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Hi Yannis/everyone,
We can do reflectance on our Zeiss LSM 800. We use the 640nm laser and collect into Ch1. For reflection, the important thing is to keep the emission band very small so that you don't also collect fluorescence. We extend the emission range only to 641nm and usually narrow the pinhole even more than the usual 0.75 AU that I would use for reflection elsewhere, e.g. 0.5 or sometimes less. It's important to watch this and the laser power because of the sensitivity of the detectors- they will shut down in protest if you hit them with too much light and obviously that's not good.
We do quite a bit of reflection imaging on the LSM 710 which has the notch filter which can be removed (you still need to narrow the band) so when we got the LSM 800, we needed to be able to do it there so got advice from our local Zeiss experts (thanks Gavin/Richard) and this works.
I hope this will help with the LSM 700 as well. It does have to be the 640nm laser because it is at the end.
Kind regards,
Jacqui
________________________________
From: Confocal Microscopy List <[log in to unmask]> on behalf of Jeremy Adler <[log in to unmask]>
Sent: Friday, 28 May 2021 8:34 PM
To: [log in to unmask] <[log in to unmask]>
Subject: Re: confocal reflectance microscopy with Zeiss LSM700
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How reflectance works on your LSM700 depends on how it is set up.
In our 4 laser version the laser lines are blocked for 3 lasers but the 405 line is not blocked,
And therefore by default generates a combined reflection image plus fluorescence image - to the great confusion of many users.
An additional bandpass filter in the emission path, to remove the laser line, is required to produce a fluorescence only image
This is not helped by the Smart Setup, which mostly forgets that the 405 channel needs a bandpass filter.
Jeremy Adler
BioVis
Uppsala U
Sweden
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Douglas Richardson
Sent: Friday, May 28, 2021 2:42 AM
To: [log in to unmask]
Subject: Re: confocal reflectance microscopy with Zeiss LSM700
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The LSM 700 has a fixed laser blocking filter that reflects/blocks all laser light from reaching the detectors. With laser power and gains maxed out (and an empty filter position in front of the detector) you may be able to see some reflection, but you won't get the quality you're used to with the 15/85 beamsplitter. The LSM 710 and 780 had a 80/20 beam splitter for reflection imaging.
We recently purchased an LSM 900 that has 100% reflectance for the 488, 561 and 638 lines, but only reflects 20% of 405 light. This lets you do reflectance imaging with the 405 laser only. Maybe upgradable?
-Doug
On Thu, May 27, 2021 at 8:26 PM Craig Brideau <[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<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> Post images on http://www.imgur.com<http://www.imgur.com> and include the link in your posting.
> *****
>
> If the lasers are turning on (confirm this by looking at your sample
> and see if you observe cyan or red laser scatter) then there is
> probably a filter blocking the laser light from reaching the
> detectors. In fluorescence mode a confocal is very particular about
> ensuring that laser scatter does not interfere with fluorescent
> signal, so to perform reflectance confocal you have to override all these safeties!
>
> Craig
>
> On Thu, May 27, 2021 at 5:37 PM Zervantonakis, Ioannis <[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<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > Post images on http://www.imgur.com<http://www.imgur.com> and include the link in your
> posting.
> > *****
> >
> > Hello,
> >
> > we are trying to image collagen fibers in 2mg/ml gels (type I rat
> > tail)
> as
> > performed in Figure 2 here:
> > https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717242/<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717242>
> >
> > We have been able to do reflectance on a Leica TCSP8 with a 15/85
> > beamsplitter after collecting light reflectance from the 488nm
> excitation,
> > but don't get any signal in the Zeiss LSM 700 (we have tried both
> > the
> 640nm
> > laser and 488nm).
> >
> > Thank you,
> > Yannis
> >
>
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