Near IR luminescence observed from Microscope Objectives.

[Arnold Estrada]

 In the process of trying to make phosphorescent lifetime measurements from a very week signal, I have observed that all of my microscope objectives seem to luminesce in the near-infrared. My research involves using two-photon excitation of phosphorescent probes with very small two-photon action cross-sections.  Consequently my excitation source is a gated Ti:Sapphire (@ 800 nm) such that the excitation reaches the sample (or objective in this case) for 20 microseconds out of every two milliseconds.  The laser power reaching the objective is about 400 mW while the gating is on.  Using a spectrometer, I have confirmed that this luminescence extends from ~ 700nm to greater than 850 nm.  The presence of the anti-stokes shifted light implies some sort of multi-photon absorption mechanism.  However, I observe this luminescence whether the laser is mode locked or not.  I have also observed that the luminescence process appears to have a lifetime of ~ 60-80 uS.  Has anyone else ever observed this?  I have observed this with other lenses (not microscope objectives) as well.  Is it possible that the high laser power used is causing a multi-step absorption process followed by luminescence from the metal dopants in the high index glass in the objective?

Any help would be greatly appreciated.

---
Arnold D. Estrada.

Doctoral Candidate
Department of Biomedical Engineering
The University of Texas at Austin
Biomedical Engineering Bldg, 1.324
107 W. Dean Keaton Street
Austin, TX 78712

Office Phone [phone]
Lab Phone [phone]
Cell Phone: [phone]

[Guy Cox-2]

Interesting.  Does the luminescence intensity scale linearly or
quadratically with excitation?

                                                  Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Arnold Estrada
Sent: Friday, 19 March 2010 9:01 AM
To: [hidden email]
Subject: Near IR luminescence observed from Microscope Objectives.

 In the process of trying to make phosphorescent lifetime measurements
from a very week signal, I have observed that all of my microscope
objectives seem to luminesce in the near-infrared. My research involves
using two-photon excitation of phosphorescent probes with very small
two-photon action cross-sections.  Consequently my excitation source is
a gated Ti:Sapphire (@ 800 nm) such that the excitation reaches the
sample (or objective in this case) for 20 microseconds out of every two
milliseconds.  The laser power reaching the objective is about 400 mW
while the gating is on.  Using a spectrometer, I have confirmed that
this luminescence extends from ~ 700nm to greater than 850 nm.  The
presence of the anti-stokes shifted light implies some sort of
multi-photon absorption mechanism.  However, I observe this luminescence
whether the laser is mode locked or not.  I have also observed that the
luminescence process appears to have a lifetime of ~ 60-80 uS.  Has
anyone else ever observed this?  I have observed this with other lenses
(not microscope objectives) as well.  Is it possible that the high laser
power used is causing a multi-step absorption process followed by
luminescence from the metal dopants in the high index glass in the
objective?

Any help would be greatly appreciated.

---
Arnold D. Estrada.

Doctoral Candidate
Department of Biomedical Engineering
The University of Texas at Austin
Biomedical Engineering Bldg, 1.324
107 W. Dean Keaton Street
Austin, TX 78712

Office Phone [phone]
Lab Phone [phone]
Cell Phone: [phone]

[Arnold Estrada]

It scales approximately linearly.

Thanks for a response
-Arnold E.
On Mar 18, 2010, at 11:32 PM, Guy Cox wrote:

Interesting.  Does the luminescence intensity scale linearly or
quadratically with excitation?

                                                 Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Arnold Estrada
Sent: Friday, 19 March 2010 9:01 AM
To: [hidden email]
Subject: Near IR luminescence observed from Microscope Objectives.

In the process of trying to make phosphorescent lifetime measurements
from a very week signal, I have observed that all of my microscope
objectives seem to luminesce in the near-infrared. My research involves
using two-photon excitation of phosphorescent probes with very small
two-photon action cross-sections.  Consequently my excitation source is
a gated Ti:Sapphire (@ 800 nm) such that the excitation reaches the
sample (or objective in this case) for 20 microseconds out of every two
milliseconds.  The laser power reaching the objective is about 400 mW
while the gating is on.  Using a spectrometer, I have confirmed that
this luminescence extends from ~ 700nm to greater than 850 nm.  The
presence of the anti-stokes shifted light implies some sort of
multi-photon absorption mechanism.  However, I observe this luminescence
whether the laser is mode locked or not.  I have also observed that the
luminescence process appears to have a lifetime of ~ 60-80 uS.  Has
anyone else ever observed this?  I have observed this with other lenses
(not microscope objectives) as well.  Is it possible that the high laser
power used is causing a multi-step absorption process followed by
luminescence from the metal dopants in the high index glass in the
objective?

Any help would be greatly appreciated.

