Two-photon microscope questions

[Heping Yuan]

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Hi everyone, I was looking to modify an old confocal system into a two-photon system. I
had two questions and would appreciate any help.

1) A section of the schematic of typical two-photon is as follows
(http://www.thorlabs.com/tutorials.cfm?tabID=32729):

Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective

where fluorescence emission is reflected off Dichroic towards a collection lens and PMT's

I'm wondering if there are some unforeseen problems in changing the order as follows:

Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective

where the tube lens can focus the fluorescence emission back to the Dichroic and directly
into the PMT's (without a collection lens).

2) What is a common procedure to image the back aperture of the objective to the input
window of the PMT? My first thought is to create a collimated source with size > than
back aperture and shine directly into the back aperture with objective off (to see spot size
at PMT input). Would this be correct? If so, practically speaking what type of source is
typically used?

Thanks,
Heping

[Gary G. Li-2]

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Hi Heping,

I recently built a multiphoton laser scanning microscope, which works
perfectly.

Please contact me, and I would be happy to discuss with you about your
technical issues.

Best regards

Gary G Li, PhD

On Sat, Nov 29, 2014 at 11:23 AM, Heping Yuan <[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.
> *****
>
> Hi everyone, I was looking to modify an old confocal system into a
> two-photon system. I
> had two questions and would appreciate any help.
>
> 1) A section of the schematic of typical two-photon is as follows
> (http://www.thorlabs.com/tutorials.cfm?tabID=32729):
>
> Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective
>
> where fluorescence emission is reflected off Dichroic towards a collection
> lens and PMT's
>
> I'm wondering if there are some unforeseen problems in changing the order
> as follows:
>
> Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective
>
> where the tube lens can focus the fluorescence emission back to the
> Dichroic and directly
> into the PMT's (without a collection lens).
>
> 2) What is a common procedure to image the back aperture of the objective
> to the input
> window of the PMT? My first thought is to create a collimated source with
> size > than
> back aperture and shine directly into the back aperture with objective off
> (to see spot size
> at PMT input). Would this be correct? If so, practically speaking what
> type of source is
> typically used?
>
> Thanks,
> Heping
>

[Mark Cannell-2]

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Hi

The only problem I foresee will be the varying angle to the dichroic which will change its cutoff over the scan area. The effect may be small and you may get away with it.

HTH

Mark
On 29/11/2014, at 4:23 pm, Heping Yuan <[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.
> *****
>
> Hi everyone, I was looking to modify an old confocal system into a two-photon system. I
> had two questions and would appreciate any help.
>
> 1) A section of the schematic of typical two-photon is as follows
> (http://www.thorlabs.com/tutorials.cfm?tabID=32729):
>
> Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective
>
> where fluorescence emission is reflected off Dichroic towards a collection lens and PMT's
>
> I'm wondering if there are some unforeseen problems in changing the order as follows:
>
> Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective
>
> where the tube lens can focus the fluorescence emission back to the Dichroic and directly
> into the PMT's (without a collection lens).
>
> 2) What is a common procedure to image the back aperture of the objective to the input
> window of the PMT? My first thought is to create a collimated source with size > than
> back aperture and shine directly into the back aperture with objective off (to see spot size
> at PMT input). Would this be correct? If so, practically speaking what type of source is
> typically used?
>
> Thanks,
> Heping

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Andrew York]

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 Short answer to (1) is, astigmatism.

 Transmission through a flat, tilted piece of glass gives only a lateral
deflection to a collimated beam, but it gives astigmatism to a focusing
beam. You could imagine using a dichroic that reflects the excitation
rather than transmitting it, but be careful about flatness (most dichroics
are quite curved, sadly) which can also give astigmatism.

 Because of some bad decisions, I ended up using a dichroic in the same
manner you describe. You can cancel a lot of the resulting astigmatism with
a second piece of glass tilted the same amount but rotated 90 degrees about
the optic axis. On the other hand, why bother solving a problem you don't
have to have?

On Sat, Nov 29, 2014 at 11:23 AM, Heping Yuan <[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.
> *****
>
> Hi everyone, I was looking to modify an old confocal system into a
> two-photon system. I
> had two questions and would appreciate any help.
>
> 1) A section of the schematic of typical two-photon is as follows
> (http://www.thorlabs.com/tutorials.cfm?tabID=32729):
>
> Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective
>
> where fluorescence emission is reflected off Dichroic towards a collection
> lens and PMT's
>
> I'm wondering if there are some unforeseen problems in changing the order
> as follows:
>
> Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective
>
> where the tube lens can focus the fluorescence emission back to the
> Dichroic and directly
> into the PMT's (without a collection lens).
>
> 2) What is a common procedure to image the back aperture of the objective
> to the input
> window of the PMT? My first thought is to create a collimated source with
> size > than
> back aperture and shine directly into the back aperture with objective off
> (to see spot size
> at PMT input). Would this be correct? If so, practically speaking what
> type of source is
> typically used?
>
> Thanks,
> Heping
>

[Michael Giacomelli]

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*****

The main problem with changing the order will be a loss of collection NA,
particularly for larger fields of view. If you take apart the thorlabs
optics you will see that they have placed the dichroic nearly touching the
back of the objective. This maximizes light collection.

This may or may not matter depending on how deep you intend to image into
samples, the field of view you want to scan, and the effective focal length
of your objective. I would measure the displacement you intend to add
between the dichroic and objective and compute the approximate collection
NA and FOV that will give you.

Mike

On Sat, Nov 29, 2014, 12:33 PM Andrew York <
[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.
> *****
>
>  Short answer to (1) is, astigmatism.
>
>  Transmission through a flat, tilted piece of glass gives only a lateral
> deflection to a collimated beam, but it gives astigmatism to a focusing
> beam. You could imagine using a dichroic that reflects the excitation
> rather than transmitting it, but be careful about flatness (most dichroics
> are quite curved, sadly) which can also give astigmatism.
>
>  Because of some bad decisions, I ended up using a dichroic in the same
> manner you describe. You can cancel a lot of the resulting astigmatism with
> a second piece of glass tilted the same amount but rotated 90 degrees about
> the optic axis. On the other hand, why bother solving a problem you don't
> have to have?
>
> On Sat, Nov 29, 2014 at 11:23 AM, Heping Yuan <[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.
> > *****
> >
> > Hi everyone, I was looking to modify an old confocal system into a
> > two-photon system. I
> > had two questions and would appreciate any help.
> >
> > 1) A section of the schematic of typical two-photon is as follows
> > (http://www.thorlabs.com/tutorials.cfm?tabID=32729):
> >
> > Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective
> >
> > where fluorescence emission is reflected off Dichroic towards a
> collection
> > lens and PMT's
> >
> > I'm wondering if there are some unforeseen problems in changing the order
> > as follows:
> >
> > Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective
> >
> > where the tube lens can focus the fluorescence emission back to the
> > Dichroic and directly
> > into the PMT's (without a collection lens).
> >
> > 2) What is a common procedure to image the back aperture of the objective
> > to the input
> > window of the PMT? My first thought is to create a collimated source with
> > size > than
> > back aperture and shine directly into the back aperture with objective
> off
> > (to see spot size
> > at PMT input). Would this be correct? If so, practically speaking what
> > type of source is
> > typically used?
> >
> > Thanks,
> > Heping
> >
>

[James Pawley]

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*****

Dear Hepping,

I think that one needs to remember that the source of "non-descanned"
signal light is not a point. It is sort of a 3D blob caused by light
that emerged from a point but then scattered off refractive features
in the specimen. As a result, strictly speaking, it cannot be
"focused" onto any remote detector.

What seems to work best is locating the sensitive element of the
detector as close as possible to the back of the objective. It would
seem to me that if you are planning to use this 2-photon system to
look at thick specimens (brain, embryoes, etc) then mounting the tube
lens between the dichroic and the objective might mean you losing
some signal you could otherwise have recorded.

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.
>*****
>
>  Short answer to (1) is, astigmatism.
>
>  Transmission through a flat, tilted piece of glass gives only a lateral
>deflection to a collimated beam, but it gives astigmatism to a focusing
>beam. You could imagine using a dichroic that reflects the excitation
>rather than transmitting it, but be careful about flatness (most dichroics
>are quite curved, sadly) which can also give astigmatism.
>
>  Because of some bad decisions, I ended up using a dichroic in the same
>manner you describe. You can cancel a lot of the resulting astigmatism with
>a second piece of glass tilted the same amount but rotated 90 degrees about
>the optic axis. On the other hand, why bother solving a problem you don't
>have to have?
>
>On Sat, Nov 29, 2014 at 11:23 AM, Heping Yuan <[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.
>>  *****
>>
>>  Hi everyone, I was looking to modify an old confocal system into a
>>  two-photon system. I
>>  had two questions and would appreciate any help.
>>
>>  1) A section of the schematic of typical two-photon is as follows
>>  (http://www.thorlabs.com/tutorials.cfm?tabID=32729):
>>
>>  Galvos -> Scan Lens -> Tube Lens -> Dichroic (690 LP) -> Objective
>>
>>  where fluorescence emission is reflected off Dichroic towards a collection
>>  lens and PMT's
>>
>>  I'm wondering if there are some unforeseen problems in changing the order
>>  as follows:
>>
>>  Galvos -> Scan Lens -> Dichroic (690 LP)  -> Tube Lens -> Objective
>>
>>  where the tube lens can focus the fluorescence emission back to the
>>  Dichroic and directly
>>  into the PMT's (without a collection lens).
>>
>>  2) What is a common procedure to image the back aperture of the objective
>>  to the input
>>  window of the PMT? My first thought is to create a collimated source with
>>  size > than
>>  back aperture and shine directly into the back aperture with objective off
>>  (to see spot size
>>  at PMT input). Would this be correct? If so, practically speaking what
>>  type of source is
>>  typically used?
>>
>>  Thanks,
>>  Heping
>>

--
               ****************************************
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

[Peter Rupprecht]

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*****

Dear Heping,

To your 2nd question:
>> 2) What is a common procedure to image the back aperture of the objective to the input window of the PMT?
>> My first thought is to create a collimated source with size > than
>> back aperture and shine directly into the back aperture with objective off (to see spot size
>> at PMT input). Would this be correct?

This sounds difficult to me. The detector size is usually so big that it would be difficult to find the optimal alignment by this method.

For a quick alignment, I would, if the excitation light path is perfectly aligned, put a homogeneous plastic fluoslide under the objective and align the detection pathway such that the picture you get looks as good as possible. Do this with the lowest zoom setting (highest scanning angle), otherwise it will always look perfect. With "good", I mean a FOV with the highest brightness in its center. Simply play around with mirror angles while imaging and check the picture live. Usually, due to aperture clipping effects while scanning, the FOV is not totally homogeneous. This also depends on the highest zoom factor allowed by your scanners or your software. -- If someone has a better idea, I'd be happy to learn them!

As already mentioned, I would also vote for putting the dichroic as close to the objective as possible.


As a sidenote, I have always been wondering why some 2P-microscopes I've seen use such long detection pathways (>15 cm) for 2P microscopy, with all the tubes and inner parts being completely black. Wouldn't it be much better to coat the full detection pathway surfaces with an aluminium or silver coating in order to trap every scattered photon until it impinges on the detector? Has anyone tried this out and compared it to non-coated surfaces for highly scattering conditions?
Best,
Peter

[Michael Giacomelli]

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Hi Peter,

With good optical design you can collect nearly the full radiance of the
objective back aperture (that is, the only place where light will hit the
sides is inside the objective).  There should be negligible signal hitting
the lens tubes, but perhaps a bit of light leakage from outside the
microscope if any part is not fully light tight.  I think this is the main
concern, although in practice I'm not sure how much it really matters.

Mike

On Sat, Nov 29, 2014 at 6:07 PM, Peter Rupprecht <[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.
> *****
>
> Dear Heping,
>
> To your 2nd question:
> >> 2) What is a common procedure to image the back aperture of the
> objective to the input window of the PMT?
> >> My first thought is to create a collimated source with size > than
> >> back aperture and shine directly into the back aperture with objective
> off (to see spot size
> >> at PMT input). Would this be correct?
>
> This sounds difficult to me. The detector size is usually so big that it
> would be difficult to find the optimal alignment by this method.
>
> For a quick alignment, I would, if the excitation light path is perfectly
> aligned, put a homogeneous plastic fluoslide under the objective and align
> the detection pathway such that the picture you get looks as good as
> possible. Do this with the lowest zoom setting (highest scanning angle),
> otherwise it will always look perfect. With "good", I mean a FOV with the
> highest brightness in its center. Simply play around with mirror angles
> while imaging and check the picture live. Usually, due to aperture clipping
> effects while scanning, the FOV is not totally homogeneous. This also
> depends on the highest zoom factor allowed by your scanners or your
> software. -- If someone has a better idea, I'd be happy to learn them!
>
> As already mentioned, I would also vote for putting the dichroic as close
> to the objective as possible.
>
>
> As a sidenote, I have always been wondering why some 2P-microscopes I've
> seen use such long detection pathways (>15 cm) for 2P microscopy, with all
> the tubes and inner parts being completely black. Wouldn't it be much
> better to coat the full detection pathway surfaces with an aluminium or
> silver coating in order to trap every scattered photon until it impinges on
> the detector? Has anyone tried this out and compared it to non-coated
> surfaces for highly scattering conditions?
> Best,
> Peter
>

[Zdenek Svindrych]

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*****

Yes,
the BFP of the objective should be the only aperture that limits your
optical system. By properly imaging this onto the detector you don't loose
any useful light. It is convenient to use black tubes, as any light leaking
into the system will degrade your image considerably.

With a high magnification objective (say 60x 1.4NA) the rays between the
objective and the tube lens are usually well collimated, so you don't need
to be concerned with 'long' relay lenses between the objective and the non-
descanned detector (this is not entirely true for scattered two-photon
fluorescence, you need to perform some sort of simulation or calculation to
get an idea about the diameter/focal length of your relay lenses necessary
to collect out-of-focus light) .

Best, Zdenek, www.kcci.virginia.edu



---------- Původní zpráva ----------
Od: Michael Giacomelli <[hidden email]>
Komu: [hidden email]
Datum: 29. 11. 2014 18:27:33
Předmět: Re: Two-photon microscope questions

"*****
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*****

Hi Peter,

With good optical design you can collect nearly the full radiance of the
objective back aperture (that is, the only place where light will hit the
sides is inside the objective). There should be negligible signal hitting
the lens tubes, but perhaps a bit of light leakage from outside the
microscope if any part is not fully light tight. I think this is the main
concern, although in practice I'm not sure how much it really matters.

