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strange images using multiphoton

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Konstantín Levitskiy

strange images using multiphoton

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Dear Microscopists.

Recently weve acquired a multiphoton system for in vivo acquisitions. We
just had it for 3 weeks and in this time we have observed some strange
things when comparing the images of famous Convallaria preparation obtained
by convencional (LED) ilumination or one-photon confocal system vs new 2P
confocal.

https://imgur.com/a/6xZwZhz

Weve used a 810nm excitation (double of 405nm). The first image (Fig.1) is
a (supposed) blue channel and its quite different to that obtained by LED
ilumination (Fig.3) or using one-photon confocal (Fig.4) system (for 405,
458 and 488nm excitation). The wide rings (in the preparation) have the same
intensity as the narrow ones on 2P system. On the contrary, there is a very
clear difference on images by LED or 1P confocal. Has this any reason for
happening in the 2P system?

On the other hand, using the same 810nm excitation we detect, in 2P system
on the second (red) channel, fluorescence similar to obtain for red-IR
excitation for this preparation. I suppose we obtain 1P detection on the
system. However, the contrast is quite poor, but when we increased the laser
power the preparation starts boiling (transmitted image). Could you help to
explain this?

Any suggestion will be appreciate.

Thanks in advance.,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: <mailto:[hidden email]> [hidden email]

Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es

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mcammer

Re: strange images using multiphoton

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We often find problems when the laser is exactly twice the wavelength of a wavelength detected by the emission filters because of unexpected 2nd harmonics. When this happens, we tune the laser to just outside the emission range of the filter to try to excite the fluorescence of interest but get rid of the harmonics. If this doesn't work, a narrower filter may be needed. There is no one perfect solution.

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
C: 914-309-3270 [hidden email] http://nyulmc.org/micros http://microscopynotes.com/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Konstantín Levitskiy
Sent: Monday, April 23, 2018 11:03 AM
To: [hidden email]
Subject: strange images using multiphoton

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Dear Microscopists.

Recently we've acquired a multiphoton system for in vivo acquisitions. We just had it for 3 weeks and in this time we have observed some strange things when comparing the images of famous Convallaria preparation obtained by convencional (LED) ilumination or one-photon confocal system vs new 2P confocal.

https://urldefense.proofpoint.com/v2/url?u=https-3A__imgur.com_a_6xZwZhz&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=OcPmQ5uTt24KWR6mKpfgNhidp38TWICFZ4dfwy2FArY&s=aLjKO167fzMQ8vQhTsTRvRBWFuenwv4q39fhBEE3gXk&e=

We've used a 810nm excitation (double of 405nm). The first image (Fig.1) is a (supposed) blue channel and it's quite different to that obtained by LED ilumination (Fig.3) or using one-photon confocal (Fig.4) system (for 405,
458 and 488nm excitation). The wide rings (in the preparation) have the same intensity as the narrow ones on 2P system. On the contrary, there is a very clear difference on images by LED or 1P confocal. Has this any reason for happening in the 2P system?

On the other hand, using the same 810nm excitation we detect, in 2P system on the second (red) channel, fluorescence similar to obtain for red-IR excitation for this preparation. I suppose we obtain 1P detection on the system. However, the contrast is quite poor, but when we increased the laser power the preparation starts boiling (transmitted image). Could you help to explain this?

Any suggestion will be appreciate.

Thanks in advance.,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: <mailto:[hidden email]> [hidden email]

Web: <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ibis-2Dsevilla.es_&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=OcPmQ5uTt24KWR6mKpfgNhidp38TWICFZ4dfwy2FArY&s=T0A2CgHyO-UPPvTJmVJJ40CIggK3Xtt1vP17xwVj2SU&e=> www.ibis-sevilla.es

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Csúcs Gábor-3

Re: strange images using multiphoton

In reply to this post by Konstantín Levitskiy

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Dear Konstantin,

Though there will be probably more detailed answers - a very simple explanation is that with the 810 excitation you are exiting different fluorophores (compared to the 405 nm case). Please remember that the two-photon (or more generally the multi-photon) excitation spectra of a fluorophore has "very little" to do with its single photon excitation spectra. In practice this means that you can't simply multiply the optimal/usual excitation wavelength of a fluorophore with 2 to get the corresponding two-photon excitation spectra.

Greetings Gabor

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Konstantín Levitskiy
Sent: Monday, April 23, 2018 5:03 PM
To: [hidden email]
Subject: strange images using multiphoton

*****
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 Microscopists.

Recently we've acquired a multiphoton system for in vivo acquisitions. We just had it for 3 weeks and in this time we have observed some strange things when comparing the images of famous Convallaria preparation obtained by convencional (LED) ilumination or one-photon confocal system vs new 2P confocal.

https://imgur.com/a/6xZwZhz

We've used a 810nm excitation (double of 405nm). The first image (Fig.1) is a (supposed) blue channel and it's quite different to that obtained by LED ilumination (Fig.3) or using one-photon confocal (Fig.4) system (for 405,
458 and 488nm excitation). The wide rings (in the preparation) have the same intensity as the narrow ones on 2P system. On the contrary, there is a very clear difference on images by LED or 1P confocal. Has this any reason for happening in the 2P system?

