More power and less exposure or vice versa – use of 100% laser

[Seb Stephens]

Hi,

100% laser for 1 ms OR 1% laser for 100ms?

This is a subject that has been bothering me and I have never got quite the
answer for it.

After speaking to someone who uses 100% laser power (sorry I don’t know
what the actual laser output is) I noted their goal was to expose for the least
amount of time possible (which was only a few millesecs). This enticed me to
check myself how my protein structures (podosomes) could handle 100% laser
power as opposed to 30% which I usually use. As expected the exposure
times went down significantly (from 320 ms to 30ms) and in fact I was able to
still get decent information from 10ms exposures taken every sec for 3 mins.
Surprisingly, I saw no detectable bleach and it did not seem any different to
long exposure times with less laser power.

Im only new to microscopy and surely others must have experimented with:
high laser+ low exp compared to low laser+high exp.

I really want to know, which one should I do (high or low power)?

It seems that there could be mathematically and practically equivalents of
high laser+low exp to low laser+high exp (ie as above 100ms 1% power pairs
with 1ms 100% laser power). So, is there a relationship between power and
exposure time exists that can sort of be mathematically related?

Cheers

Seb

[Shalin Mehta]

Hi Sebastian,

You are nearly correct in saying that the product of excitation power
and exposure time (i.e. total energy put in the sample) is invariant.
This linear relationship breaks down when you increase power too much,
so that fluorophore gets 'saturated' from too many photons trying to
excite it in short time.

The energy (E) of the photon and wavelength (L) are related by
Planck's constant (h) and speed of light (c): E=hc/L. The basic model
of fluorescence is that the fluorophore absorbs a photon (with varying
probabilities) across a range of wavelengths and after some time
(called lifetime ) typically of pico to nano second values emits a
lower energy photon.

Ideally, you get one emission photon per excitation photon. When you
put in certain power, you are imparting certain energy per second,
i.e., putting in a given number of photons per second. As long as the
time-rate of photons hitting the fluorophore is low you should get an
emission photon per excitation photon. But at very high power ( high
pohton-rates) this relationship becomes non-linear, and you get less
number of emission photons per excitation photon.

You may find introductory chapters of "Principles of fluorescence
spectroscopy" by Joseph Lakowicz illuminating in this regard.

best
shalin

On Mon, Jul 13, 2009 at 2:18 AM, Sebastien
Stephens<[hidden email]> wrote:

> Hi,
>
> 100% laser for 1 ms OR 1% laser for 100ms?
>
> This is a subject that has been bothering me and I have never got quite the
> answer for it.
>
> After speaking to someone who uses 100% laser power (sorry I don’t know
> what the actual laser output is) I noted their goal was to expose for the least
> amount of time possible (which was only a few millesecs). This enticed me to
> check myself how my protein structures (podosomes) could handle 100% laser
> power as opposed to 30% which I usually use. As expected the exposure
> times went down significantly (from 320 ms to 30ms) and in fact I was able to
> still get decent information from 10ms exposures taken every sec for 3 mins.
> Surprisingly, I saw no detectable bleach and it did not seem any different to
> long exposure times with less laser power.
>
> Im only new to microscopy and surely others must have experimented with:
> high laser+ low exp compared to low laser+high exp.
>
> I really want to know, which one should I do (high or low power)?
>
> It seems that there could be mathematically and practically equivalents of
> high laser+low exp to low laser+high exp (ie as above 100ms 1% power pairs
> with 1ms 100% laser power). So, is there a relationship between power and
> exposure time exists that can sort of be mathematically related?
>
> Cheers
>
> Seb
>

[John Oreopoulos]