Mike

On Sat, Nov 29, 2014 at 6:07 PM, Peter Rupprecht <[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.
> *****
>
> Dear Heping,
>
> To your 2nd question:
> >> 2) What is a common procedure to image the back aperture of the
> objective to the input window of the PMT?
> >> My first thought is to create a collimated source with size > than
> >> back aperture and shine directly into the back aperture with objective
> off (to see spot size
> >> at PMT input). Would this be correct?
>
> This sounds difficult to me. The detector size is usually so big that it
> would be difficult to find the optimal alignment by this method.
>
> For a quick alignment, I would, if the excitation light path is perfectly
> aligned, put a homogeneous plastic fluoslide under the objective and align
> the detection pathway such that the picture you get looks as good as
> possible. Do this with the lowest zoom setting (highest scanning angle),
> otherwise it will always look perfect. With "good", I mean a FOV with the
> highest brightness in its center. Simply play around with mirror angles
> while imaging and check the picture live. Usually, due to aperture

clipping

> effects while scanning, the FOV is not totally homogeneous. This also
> depends on the highest zoom factor allowed by your scanners or your
> software. -- If someone has a better idea, I'd be happy to learn them!
>
> As already mentioned, I would also vote for putting the dichroic as close
> to the objective as possible.
>
>
> As a sidenote, I have always been wondering why some 2P-microscopes I've
> seen use such long detection pathways (>15 cm) for 2P microscopy, with all
> the tubes and inner parts being completely black. Wouldn't it be much
> better to coat the full detection pathway surfaces with an aluminium or
> silver coating in order to trap every scattered photon until it impinges

on
> the detector? Has anyone tried this out and compared it to non-coated
> surfaces for highly scattering conditions?
> Best,
> Peter
>"

[Tobias Rose]

Just in case this has not been posted yet:

P.S. Tsai, and D. Kleinfeld. In vivo two-photon laser scanning microscopy with concurrent plasma-mediated ablation: Principles and hardware realization in Methods for In Vivo Optical Imaging, Second Edition, R. D. Frostig, editor, CRC Press, 3:59-115 (2009)

http://www.ncbi.nlm.nih.gov/books/NBK20231/

This covers pretty much all questions concerning DIY 2p building.

Best,
Tobias

--  
Tobias Rose, PhD
Max-Planck-Institute of Neurobiology
Department: Synapses - Circuits - Plasticity
Am Klopferspitz 18
D-82152 Martinsried
Phone: [phone] - 3684
Phone2: [phone] - 3782
Fax: [phone] - 2481
[hidden email]
http://www.neuro.mpg.de/37634/rose

> On 30 Nov 2014, at 00:41, Zdenek Svindrych <[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.
> *****
>
> Yes,
> the BFP of the objective should be the only aperture that limits your
> optical system. By properly imaging this onto the detector you don't loose
> any useful light. It is convenient to use black tubes, as any light leaking
> into the system will degrade your image considerably.
>
> With a high magnification objective (say 60x 1.4NA) the rays between the
> objective and the tube lens are usually well collimated, so you don't need
> to be concerned with 'long' relay lenses between the objective and the non-
> descanned detector (this is not entirely true for scattered two-photon
> fluorescence, you need to perform some sort of simulation or calculation to
> get an idea about the diameter/focal length of your relay lenses necessary
> to collect out-of-focus light) .
>
> Best, Zdenek, www.kcci.virginia.edu
>
>
>
> ---------- Původní zpráva ----------
> Od: Michael Giacomelli <[hidden email]>
> Komu: [hidden email]
> Datum: 29. 11. 2014 18:27:33
> Předmět: Re: Two-photon microscope questions
>
> "*****
> 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 Peter,
>
> With good optical design you can collect nearly the full radiance of the
> objective back aperture (that is, the only place where light will hit the
> sides is inside the objective). There should be negligible signal hitting
> the lens tubes, but perhaps a bit of light leakage from outside the
> microscope if any part is not fully light tight. I think this is the main
> concern, although in practice I'm not sure how much it really matters.
>
> Mike
>
> On Sat, Nov 29, 2014 at 6:07 PM, Peter Rupprecht <[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.
>> *****
>>
>> Dear Heping,
>>
>> To your 2nd question:
>>>> 2) What is a common procedure to image the back aperture of the
>> objective to the input window of the PMT?
>>>> My first thought is to create a collimated source with size > than
>>>> back aperture and shine directly into the back aperture with objective
>> off (to see spot size
>>>> at PMT input). Would this be correct?
>>
>> This sounds difficult to me. The detector size is usually so big that it
>> would be difficult to find the optimal alignment by this method.
>>
>> For a quick alignment, I would, if the excitation light path is perfectly
>> aligned, put a homogeneous plastic fluoslide under the objective and align
>> the detection pathway such that the picture you get looks as good as
>> possible. Do this with the lowest zoom setting (highest scanning angle),
>> otherwise it will always look perfect. With "good", I mean a FOV with the
>> highest brightness in its center. Simply play around with mirror angles
>> while imaging and check the picture live. Usually, due to aperture
> clipping
>> effects while scanning, the FOV is not totally homogeneous. This also
>> depends on the highest zoom factor allowed by your scanners or your
>> software. -- If someone has a better idea, I'd be happy to learn them!
>>
>> As already mentioned, I would also vote for putting the dichroic as close
>> to the objective as possible.
>>
>>
>> As a sidenote, I have always been wondering why some 2P-microscopes I've
>> seen use such long detection pathways (>15 cm) for 2P microscopy, with all
>> the tubes and inner parts being completely black. Wouldn't it be much
>> better to coat the full detection pathway surfaces with an aluminium or
>> silver coating in order to trap every scattered photon until it impinges
> on
>> the detector? Has anyone tried this out and compared it to non-coated
>> surfaces for highly scattering conditions?
>> Best,
>> Peter
>> "

[Heping Yuan]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

I would like to thank everyone for their insightful comments. They have helped me much
better understand some system design concepts.