---
Arnold D. Estrada.

Doctoral Candidate
Department of Biomedical Engineering
The University of Texas at Austin
Biomedical Engineering Bldg, 1.324
107 W. Dean Keaton Street
Austin, TX 78712

Office Phone [phone]
Lab Phone [phone]
Cell Phone: [phone]

[Vladimir Ghukasyan-2]

Hello Arnold,

I can just share a similar obervation on our system. We were using
multiphoton excitation in the range of 800 and 960 nm when we were
trying to calibrate the system for the FCS experiments. What we
observed was that there was a signal in case of no sample above the
objective, (inverted) there was a well - expressed fluorescence there.
However, whenever just a glass slide was put, the signal was
diminished almost to the background level. We still don't know what
was the exact source of this phenomena.

With regards,
Vladimir

On Fri, Mar 19, 2010 at 1:36 AM, Arnold Estrada <[hidden email]> wrote:

> It scales approximately linearly.
>
> Thanks for a response
> -Arnold E.
> On Mar 18, 2010, at 11:32 PM, Guy Cox wrote:
>
> Interesting.  Does the luminescence intensity scale linearly or
> quadratically with excitation?
>
>                                                 Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Arnold Estrada
> Sent: Friday, 19 March 2010 9:01 AM
> To: [hidden email]
> Subject: Near IR luminescence observed from Microscope Objectives.
>
> In the process of trying to make phosphorescent lifetime measurements
> from a very week signal, I have observed that all of my microscope
> objectives seem to luminesce in the near-infrared. My research involves
> using two-photon excitation of phosphorescent probes with very small
> two-photon action cross-sections.  Consequently my excitation source is
> a gated Ti:Sapphire (@ 800 nm) such that the excitation reaches the
> sample (or objective in this case) for 20 microseconds out of every two
> milliseconds.  The laser power reaching the objective is about 400 mW
> while the gating is on.  Using a spectrometer, I have confirmed that
> this luminescence extends from ~ 700nm to greater than 850 nm.  The
> presence of the anti-stokes shifted light implies some sort of
> multi-photon absorption mechanism.  However, I observe this luminescence
> whether the laser is mode locked or not.  I have also observed that the
> luminescence process appears to have a lifetime of ~ 60-80 uS.  Has
> anyone else ever observed this?  I have observed this with other lenses
> (not microscope objectives) as well.  Is it possible that the high laser
> power used is causing a multi-step absorption process followed by
> luminescence from the metal dopants in the high index glass in the
> objective?
>
> Any help would be greatly appreciated.
>
> ---
> Arnold D. Estrada.
>
> Doctoral Candidate
> Department of Biomedical Engineering
> The University of Texas at Austin
> Biomedical Engineering Bldg, 1.324
> 107 W. Dean Keaton Street
> Austin, TX 78712
>
> Office Phone    [phone]
> Lab Phone       [phone]
> Cell Phone:     [phone]
>

[Martin Wessendorf-2]

What happens if you remove the transmitted-light condenser?

Vladimir Gukassyan wrote:

> Hello Arnold,
>
> I can just share a similar obervation on our system. We were using
> multiphoton excitation in the range of 800 and 960 nm when we were
> trying to calibrate the system for the FCS experiments. What we
> observed was that there was a signal in case of no sample above the
> objective, (inverted) there was a well - expressed fluorescence there.
> However, whenever just a glass slide was put, the signal was
> diminished almost to the background level. We still don't know what
> was the exact source of this phenomena.
>
> With regards,
> Vladimir
>
> On Fri, Mar 19, 2010 at 1:36 AM, Arnold Estrada <[hidden email]> wrote:
>> It scales approximately linearly.
>>
>> Thanks for a response
>> -Arnold E.
>> On Mar 18, 2010, at 11:32 PM, Guy Cox wrote:
>>
>> Interesting.  Does the luminescence intensity scale linearly or
>> quadratically with excitation?
>>
>>                                                 Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Arnold Estrada
>> Sent: Friday, 19 March 2010 9:01 AM
>> To: [hidden email]
>> Subject: Near IR luminescence observed from Microscope Objectives.
>>
>> In the process of trying to make phosphorescent lifetime measurements
>> from a very week signal, I have observed that all of my microscope
>> objectives seem to luminesce in the near-infrared. My research involves
>> using two-photon excitation of phosphorescent probes with very small
>> two-photon action cross-sections.  Consequently my excitation source is
>> a gated Ti:Sapphire (@ 800 nm) such that the excitation reaches the
>> sample (or objective in this case) for 20 microseconds out of every two
>> milliseconds.  The laser power reaching the objective is about 400 mW
>> while the gating is on.  Using a spectrometer, I have confirmed that
>> this luminescence extends from ~ 700nm to greater than 850 nm.  The
>> presence of the anti-stokes shifted light implies some sort of
>> multi-photon absorption mechanism.  However, I observe this luminescence
>> whether the laser is mode locked or not.  I have also observed that the
>> luminescence process appears to have a lifetime of ~ 60-80 uS.  Has
>> anyone else ever observed this?  I have observed this with other lenses
>> (not microscope objectives) as well.  Is it possible that the high laser
>> power used is causing a multi-step absorption process followed by
>> luminescence from the metal dopants in the high index glass in the
>> objective?
>>
>> Any help would be greatly appreciated.
>>
>> ---
>> Arnold D. Estrada.
>>
>> Doctoral Candidate
>> Department of Biomedical Engineering
>> The University of Texas at Austin
>> Biomedical Engineering Bldg, 1.324
>> 107 W. Dean Keaton Street
>> Austin, TX 78712
>>
>> Office Phone    [phone]
>> Lab Phone       [phone]
>> Cell Phone:     [phone]
>>

--
Martin Wessendorf, PhD   [phone] (office)
Associate Professor      [phone] (lab)
Dept Neuroscience      [phone] (FAX)
Univ Minnesota        e-mail: martinw(at)umn.edu

[Vladimir Ghukasyan-2]

We did check that by putting a black box on the stage. This wouldn't
eliminate the effect.



On Sat, Mar 27, 2010 at 12:02 AM, Martin Wessendorf <[hidden email]> wrote:

> What happens if you remove the transmitted-light condenser?
>
> Vladimir Gukassyan wrote:
>>
>> Hello Arnold,
>>
>> I can just share a similar obervation on our system. We were using
>> multiphoton excitation in the range of 800 and 960 nm when we were
>> trying to calibrate the system for the FCS experiments. What we
>> observed was that there was a signal in case of no sample above the
>> objective, (inverted) there was a well - expressed fluorescence there.
>> However, whenever just a glass slide was put, the signal was
>> diminished almost to the background level. We still don't know what
>> was the exact source of this phenomena.
>>
>> With regards,
>> Vladimir
>>
>> On Fri, Mar 19, 2010 at 1:36 AM, Arnold Estrada <[hidden email]>
>> wrote:
>>>
>>> It scales approximately linearly.
>>>
>>> Thanks for a response
>>> -Arnold E.
>>> On Mar 18, 2010, at 11:32 PM, Guy Cox wrote:
>>>
>>> Interesting.  Does the luminescence intensity scale linearly or
>>> quadratically with excitation?
>>>
>>>                                                Guy
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>> On Behalf Of Arnold Estrada
>>> Sent: Friday, 19 March 2010 9:01 AM
>>> To: [hidden email]
>>> Subject: Near IR luminescence observed from Microscope Objectives.
>>>
>>> In the process of trying to make phosphorescent lifetime measurements
>>> from a very week signal, I have observed that all of my microscope
>>> objectives seem to luminesce in the near-infrared. My research involves
>>> using two-photon excitation of phosphorescent probes with very small
>>> two-photon action cross-sections.  Consequently my excitation source is
>>> a gated Ti:Sapphire (@ 800 nm) such that the excitation reaches the
>>> sample (or objective in this case) for 20 microseconds out of every two
>>> milliseconds.  The laser power reaching the objective is about 400 mW
>>> while the gating is on.  Using a spectrometer, I have confirmed that
>>> this luminescence extends from ~ 700nm to greater than 850 nm.  The
>>> presence of the anti-stokes shifted light implies some sort of
>>> multi-photon absorption mechanism.  However, I observe this luminescence
>>> whether the laser is mode locked or not.  I have also observed that the
>>> luminescence process appears to have a lifetime of ~ 60-80 uS.  Has
>>> anyone else ever observed this?  I have observed this with other lenses
>>> (not microscope objectives) as well.  Is it possible that the high laser
>>> power used is causing a multi-step absorption process followed by
>>> luminescence from the metal dopants in the high index glass in the
>>> objective?
>>>
>>> Any help would be greatly appreciated.
>>>
>>> ---
>>> Arnold D. Estrada.
>>>
>>> Doctoral Candidate
>>> Department of Biomedical Engineering
>>> The University of Texas at Austin
>>> Biomedical Engineering Bldg, 1.324
>>> 107 W. Dean Keaton Street
>>> Austin, TX 78712
>>>
>>> Office Phone    [phone]
>>> Lab Phone       [phone]
>>> Cell Phone:     [phone]
>>>
>
>
> --
> Martin Wessendorf, PhD     [phone] (office)
> Associate Professor           [phone] (lab)
> Dept Neuroscience             [phone] (FAX)
> Univ Minnesota          e-mail: martinw(at)umn.edu
>