a very simple "tube-scope": an old finite focus objective plus camera separated by a tube. Edmunds is selling something like this or you could use Thorlabs 2 inch tubes with adapters for c-mount and RMS/M25 on respective ends.
This would take care of the straylight problem and maximize light collection as there are no other elements in the light path. Cost should be pretty low.

Maybe that helps.
Best,

Jens



Jens-B. Bosse
[phone]

> On Dec 14, 2014, at 13:06, Menelaos Symeonides <[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.
> *****
>
> 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
> [hidden email]
> Phone: [phone]

[samjlord]

*****
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.
*****

On Mon, 15 Dec 2014 11:31:00 +0000, Guy Cox <[hidden email]>
wrote:
>Finally, people have been doing luciferin - luciferase imaging successfully long
before EMCCD and SCMOS and the latest high-brightness probes.  So it can't be
that hard.

People have been imaging luciferin, but it is known to be very difficult to image on
the single-cell level. From the papers I've read for single-cell luminescence
imaging, they used intensified CCDs (cooled have very low background) in
digital/photon-counting mode and integrate for tens of minutes.

I tried luciferin imaging once for single cells. I gave up before I got it working.

It's worth thinking about the rates. Fluorescence imaging involves approximately
the same number of probes, but the excitation/fluorescence cycling rate is going to
be WAY higher than the chemical turnover. And each turnover gives at most one
photon. It's not surprising that luminescence signal is orders of magnitude dimmer
than fluorescence.

The real benefit to luminescence imaging is zero background. But to take
advantage of that, you need to block absolutely all room light (and LED lights on
the scope and other electronic equipment). Also, you need a way to integrate for
minutes without filling up your entire CCD well depth with dark counts.

Don't feel ashamed if you're having trouble. :)

(That said, the suggestion from Guy Cox and others to confirm that light is able to
reach the camera and that there is not a problem with the hardware is an excellent
point!)

[Alison J. North]

*****
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 Menelaos,

We tried luminescence imaging of cell monolayers a few times a couple of
years ago.  I moved around between various microscopes to try to get it
to work.  I remember starting on the DelltaVision but that was not the
system that finally worked for us - I wish for the life of me I could
remember why!  We got it to work vaguely using another old Olympus IX71
with an EMCCD camera on it - the trick was indeed (as others have said)
to use a low magnification lens with as high an NA as possible.  I think
the 4x was actually the best on that system.  On most microscopes the
issue was the internal LEDs that seem to be hidden inside the microscope
stand and so you can't do anything about them.  In the end, much to our
surprise, we had the best luck on our Olympus VivaView system, with an
empty filter cube position (that was essential - we saw nothing through
the DAPI, CFP or GFP cubes), the 20x objective (which is the only option
in there), and the Orca R2 camera (which, BTW, is not intensified). The
researcher was using Nanoluc and I think he only had to use exposures of
around 30 secs to see a signal.  The nice thing about the VivaView is
that it is entirely enclosed in the little incubator so there was no
problem regarding stray light.

Sadly though, the researcher never got enough data to publish on our
system because of an entirely different limitation - the Nanoluc
substrate is very unstable under culture conditions (half life ~ 0.5 h)
so he would have needed to apply fresh substrate before every
acquisition, which would have been a bit of a pain since he was imaging
for several days at a time.  He said they got it to work elsewhere with
a microfluidic chamber, but that chamber wasn't compatible with the
VivaView system.

So - don't give up yet!  But it certainly is a challenge.
Best,
Alison



On 12/15/2014 1:37 PM, Sam Lord 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.
> *****
>
> On Mon, 15 Dec 2014 11:31:00 +0000, Guy Cox <[hidden email]>
> wrote:
>> Finally, people have been doing luciferin - luciferase imaging successfully long
> before EMCCD and SCMOS and the latest high-brightness probes.  So it can't be
> that hard.
>
> People have been imaging luciferin, but it is known to be very difficult to image on
> the single-cell level. From the papers I've read for single-cell luminescence
> imaging, they used intensified CCDs (cooled have very low background) in
> digital/photon-counting mode and integrate for tens of minutes.
>
> I tried luciferin imaging once for single cells. I gave up before I got it working.
>
> It's worth thinking about the rates. Fluorescence imaging involves approximately
> the same number of probes, but the excitation/fluorescence cycling rate is going to
> be WAY higher than the chemical turnover. And each turnover gives at most one
> photon. It's not surprising that luminescence signal is orders of magnitude dimmer
> than fluorescence.
>
> The real benefit to luminescence imaging is zero background. But to take
> advantage of that, you need to block absolutely all room light (and LED lights on
> the scope and other electronic equipment). Also, you need a way to integrate for
> minutes without filling up your entire CCD well depth with dark counts.
>
> Don't feel ashamed if you're having trouble. :)
>
> (That said, the suggestion from Guy Cox and others to confirm that light is able to
> reach the camera and that there is not a problem with the hardware is an excellent
> point!)

--
Alison J. North, Ph.D.,
Senior Director of the Bio-Imaging Resource Center and
Research Associate Professor,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab    ++ 212 327 7486
Fax:        ++ 212 327 7489