It's a good question and I think Shalin and Claire have given good  
answers already. Perhaps there is a way to determine on the fly  
during an experiment if you are indeed saturating the fluorescence  
signal in any parts of the cell, but without that knowledge you could  
be doing damage to the cells. Generally, it has been my experience,  
and also it was emphasized many times at Jim Pawley's live-cell  
microscopy course that I attended a few years ago, that the products  
of photobleaching are toxic to the cell, and so using as little light  
as possible is preferred so long as you can compromise with a longer  
exposure time or increased detector gain and still get an acceptable  
SNR for the phenomenon you're trying to observe. That being said,  
there may be situations where you are trying to observe a fast  
dynamic process in the cell by time-lapse imaging which necessitates  
short exposure times and using a larger laser power to get a good SNR  
again. As I understand it, some of the super-resolution imaging  
techniques even depend on the non-linear effects of fluorescence  
saturation to achieve imaging resolution below the diffraction limit,  
so there are a few times when a larger laser power might be called for.
So, I would say there is no one definite recipe for all imaging  
situations, but keeping the cells alive on the microscope stage is a  
priority. Jennifer Waters has a very nice recent review on "Accuracy  
and Precision in Light Microscopy" in the Journal of Cell Biology  
where  she rightly points out that it's up to the experimenter to  
test and figure out the optimum settings of acquisition and perform  
the proper control experiments to back up the interpretation results  
of an imaging experiment.

John Oreopoulos


On 13-Jul-09, at 4:01 AM, Shalin Mehta wrote:

> Hi Sebastian,
>
> You are nearly correct in saying that the product of excitation power
> and exposure time (i.e. total energy put in the sample) is invariant.
> This linear relationship breaks down when you increase power too much,
> so that fluorophore gets 'saturated' from too many photons trying to
> excite it in short time.
>
> The energy (E) of the photon and wavelength (L) are related by
> Planck's constant (h) and speed of light (c): E=hc/L. The basic model
> of fluorescence is that the fluorophore absorbs a photon (with varying
> probabilities) across a range of wavelengths and after some time
> (called lifetime ) typically of pico to nano second values emits a
> lower energy photon.
>
> Ideally, you get one emission photon per excitation photon. When you
> put in certain power, you are imparting certain energy per second,
> i.e., putting in a given number of photons per second. As long as the
> time-rate of photons hitting the fluorophore is low you should get an
> emission photon per excitation photon. But at very high power ( high
> pohton-rates) this relationship becomes non-linear, and you get less
> number of emission photons per excitation photon.
>
> You may find introductory chapters of "Principles of fluorescence
> spectroscopy" by Joseph Lakowicz illuminating in this regard.
>
> best
> shalin
>
> On Mon, Jul 13, 2009 at 2:18 AM, Sebastien
> Stephens<[hidden email]> wrote:
>> Hi,
>>
>> 100% laser for 1 ms OR 1% laser for 100ms?
>>
>> This is a subject that has been bothering me and I have never got  
>> quite the
>> answer for it.
>>
>> After speaking to someone who uses 100% laser power (sorry I don’t  
>> know
>> what the actual laser output is) I noted their goal was to expose  
>> for the least
>> amount of time possible (which was only a few millesecs). This  
>> enticed me to
>> check myself how my protein structures (podosomes) could handle  
>> 100% laser
>> power as opposed to 30% which I usually use. As expected the exposure
>> times went down significantly (from 320 ms to 30ms) and in fact I  
>> was able to
>> still get decent information from 10ms exposures taken every sec  
>> for 3 mins.
>> Surprisingly, I saw no detectable bleach and it did not seem any  
>> different to
>> long exposure times with less laser power.
>>
>> Im only new to microscopy and surely others must have experimented  
>> with:
>> high laser+ low exp compared to low laser+high exp.
>>
>> I really want to know, which one should I do (high or low power)?
>>
>> It seems that there could be mathematically and practically  
>> equivalents of
>> high laser+low exp to low laser+high exp (ie as above 100ms 1%  
>> power pairs
>> with 1ms 100% laser power). So, is there a relationship between  
>> power and
>> exposure time exists that can sort of be mathematically related?
>>
>> Cheers
>>
>> Seb
>>

[Seb Stephens]

Dear Tom, John, Claire, Esteban and Shalin,
 
Thank you all very much for your input.

I am now very clear on how I will approach my image sampling.
 
