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confocal reflectance microscopy with Zeiss LSM700

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Zervantonakis, Ioannis

confocal reflectance microscopy with Zeiss LSM700

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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/

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

Craig Brideau

Re: confocal reflectance microscopy with Zeiss LSM700

*****
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.
*****

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 <[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.
> *****
>
> 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/
>
> 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
>

Douglas Richardson

Re: confocal reflectance microscopy with Zeiss LSM700

*****
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.
*****

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 <[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.
> *****
>
> 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 <[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.
> > *****
> >
> > 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/
> >
> > 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
> >
>

mmodel

Re: EXT: Re: confocal reflectance microscopy with Zeiss LSM700

*****
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 never had LSM700 but I wonder if it might be possible to trick it by scanning with two lasers simultaneously... At least one of them must get past the filter. (Apologies for talking without knowing)

Mike

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Douglas Richardson
Sent: Thursday, May 27, 2021 8:42 PM
To: [hidden email]
Subject: EXT: Re: confocal reflectance microscopy with Zeiss LSM700

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2BOC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0 and include the link in your posting.
*****

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 <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists
> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cm
> model%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d01
> 8f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8
> eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3
> 000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY4%3D&res
> erved=0 Post images on
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2BOC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0 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 <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flis
> > ts.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01
> > %7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1e
> > c44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFp
> > bGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
> > Mn0%3D%7C3000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY
> > 4%3D&reserved=0 Post images on
> > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww
> > .imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401
> > e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63757
> > 7594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2
> > luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2B
> > OC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fww
> > w.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2717242%2F&data=04%7C01
> > %7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1e
> > c44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFp
> > bGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
> > Mn0%3D%7C3000&sdata=TPhNB6pv3OCwFwLWMxtwGDgDoho8%2FcqOJg%2FV6rXV
> > w6o%3D&reserved=0
> >
> > 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
> >
>

CAUTION: EXTERNAL SENDER Do not click any links, open any attachments, or REPLY to the message unless you trust the sender and know the content is safe.

Douglas Richardson

Re: EXT: Re: confocal reflectance microscopy with Zeiss LSM700

*****
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.
*****

Unfortunately not. It's a single fixed mirror that blocks all 4 laser lines.

Component #4 in diagram on Page 4 here:
https://bcf.technion.ac.il/wp-content/uploads/2017/12/Instruction-LSM700-2017-12-07.pdf

-Doug

On Thu, May 27, 2021 at 8:57 PM Model, Michael <[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 never had LSM700 but I wonder if it might be possible to trick it by
> scanning with two lasers simultaneously... At least one of them must get
> past the filter. (Apologies for talking without knowing)
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Douglas Richardson
> Sent: Thursday, May 27, 2021 8:42 PM
> To: [hidden email]
> Subject: EXT: Re: confocal reflectance microscopy with Zeiss LSM700
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY4%3D&reserved=0
> Post images on
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2BOC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0
> and include the link in your posting.
> *****
>
> 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 <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists
> > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cm
> > model%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d01
> > 8f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8
> > eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3
> > 000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY4%3D&res
> > erved=0 Post images on
> >
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2BOC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0
> 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 <[hidden email]>
> > wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flis
> > > ts.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01
> > > %7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1e
> > > c44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFp
> > > bGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
> > > Mn0%3D%7C3000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY
> > > 4%3D&reserved=0 Post images on
> > > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww
> > > .imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401
> > > e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63757
> > > 7594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2
> > > luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2B
> > > OC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fww
> > > w.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2717242%2F&data=04%7C01
> > > %7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1e
> > > c44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFp
> > > bGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
> > > Mn0%3D%7C3000&sdata=TPhNB6pv3OCwFwLWMxtwGDgDoho8%2FcqOJg%2FV6rXV
> > > w6o%3D&reserved=0
> > >
> > > 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
> > >
> >
> CAUTION: EXTERNAL SENDER Do not click any links, open any attachments, or
> REPLY to the message unless you trust the sender and know the content is
> safe.
>

