Increased background near coverslip w/2-photon excitation

[Jen Jackson]

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Hello,

I've been having trouble imaging near the bottom membrane of cells due to a
near doubling of background when focused near the coverslip.  Moving the
objective up a few microns into the cell results in a very noticeable SNR
improvement (but is not the imaging plane I need).

Same deal with beads and control samples:
Increased background is observed in a variety of imaging medias (glycerol,
cell media, di water) & coverslip types (no. 1.5: glass ones & so called
"optical quality plastic").  

We are using a 100x nikon plan apo 1.4, w/ anti-reflection coating for
~400-1100nm; exciting samples with a pulsed source in the 790-850nm range.

Is this a common issue in 2-photon microscopy (or single photon, for that
matter)?

[Cameron Nowell-3]

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Hi Jen,

Is it possible you are collecting reflection of your excitation light? What wavelength are you collecting signal at? Is your pulsed source tunable? If so does the issue go away at different wavelengths?


Cheers

Cam


Cameron J. Nowell
Research Facilities Manager

Monash Institute of Pharmaceutical Sciences
Monash University
399 Royal Parade
(Mail address: 381 Royal Parade)
Parkville, VIC, 3052
Australia

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jen Jackson
Sent: Wednesday, 24 July 2013 5:58 AM
To: [hidden email]
Subject: Increased background near coverslip w/2-photon excitation

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Hello,

I've been having trouble imaging near the bottom membrane of cells due to a near doubling of background when focused near the coverslip.  Moving the objective up a few microns into the cell results in a very noticeable SNR improvement (but is not the imaging plane I need).

Same deal with beads and control samples:
Increased background is observed in a variety of imaging medias (glycerol, cell media, di water) & coverslip types (no. 1.5: glass ones & so called "optical quality plastic").  

We are using a 100x nikon plan apo 1.4, w/ anti-reflection coating for ~400-1100nm; exciting samples with a pulsed source in the 790-850nm range.

Is this a common issue in 2-photon microscopy (or single photon, for that matter)?  

[Jen Jackson]

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Hello,

Thanks for your response,

Our filter does a good job of eliminating excitation light above 650nm.  A
good amount of time was spent making sure of that- but if this coverslip
background is not a common problem then the filter may yet be suspect.
However when exciting at 790 vs 850nm, I still get similar background
issues, again, only when focused near the coverslip.

We collect fluorescence emission from ~400 to 600nm, spectrally resolved via
difraction grating + EMCCD.  (The "spectrum" of this background is rather
broad and boring, by the way).  

Any input welcome!  

Thank you,

-Jen

[Jeff Reece]

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Reflection can be more an issue with 2PE than with single photon excitation.
Can you add an additional emission filter that provides further blocking of the laser wavelengths?


Kind Regards,
Jeff

>________________________________
> From: Jen Jackson <[hidden email]>
>To: [hidden email]
>Sent: Tuesday, July 23, 2013 8:34 PM
>Subject: Re: Increased background near coverslip w/2-photon excitation
>
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello,
>
>Thanks for your response,
>
>Our filter does a good job of eliminating excitation light above 650nm.  A
>good amount of time was spent making sure of that- but if this coverslip
>background is not a common problem then the filter may yet be suspect.
>However when exciting at 790 vs 850nm, I still get similar background
>issues, again, only when focused near the coverslip.
>
>We collect fluorescence emission from ~400 to 600nm, spectrally resolved via
>difraction grating + EMCCD.  (The "spectrum" of this background is rather
>broad and boring, by the way). 
>
>Any input welcome! 
>
>Thank you,
>
>-Jen
>
>

[Julio Vazquez]

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Hi Jen,

Just a few thoughts (with a warning that I am not a physicist).When light hits an interface between two optical media, there will always be a certain amount of reflection, which is why you always see a reflection on a window if the background is darker than the reflected scene. I think between glass and air, you always get at least 3% reflection, if I remember my Feinman lectures correctly. This property is used in laser based autofocus systems, which track the reflection of a laser off the coverslip surface. If the angle of incidence is above a critical value, you get 100% reflection (total internal reflection). With a 1.4 NA objective, you are not very far from this angle, and you would expect a good amount of reflection. We often observe this reflection in all modes of microscopy, and the more so as the true signal gets dimmer. I am guessing that either your two photon laser, in addition to the tuned wavelength, is also emitting a small amount of other wavelengths. I don't know enough about the physics of such lasers to know if this is to be expected. Alternatively, if you are using a camera for imaging, as you mention, you may just be getting background from the infra-red laser, although this seems less likely since you are using a diffraction grating to separate a defined spectral band. Just guesses. Adding filters as has been suggested is probably the solution. The problem with two photon is that you will typically be working at very high power levels (we tend to need in the order of 5 mW, which is 500 times more power than the  ~ 10 micro-Watts we typically use for confocal imaging. At such power levels, even a 0.1 % reflection will be orders of magnitude greater than your signal, and you need very good filters to block those stray signals. If the reflection is from minor spectral lines present in your two photon beam, then I wouldn't know what to do about it, other than checking with the vendor to see if they are within specification. If what you are seeing is a reflection of the laser, then your background signal near the coverslip should very closely follow changes in laser power.