[Ke Peng]

Dear lister:
I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
I'm looking forward and I'll be very grateful to your opinions.
Best wishes,
Aro    

[Jens]

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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,

2x the SD over mean background is a good rule of thumb.

Jens

Jens-B. Bosse
[phone]

> On Nov 30, 2014, at 12:55, PengKe <[hidden email]> wrote:
>
> Dear lister:
> I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
> Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
> To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
> A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
> I'm looking forward and I'll be very grateful to your opinions.
> Best wishes,
> Aro                        

[Ke Peng]

Thanks a lot, Jens. You mean the intensity should be >= (mean background + 2*SD of the background)? So the difference will be statistically significant?
Is there any reference I can refer to?
Best,
Aro

> Date: Sun, 30 Nov 2014 13:24:48 -0500
> From: [hidden email]
> Subject: Re: How to define the presence/absence of an object in immunofluorescence
> To: [hidden email]
>
> *****
> 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,
>
> 2x the SD over mean background is a good rule of thumb.
>
> Jens
>
> Jens-B. Bosse
> [phone]
>
> > On Nov 30, 2014, at 12:55, PengKe <[hidden email]> wrote:
> >
> > Dear lister:
> > I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
> > Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
> > To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
> > A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
> > I'm looking forward and I'll be very grateful to your opinions.
> > Best wishes,
> > Aro                        

     

[Jens]

*****
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*****

Yes, however I do not have a reference of the top of my head.

Jens

Jens-B. Bosse
[phone]

> On Nov 30, 2014, at 13:38, AroPro <[hidden email]> wrote:
>
> Thanks a lot, Jens. You mean the intensity should be >= (mean background + 2*SD of the background)? So the difference will be statistically significant?
> Is there any reference I can refer to?
> Best,
> Aro
>
>> Date: Sun, 30 Nov 2014 13:24:48 -0500
>> From: [hidden email]
>> Subject: Re: How to define the presence/absence of an object in immunofluorescence
>> To: [hidden email]
>>
>> *****
>> 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,
>>
>> 2x the SD over mean background is a good rule of thumb.
>>
>> Jens
>>
>> Jens-B. Bosse
>> [phone]
>>
>>> On Nov 30, 2014, at 12:55, PengKe <[hidden email]> wrote:
>>>
>>> Dear lister:
>>> I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
>>> Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
>>> To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
>>> A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
>>> I'm looking forward and I'll be very grateful to your opinions.
>>> Best wishes,
>>> Aro                        
>                        

[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 Aro,

Intensity 2200 of feature of interest vs 2000 of background. Camera
maximum 65535. Not enough information. Also, N=1, so more cells, and
proper controls will help. If you increase exposure time 10x you might
get values of ~22,000 vs 20,000, which would help. Personally, I would:
1. acquire more data, including no primary antibody controls.
2. acquire Z-series and deconvolve (whether widefield or confocal ).
3. acquire other fluorescence channel(s) to understand how
autofluorescent your cells and/or tissue are. I routinely acquire at
least one channel that is not supposed to have any fluorophores in it
(440nm or 458 nm excitation and "Cyan FP" emission band works well for
many kinds of cellular autofluorescence).
4. If it is a protein antigen that is supposed to be synthesized by the
cells you are detecting the antigen in or on, corroborate with RNA FISH
(more on this later).


The imaging hardware we have is able to discriminate single molecules
even in brightfield - see
http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf
(a reference I am recycling from the recent single microtubules thread).
On a clean background, a few counts above the camera offset can be
enough. In or on cells that have autofluorescence, this is much harder.


For increasing signal over background -- when using 'clean' antibodies
-- I recommend tyramide signal amplification (TSA). This is commercially
available from both Molecular Probes/Invitrogen/LifeTech/ThermoFisher
and from PerkinElmer. Perkin Elmer is making a push with TSA for 3plex
and 6plex immunofluorescence,
http://www.perkinelmer.com/catalog/category/id/opal-multiplex-tissue-staining

especially in conjunction with their Vectra spectral imager. Good ideas
though I recommend PeroxAbolish instead of microwaves to kill
horseradish peroxidase - my former UMiami colleagues, Dr. Takahashi and
Prof. Ichii, had outstanding CD4 and CD8 TSA immunofluorescence in their
work published in
http://www.ncbi.nlm.nih.gov/pubmed/21929847
(the published images do not do justice to how bright and specific their
staining was).