Douglas Richardson

Re: EXT: Re: confocal reflectance microscopy with Zeiss LSM700

> Unfortunately not. It's a single fixed mirror that blocks all 4 laser
> lines.
>
> Component #4 in diagram on Page 4 here:
> https://bcf.technion.ac.il/wp-content/uploads/2017/12/Instruction-LSM700-2017-12-07.pdf
>
>
> -Doug
>
> On Thu, May 27, 2021 at 8:57 PM Model, Michael <[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 never had LSM700 but I wonder if it might be possible to trick it by
>> scanning with two lasers simultaneously... At least one of them must get
>> past the filter. (Apologies for talking without knowing)
>>
>> Mike
>>
>> -----Original Message-----
>> From: Confocal Microscopy List <[hidden email]> On
>> Behalf Of Douglas Richardson
>> Sent: Thursday, May 27, 2021 8:42 PM
>> To: [hidden email]
>> Subject: EXT: Re: confocal reflectance microscopy with Zeiss LSM700
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY4%3D&reserved=0
>> Post images on
>> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2BOC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0
>> and include the link in your posting.
>> *****
>>
>> 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 <[hidden email]>
>> wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists
>> > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cm
>> > model%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d01
>> > 8f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8
>> > eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3
>> > 000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY4%3D&res
>> > erved=0 Post images on
>> >
>> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2BOC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0
>> 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 <[hidden email]>
>> > wrote:
>> >
>> > > *****
>> > > To join, leave or search the confocal microscopy listserv, go to:
>> > > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flis
>> > > ts.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01
>> > > %7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1e
>> > > c44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFp
>> > > bGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
>> > > Mn0%3D%7C3000&sdata=3SpQo%2FLufyqh2WU3u7haFgLtgeT4kY5MXh6Px1XmfY
>> > > 4%3D&reserved=0 Post images on
>> > > https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww
>> > > .imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7C49473550fec1401
>> > > e87bb08d921718851%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63757
>> > > 7594030733559%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2
>> > > luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=uUREpBI7nba%2B
>> > > OC7Cs45RWROXbYaMDjCea9KBWZfm4zM%3D&reserved=0 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fww
>> > > w.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2717242%2F&data=04%7C01
>> > > %7Cmmodel%40KENT.EDU%7C49473550fec1401e87bb08d921718851%7Ce5a06f4a1e
>> > > c44d018f73e7dd15f26134%7C1%7C0%7C637577594030733559%7CUnknown%7CTWFp
>> > > bGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6
>> > > Mn0%3D%7C3000&sdata=TPhNB6pv3OCwFwLWMxtwGDgDoho8%2FcqOJg%2FV6rXV
>> > > w6o%3D&reserved=0
>> > >
>> > > 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
>> > >
>> >
>> CAUTION: EXTERNAL SENDER Do not click any links, open any attachments, or
>> REPLY to the message unless you trust the sender and know the content is
>> safe.
>>
>

Cammer, Michael-2

Re: EXT: Re: confocal reflectance microscopy with Zeiss LSM700

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The Zeiss 700 and 800 confocals cannot do reflectance. There is no user option to trick this. We have a few of these scopes and have tried.

The 710, 780, 880 can. We do it routinely on the 710 and 880.

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016

[hidden email]<mailto:[hidden email]> http://nyulmc.org/micros http://microscopynotes.com/

Voice direct only, no text or messages: 1-914-309-3270 and 1-646-501-0567

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Douglas Richardson <[hidden email]>
Sent: Thursday, May 27, 2021 9:06:32 PM
To: [hidden email]
Subject: Re: EXT: Re: confocal reflectance microscopy with Zeiss LSM700

[EXTERNAL]

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Sorry, page 3.

On Thu, May 27, 2021 at 9:05 PM Douglas Richardson <[hidden email]>
wrote:

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Benjamin Smith

Re: confocal reflectance microscopy with Zeiss LSM700

In reply to this post by Douglas Richardson

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If I remember correctly, I think the pin filter Doug is referring to can be
removed from the path on the 880 (it is a check box option) so maybe there
is a similar feature for the 700? I would be shocked if it was impossible
to remove the filter, as reflected confocal microscopy is a pretty standard
imaging modality.