Out of curiosity, why are you using two photon for your application? If you are imaging cells on a coverslip, other types of microscopy might seem more appropriate (unless you specifically need two photon, that is...)

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109

http://www.fhcrc.org/en.html

==



On Jul 23, 2013, at 12:57 PM, Jen Jackson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I've been having trouble imaging near the bottom membrane of cells due to a
> near doubling of background when focused near the coverslip.  Moving the
> objective up a few microns into the cell results in a very noticeable SNR
> improvement (but is not the imaging plane I need).
>
> Same deal with beads and control samples:
> Increased background is observed in a variety of imaging medias (glycerol,
> cell media, di water) & coverslip types (no. 1.5: glass ones & so called
> "optical quality plastic").  
>
> We are using a 100x nikon plan apo 1.4, w/ anti-reflection coating for
> ~400-1100nm; exciting samples with a pulsed source in the 790-850nm range.
>
> Is this a common issue in 2-photon microscopy (or single photon, for that
> matter)?

[Guy Cox-2]

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One possibility nobody seems to have considered yet is second harmonic generation at the interface.  If I recall correctly quite wide-band detection was being used.  

                                            Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Thursday, 25 July 2013 7:55 AM
To: [hidden email]
Subject: Re: Increased background near coverslip w/2-photon excitation

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Hi Jen,

Just a few thoughts (with a warning that I am not a physicist).When light hits an interface between two optical media, there will always be a certain amount of reflection, which is why you always see a reflection on a window if the background is darker than the reflected scene. I think between glass and air, you always get at least 3% reflection, if I remember my Feinman lectures correctly. This property is used in laser based autofocus systems, which track the reflection of a laser off the coverslip surface. If the angle of incidence is above a critical value, you get 100% reflection (total internal reflection). With a 1.4 NA objective, you are not very far from this angle, and you would expect a good amount of reflection. We often observe this reflection in all modes of microscopy, and the more so as the true signal gets dimmer. I am guessing that either your two photon laser, in addition to the tuned wavelength, is also emitting a small amount of other wavelengths. I don't know enough about the physics of such lasers to know if this is to be expected. Alternatively, if you are using a camera for imaging, as you mention, you may just be getting background from the infra-red laser, although this seems less likely since you are using a diffraction grating to separate a defined spectral band. Just guesses. Adding filters as has been suggested is probably the solution. The problem with two photon is that you will typically be working at very high power levels (we tend to need in the order of 5 mW, which is 500 times more power than the  ~ 10 micro-Watts we typically use for confocal imaging. At such power levels, even a 0.1 % reflection will be orders of magnitude greater than your signal, and you need very good filters to block those stray signals. If the reflection is from minor spectral lines present in your two photon beam, then I wouldn't know what to do about it, other than checking with the vendor to see if they are within specification. If what you are seeing is a reflection of the laser, then your background signal near the coverslip should very closely follow changes in laser power.

Out of curiosity, why are you using two photon for your application? If you are imaging cells on a coverslip, other types of microscopy might seem more appropriate (unless you specifically need two photon, that is...)

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109

http://www.fhcrc.org/en.html

==



On Jul 23, 2013, at 12:57 PM, Jen Jackson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I've been having trouble imaging near the bottom membrane of cells due
> to a near doubling of background when focused near the coverslip.  
> Moving the objective up a few microns into the cell results in a very
> noticeable SNR improvement (but is not the imaging plane I need).
>
> Same deal with beads and control samples:
> Increased background is observed in a variety of imaging medias
> (glycerol, cell media, di water) & coverslip types (no. 1.5: glass
> ones & so called "optical quality plastic").
>
> We are using a 100x nikon plan apo 1.4, w/ anti-reflection coating for
> ~400-1100nm; exciting samples with a pulsed source in the 790-850nm range.
>
> Is this a common issue in 2-photon microscopy (or single photon, for
> that matter)?

[Jen Jackson]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

To add a twist-
I've recently observed what looks like bleaching of this background- whilst
doing repeated scans to small regions of a non-coated dish filled with di
water, focused near the water/glass interface.  It's not a dramatic decrease
in signal level but, it has crisp & well defined edges as I'd expect when
FRAPping a fluorescent thing, as well as being able to be defocused and
magnified w/an optovar.  Granted, I used rather high power, longish dwell
times.. (heating could be an issue?)  No "recovery" though- 10 minutes later
the bleached pattern was still intact. Quite puzzling...


To respond to a few others:
Our detection covers a wide band but spectrally resolved to within a few nm-
the spectrum of this background is very broad so SHG is not likely (also our
coverslips aren't coated, typically).

Reflection is an issue however it usually presents on our camera as an
overall increase in background over the entire camera array, as opposed to a
broad spectrum as seen in this case- the while the spectrum is wide (similar
to some autofluorescence), it is confined quite well to the subregion on the
camera we use for spectral imaging (and has something of a peak around
510nm, atleast when exciting plain glass coverslips + water @ 840nm).


...any further thoughts?  Thanks,

jen