*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****
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:[log in to unmask]] On Behalf Of Julio Vazquez
Sent: Thursday, 25 July 2013 7:55 AM
To: [log in to unmask]
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
*****
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)?
|