On Thu, May 27, 2021 at 5:43 PM Douglas Richardson <[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.
> *****
>
> 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 <[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.
> > *****
> >
> > 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 <[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.
> > > *****
> > >
> > > 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/
> > >
> > > 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
> > >
> >
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Weill Hall
Berkeley, CA 94720-3200
Tel (510) 642-9712
Fax (510) 643-6791
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

Douglas Richardson

Re: confocal reflectance microscopy with Zeiss LSM700

*****
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No, I was not referring to the pin filter. The pin filter only exists on
the 780, 880, 980 models with a meta detector. The 700 does not use pins.
Just a plain old filter that blocks 4 wavelengths of light: 405, 488, 555
and 638 (+/- a few nm each). There are actually two of them in the system.
One is in the excitation path to combine the lasers and one in the emission
path to block reflected laser light.

On Thu, May 27, 2021 at 10:04 PM Benjamin Smith <[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.
> *****
>
> If I remember correctly, I think the pin filter Doug is referring to can be
> removed from the path on the 880 (it is a check box option) so maybe there
> is a similar feature for the 700? I would be shocked if it was impossible
> to remove the filter, as reflected confocal microscopy is a pretty standard
> imaging modality.
>
> On Thu, May 27, 2021 at 5:43 PM Douglas Richardson <
> [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.
> > *****
> >
> > 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 <[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.
> > > *****
> > >
> > > 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 <[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.
> > > > *****
> > > >
> > > > 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/
> > > >
> > > > 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
> > > >
> > >
> >
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Weill Hall
> Berkeley, CA 94720-3200
> Tel (510) 642-9712
> Fax (510) 643-6791
> e-mail: [hidden email]
>
> https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/
>

Jeremy Adler-4

Re: confocal reflectance microscopy with Zeiss LSM700

In reply to this post by Douglas Richardson

*****
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.
*****

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 <[hidden email]> On Behalf Of Douglas Richardson
Sent: Friday, May 28, 2021 2:42 AM
To: [hidden email]
Subject: Re: confocal reflectance microscopy with Zeiss LSM700

*****
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.
*****

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 <[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.
> *****
>
> 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 <[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.
> > *****
> >
> > 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/
> >
> > 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
> >
>

När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/

E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy

Jacqueline Ross

Re: confocal reflectance microscopy with Zeiss LSM700

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

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 <[hidden email]> on behalf of Jeremy Adler <[hidden email]>
Sent: Friday, 28 May 2021 8:34 PM
To: [hidden email] <[hidden email]>
Subject: Re: confocal reflectance microscopy with Zeiss LSM700

*****
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.
*****

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 <[hidden email]> On Behalf Of Douglas Richardson
Sent: Friday, May 28, 2021 2:42 AM
To: [hidden email]
Subject: Re: confocal reflectance microscopy with Zeiss LSM700

*****
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.
*****

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 <[hidden email]>
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 <[hidden email]>
> 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
> >
>

När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/<http://www.uu.se/om-uu/dataskydd-personuppgifter>

E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy<http://www.uu.se/en/about-uu/data-protection-policy>

Jacqueline Ross

Re: confocal reflectance microscopy with Zeiss LSM700

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi all,

I forgot to say that the reason this works for the LSM 800 is because the beam splitter for the 640nm is a T80/R20. Also I just checked the original email I had received and you can extend the range a bit further depending in how far it goes but I wouldn't go too much more myself because of the risk of getting fluorecence even if there isn't a fluorophore in that range. We sometimes do get a bit of autofluorescence there, e.g. plants but it's not common.

Cheers,

Jacqui

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Jacqueline Ross <[hidden email]>
Sent: Saturday, 29 May 2021 7:22 AM
To: [hidden email] <[hidden email]>
Subject: Re: confocal reflectance microscopy with Zeiss LSM700

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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 <[hidden email]> on behalf of Jeremy Adler <[hidden email]>
Sent: Friday, 28 May 2021 8:34 PM
To: [hidden email] <[hidden email]>
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 <[hidden email]> On Behalf Of Douglas Richardson
Sent: Friday, May 28, 2021 2:42 AM
To: [hidden email]
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 <[hidden email]>
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><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><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 <[hidden email]>
> 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><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><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><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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