There is a famous quote by Arthur Kornberg, "Thou shalt Not waste clean
thinking on dirty enzymes"
http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.html

To which I'll add: "do not waste time or money on stupid antibodies", so
if you are using polyclonal antibodies (not reproducible, the rabbit is
probably dead already), reagents from companies like 'Santa Crap' that
don't do quality control, and especially companies that the USDA has
cited for violating the US animal welfare act,
https://awionline.org/sites/default/files/uploads/documents/SanatCruz-1-15-0023COMPLAINTRedacted.pdf


The first two papers in what will be a series were recently published in
Journal of Histochemistry and Cytochemistry, are open access, and
provide good advice:

http://jhc.sagepub.com/content/62/10.toc

    * Denis G. Baskin and
    * Stephen M. Hewitt


        Improving the State of the Science of Immunohistochemistry: The
        Histochemical Society’s Standards of Practice

J Histochem Cytochem October 2014 62: 691-692 doi:10.1369/0022155414538453

    * Free Full Text <http://jhc.sagepub.com/content/62/10/691.full>
    * Free Full (PDF)
      <http://jhc.sagepub.com/content/62/10/691.full.pdf+html>


    * Stephen M. Hewitt,
    * Denis G. Baskin,
    * Charles W. Frevert,
    * William L. Stahl,
    * and Eduardo Rosa-Molinar


        Controls for Immunohistochemistry: The Histochemical Society’s
        Standards of Practice for Validation of Immunohistochemical Assays

J Histochem Cytochem October 2014 62: 693-697 doi:10.1369/0022155414545224

    * Abstract <http://jhc.sagepub.com/content/62/10/693.abstract>
    * Free Full Text <http://jhc.sagepub.com/content/62/10/693.full>
    * Free Full (PDF)
      <http://jhc.sagepub.com/content/62/10/693.full.pdf+html>


For protein antigens that you expect are being made in the same cells
that you are measuring them in, the 'Central Dogma' of DNA codes for
mRNA codes for proteins, means that you could use:
1) gene knockout (more definitive) or RNAi (less definitive, but still
pretty good) to eliminate the gene or expression. ZFNs, TALENs and
CRISPR/Cas9 all work. Humin Zhao has published TALENs at $5 each
http://www.ncbi.nlm.nih.gov/pubmed/24237314 -- which is cost competitive
with the guide RNAs needed for Cas9. CRISPR Cas9 is now over 300
references for just 2014 (with one month left), for a tool only
published in August 2012 (http://www.ncbi.nlm.nih.gov/pubmed/22745249)
-- this decade's answer to previous revolutions in PCR and RNAi. These
also intersect with light microscopy as in, for entry points,
http://www.ncbi.nlm.nih.gov/pubmed/25307933
http://www.ncbi.nlm.nih.gov/pubmed/24556431
http://works.bepress.com/gmcnamara/63
http://works.bepress.com/gmcnamara/42


2. RNA detection
a. in live cells by molecular beacons, or SmartFlares (commercial name
from EMD Millipore) = NanoFlares (academic name,
http://www.ncbi.nlm.nih.gov/pubmed/25404304 and
http://www.ncbi.nlm.nih.gov/pubmed/18034495).
b. single molecule RNA FISH, for examples:
Stellaris FISH ... http://stellarisfish.smugmug.com ... one of the
galleries is a bit of a tease with respect to new hardware,
http://stellarisfish.smugmug.com/StellarVision/
RNAscope
QuantiGene
http://www.panomics.com/products/gene-expression/single-plex-assay/overview
RNAscope and QuantiGene are the same branched DNA technology, from two
different vendors.

I've been doing Stellaris FISH experiments for the past several months,
and posted some of the data online, for example
http://works.bepress.com/gmcnamara/64/

George
p.s. All of Kornberg's commandments at/
http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.html
/Biochemists also know him for creating the Ten Commandments of
Enzymology. Unlike the author of the original ten commandments, Kornberg
was able to modify and amend his commandments as new developments came
along (Kornberg, 2003).
/Thou shalt…

    * I. Rely on enzymology to resolve and reconstitute biologic events
    * II. Trust the universality of biochemistry and the power of
      microbiology
    * III. Not believe something just because you can explain it
    * IV. Not waste clean thinking on dirty enzymes
    * V. Not waste clean enzymes on dirty substrates
    * VI. Use genetics and genomics
    * VII. Be aware that cells are molecularly crowded
    * VIII. Depend on viruses to open windows
    * IX. Remain mindful of the power of radioactive tracers
    * X. Employ enzymes as unique reagents

/
My email signature line used to be:
"Old Soldier's never die, they just fade away" - Douglas Macarthur.
"Old antibodies die, please throw them away" - GM.
/

/

On 11/30/2014 11:55 AM, PengKe wrote:
> Dear lister:
> I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
> Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
> To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
> A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
> I'm looking forward and I'll be very grateful to your opinions.
> Best wishes,
> Aro    


--



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 

[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 Aro,
if you want to be certain that the feature is there, you perform hypothesis
testing. Your null hypothesis is that 'there is nothing' and you want to
calculate whether you can reject this hypothesis (that is, there  'must be
something' with quite high probability, e.g. 95 percent when the 2*SD rule
is followed). However, when it comes to images, it's hard to define
'nothing' and something'...

But nobody is really doing this. The biologic (e.g. cell to cell)
variability is usually the major contribution to your statistics, so what
you normally do is: measure the 'intensity' of your feature in some
consistent way (preferably relative to other intensity-based measure of your
image) on many cells from many 'control' and 'treated' samples and perform
the null hypothesis testing on these numbers. Since you have many numbers
now, you can easily calculate their standard deviations, both 'pixel image'
contribution and biological variability is accounted for without any image-
processing theories...

I believe this is how most assays work, though I'm not a biologist...

Best, Zdenek

www.kcci.virginia.edu



---------- Původní zpráva ----------
Od: AroPro <[hidden email]>
Komu: [hidden email]
Datum: 30. 11. 2014 13:42:34
Předmět: Re: How to define the presence/absence of an object in
immunofluorescence

"Thanks a lot, Jens. You mean the intensity should be >= (mean background +
2*SD of the background)? So the difference will be statistically
significant?
Is there any reference I can refer to?
Best,
Aro

> Date: Sun, 30 Nov 2014 13:24:48 -0500
> From: [hidden email]
> Subject: Re: How to define the presence/absence of an object in
immunofluorescence

> To: [hidden email]
>
> *****
> 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,
>
> 2x the SD over mean background is a good rule of thumb.
>
> Jens
>
> Jens-B. Bosse
> [phone]
>
> > On Nov 30, 2014, at 12:55, PengKe <[hidden email]> wrote:
> >
> > Dear lister:
> > I'm recently puzzled by a question that I felt might have puzzled some

of you as well and I would like to receive some suggestions and opinions
from you.
> > Question: In immunofluorescence analysis, sometimes we detected objects
that showed very weak signals, so how can we decide whether the signal is
really there or not?
> > To make the question a little more specific, I will give some artificial
numbers. Let's say the background has the signal intensity of 2000 and the
potential object showed a signal intensity of 2200. The saturating intensity
of the camera is 65535. In this case can one claim the 2200 signal represent
a real object?
> > A related question might be: is there a golden standard about how much
higher a signal intensity needs to be above the background to be defined as
an object?
> > I'm looking forward and I'll be very grateful to your opinions.
> > Best wishes,
> > Aro"

[Ke Peng]

Dear George:
Let me first thank you for this luxury reply and it took me already some time to run through it. Very helpful and insightful.
I will:1) include the no primary control (used to do it but then stopped as we are satisfied by the specificity of the Ab. We did not analyze the very weak signal at that time though.)2) I always acquire z-series and will start to do deconvolution (will learn this from our image specialist. We are using Autoquant. This might again be a game that somehow needs golden standards.)3) I have 4 kinds of fluophores in my samples now (405, 488, 561, 640). Inspecting another channel is very likely to give some crosstalk singles ...4) Its a protein antigen and will try KD.5) Will read the interesting references.6) I particularly like the quote ''Depend on viruses to open windows''7) We generally stick with Sigma and Abcam and overall the quality of their Abs are quite good.
Many thanks for these very helpful and interesting information.
Aro
Date: Sun, 30 Nov 2014 13:01:26 -0600
From: [hidden email]
To: [hidden email]
CC: [hidden email]
Subject: Re: How to define the presence/absence of an object in immunofluorescence




 


Hi Aro,



Intensity 2200 of feature of interest vs 2000 of background. Camera
maximum 65535. Not enough information. Also, N=1, so more cells, and
proper controls will help. If you increase exposure time 10x you might
get values of ~22,000 vs 20,000, which would help. Personally, I would:

1. acquire more data, including no primary antibody controls.

2. acquire Z-series and deconvolve (whether widefield or confocal ).

3. acquire other fluorescence channel(s) to understand how
autofluorescent your cells and/or tissue are. I routinely acquire at
least one channel that is not supposed to have any fluorophores in it
(440nm or 458 nm excitation and "Cyan FP" emission band works well for
many kinds of cellular autofluorescence).

4. If it is a protein antigen that is supposed to be synthesized by the
cells you are detecting the antigen in or on, corroborate with RNA FISH
(more on this later).





The imaging hardware we have is able to discriminate single molecules
even in brightfield - see

http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf

(a reference I am recycling from the recent single microtubules
thread). On a clean background, a few counts above the camera offset
can be enough. In or on cells that have autofluorescence, this is much
harder.





For increasing signal over background -- when using 'clean' antibodies
-- I recommend tyramide signal amplification (TSA). This is
commercially available from both Molecular
Probes/Invitrogen/LifeTech/ThermoFisher and from PerkinElmer. Perkin
Elmer is making a push with TSA for 3plex and 6plex immunofluorescence,


http://www.perkinelmer.com/catalog/category/id/opal-multiplex-tissue-staining


especially in conjunction with their Vectra spectral imager. Good ideas
though I recommend PeroxAbolish instead of microwaves to kill
horseradish peroxidase - my former UMiami colleagues, Dr. Takahashi and
Prof. Ichii, had outstanding CD4 and CD8 TSA immunofluorescence in
their work published in

http://www.ncbi.nlm.nih.gov/pubmed/21929847

(the published images do not do justice to how bright and specific
their staining was).







There is a famous quote by Arthur Kornberg, "Thou shalt Not waste clean
thinking on dirty enzymes"

http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.html



To which I'll add: "do not waste time or money on stupid antibodies",
so if you are using polyclonal antibodies (not reproducible, the rabbit
is probably dead already), reagents from companies like 'Santa Crap'
that don't do quality control, and especially companies that the USDA
has cited for violating the US animal welfare act,
https://awionline.org/sites/default/files/uploads/documents/SanatCruz-1-15-0023COMPLAINTRedacted.pdf 




The first two papers in what will be a series were recently published
in Journal of Histochemistry and Cytochemistry, are open access, and
provide good advice:



http://jhc.sagepub.com/content/62/10.toc






  Denis
G. Baskin and
   
  Stephen
M. Hewitt

Improving
the State of the Science of Immunohistochemistry: The
Histochemical Society’s Standards of Practice
J
Histochem Cytochem October
2014 62: 691-692
 doi:10.1369/0022155414538453


  Free
Full Text
   
  Free
Full (PDF)
 






  Stephen
M. Hewitt,
  Denis
G. Baskin,
  Charles
W. Frevert,
  William
L. Stahl,
  and Eduardo
Rosa-Molinar

Controls
for Immunohistochemistry: The
Histochemical Society’s Standards of Practice for Validation of
Immunohistochemical Assays
J
Histochem Cytochem October
2014 62: 693-697
doi:10.1369/0022155414545224


  Abstract
   
  Free
Full Text
   
  Free
Full (PDF)



For protein antigens that you expect are being made in the same
cells that you are measuring them in, the 'Central Dogma' of DNA codes
for mRNA codes for proteins, means that you could use:

1) gene knockout (more definitive) or RNAi (less definitive, but still
pretty good) to eliminate the gene or expression. ZFNs, TALENs and
CRISPR/Cas9 all work. Humin Zhao has published TALENs at $5 each
http://www.ncbi.nlm.nih.gov/pubmed/24237314 -- which is cost
competitive with the guide RNAs needed for Cas9. CRISPR Cas9 is now
over 300 references for just 2014 (with one month left), for a tool
only published in August 2012
(http://www.ncbi.nlm.nih.gov/pubmed/22745249) -- this decade's answer
to previous revolutions in PCR and RNAi. These also intersect with
light microscopy as in, for entry points,

http://www.ncbi.nlm.nih.gov/pubmed/25307933 

http://www.ncbi.nlm.nih.gov/pubmed/24556431

http://works.bepress.com/gmcnamara/63

http://works.bepress.com/gmcnamara/42





2. RNA detection

  a. in live cells by molecular beacons, or SmartFlares (commercial
name from EMD Millipore) = NanoFlares (academic name,
http://www.ncbi.nlm.nih.gov/pubmed/25404304 and
http://www.ncbi.nlm.nih.gov/pubmed/18034495).

  b. single molecule RNA FISH, for examples:

      Stellaris FISH ... http://stellarisfish.smugmug.com ... one of
the galleries is a bit of a tease with respect to new hardware,
http://stellarisfish.smugmug.com/StellarVision/ 

      RNAscope

      QuantiGene
http://www.panomics.com/products/gene-expression/single-plex-assay/overview

  RNAscope and QuantiGene are the same branched DNA technology, from
two different vendors.




I've been doing Stellaris FISH experiments for the past several months,
and posted some of the data online, for example
http://works.bepress.com/gmcnamara/64/ 



George

p.s. All of Kornberg's commandments at

http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.html

Biochemists
also know him for creating the Ten Commandments of Enzymology. Unlike
the author of the original ten commandments, Kornberg was able to
modify and amend his commandments as new developments came along
(Kornberg, 2003).

Thou
shalt…

  I.
Rely on enzymology to resolve and reconstitute biologic events
  II.
Trust the universality of biochemistry and the power of microbiology
  III.
Not believe something just because you can explain it
  IV.
Not waste clean thinking on dirty enzymes
  V.
Not waste clean enzymes on dirty substrates
  VI.
Use genetics and genomics
  VII.
Be aware that cells are molecularly crowded
  VIII.
Depend on viruses to open windows
  IX.
Remain mindful of the power of radioactive tracers
  X.
Employ enzymes as unique reagents



My email signature line used
to be:

"Old Soldier's never die, they just fade away" - Douglas Macarthur.

"Old antibodies die, please throw them away" - GM.









On 11/30/2014 11:55 AM, PengKe wrote:

  Dear lister:
I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
I'm looking forward and I'll be very grateful to your opinions.
Best wishes,
Aro    






--



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