My best regards

 

 

Sébastien Stephens

Harvard University School of Dental Medicine
188 Longwood Ave REB 313
Boston, MA 02115
Ph: 617-4327328
Fax: [phone]
[hidden email]

---

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[kspencer007]

Hello all;

>
> I have a colleague who is unhappy with the images of td-Tomato
> AI9 mice on our Fluoview 500. They first view their brain sections
> on our AX70, with mercury lamp illumination, a TRITC 535/50 EX filter,
> 610/75 EM filter, and like what they see. On our FV500 with 543 laser
> illumination (HeNe-Green 50% default AOTF increased to 90%), we have
> switched to a 560LP filter from the 560-600EM filter. This improved
> their results, but they still are disappointed in signal intensity. I
> recently had our system checked over by Olympus service, and we
> measured the laser output as 0.45mW out of the fiber (non-wavelength
> specific power meter), which is just off the best it's ever been. I
> have suggested opening the pinhole and sacrificing Z resolution for
> brightness.
>
> Would you have any other suggestions for increasing the intensity of
> their signal? I have explained filters and Z-thickness versus
> brightness. I don't know what to tell them, and they are faulting the
> system. From all spectra I can find, our system should be great as seeing this construct.

 Kathy Spencer

[Mark Cannell]

Are they using an index-matched mounting medium?

Do they understand that a wide field "like what they see" is quite
different to a confocal section?

Cheers

>> I have a colleague who is unhappy with the images of td-Tomato
>> AI9 mice on our Fluoview 500. They first view their brain sections
>> on our AX70, with mercury lamp illumination, a TRITC 535/50 EX filter,
>> 610/75 EM filter, and like what they see. On our FV500 with 543 laser
>> illumination (HeNe-Green 50% default AOTF increased to 90%), we have
>> switched to a 560LP filter from the 560-600EM filter. This improved
>> their results, but they still are disappointed in signal intensity. I
>> recently had our system checked over by Olympus service, and we
>> measured the laser output as 0.45mW out of the fiber (non-wavelength
>> specific power meter), which is just off the best it's ever been. I
>> have suggested opening the pinhole and sacrificing Z resolution for
>> brightness.
>>
>> Would you have any other suggestions for increasing the intensity of
>> their signal? I have explained filters and Z-thickness versus
>> brightness. I don't know what to tell them, and they are faulting the
>> system. From all spectra I can find, our system should be great as seeing this construct.
>>    
>
>  Kathy Spencer
>  

[Rietdorf, Jens]

Dear Kathy,

your mercury arc has a nice bright peak at that ex and the HeNe 543 is
the weakest laser on the list. Likewise the PMT has a QE below 10% in
that range of the em spectrum, your camera may have up to 90%.
 
What happens if you use the arc & a camera on the FV500?
Do they use lenses with similar NA on both systems?
Is the pixel size x,y similar?
Is the pinhole aligned?
Is the PSF of the confocal ok?

Just a few points to check. Good luck, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Kathryn Spencer
Sent: Wednesday, November 04, 2009 6:57 PM
To: [hidden email]
Subject: tdTomato and LSM confocal

Hello all;

>
> I have a colleague who is unhappy with the images of td-Tomato
> AI9 mice on our Fluoview 500. They first view their brain sections
> on our AX70, with mercury lamp illumination, a TRITC 535/50 EX filter,
> 610/75 EM filter, and like what they see. On our FV500 with 543 laser
> illumination (HeNe-Green 50% default AOTF increased to 90%), we have
> switched to a 560LP filter from the 560-600EM filter. This improved
> their results, but they still are disappointed in signal intensity. I
> recently had our system checked over by Olympus service, and we
> measured the laser output as 0.45mW out of the fiber (non-wavelength
> specific power meter), which is just off the best it's ever been. I
> have suggested opening the pinhole and sacrificing Z resolution for
> brightness.
>
> Would you have any other suggestions for increasing the intensity of
> their signal? I have explained filters and Z-thickness versus
> brightness. I don't know what to tell them, and they are faulting the
> system. From all spectra I can find, our system should be great as

seeing this construct.

 Kathy Spencer