On the other hand, using the same 810nm excitation we detect, in 2P system on the second (red) channel, fluorescence similar to obtain for red-IR excitation for this preparation. I suppose we obtain 1P detection on the system. However, the contrast is quite poor, but when we increased the laser power the preparation starts boiling (transmitted image). Could you help to explain this?

Any suggestion will be appreciate.

Thanks in advance.,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: <mailto:[hidden email]> [hidden email]

Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es

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Steffen Dietzel

Re: strange images using multiphoton

In reply to this post by Konstantín Levitskiy

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Konstantin,

I am not sure to which of the problems you are referring to exactly. It
looks like you have a good deal of vibration, or with bidirectional
scanning the phase is not in sync. Then, you have some darker lines in
the image at irregular intervals which may come from the electronics. On
the second image, the offset seems to be way to low.

In general, compared to the wide field, it seems you have collected not
a lot of photons and thus a low dynamic range. Some of the problems
might be less obvious with accumulation or averaging.

Depending on your filter, you might see SHG from the cellulose, and not
(mainly) fluorescence. But I don't really think so.

Which system/brand are you working with, and with PMTs, avalanche diodes
or hybrids?

Steffen

Am 23.04.2018 um 17:03 schrieb Konstantín Levitskiy:

> *****
> 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 Microscopists.
>
>
>
> Recently we’ve acquired a multiphoton system for in vivo acquisitions. We
> just had it for 3 weeks and in this time we have observed some strange
> things when comparing the images of famous Convallaria preparation obtained
> by convencional (LED) ilumination or one-photon confocal system vs new 2P
> confocal.
>
>
>
> https://imgur.com/a/6xZwZhz
>
> We’ve used a 810nm excitation (double of 405nm). The first image (Fig.1) is
> a (supposed) blue channel and it’s quite different to that obtained by LED
> ilumination (Fig.3) or using one-photon confocal (Fig.4) system (for 405,
> 458 and 488nm excitation). The wide rings (in the preparation) have the same
> intensity as the narrow ones on 2P system. On the contrary, there is a very
> clear difference on images by LED or 1P confocal. Has this any reason for
> happening in the 2P system?
>
> On the other hand, using the same 810nm excitation we detect, in 2P system
> on the second (red) channel, fluorescence similar to obtain for red-IR
> excitation for this preparation. I suppose we obtain 1P detection on the
> system. However, the contrast is quite poor, but when we increased the laser
> power the preparation starts boiling (transmitted image). Could you help to
> explain this?
>
>
>
> Any suggestion will be appreciate.
>
>
>
> Thanks in advance.,
>
> Dr. Konstantín Levitskiy
>
> Servicio de Microscopía
>
> InstitutodeBiomedicinadeSevilla - IBiS
>
> Campus del Hospital Universitario Virgen del Rocío
>
> Avda. Manuel Siurot s/nº
>
> 41013 Sevilla
>
> Tlfno: 955 92 3030
>
> Email: <mailto:[hidden email]> [hidden email]
>
> Web: <http://www.ibis-sevilla.es/> www.ibis-sevilla.es
>
>
>
>
>
> ---
> El software de antivirus Avast ha analizado este correo electrónico en busca de virus.
> https://www.avast.com/antivirus
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

Konstantín Levitskiy

Re: strange images using multiphoton

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Hi again.

We found one of the troubles at the moment: cable connector is bad.
https://imgur.com/a/KIFTgkV
When we get the new one I'll contact you again, I'm afraid. Thanks a lot for suggestions.

Best regards,
Konstantin

-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Steffen Dietzel
Enviado el: viernes, 27 de abril de 2018 15:18
Para: [hidden email]
Asunto: Re: strange images using multiphoton

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*****

Konstantin,

I am not sure to which of the problems you are referring to exactly. It
looks like you have a good deal of vibration, or with bidirectional
scanning the phase is not in sync. Then, you have some darker lines in
the image at irregular intervals which may come from the electronics. On
the second image, the offset seems to be way to low.

In general, compared to the wide field, it seems you have collected not
a lot of photons and thus a low dynamic range. Some of the problems
might be less obvious with accumulation or averaging.

Depending on your filter, you might see SHG from the cellulose, and not
(mainly) fluorescence. But I don't really think so.

Which system/brand are you working with, and with PMTs, avalanche diodes
or hybrids?

Steffen

Am 23.04.2018 um 17:03 schrieb Konstantín Levitskiy: