Feedback on TIRF systems

[Bryon Grove]

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Dear All,



We are currently looking at TIRF/widefield fluorescence microscopy
systems from Olympus, Zeiss and DeltaVision and are seeking comments
from users of these systems. We would greatly appreciate any feedback
that users could provide. Since this is not directly a confocal microscopy
question, I would be happy to receive comments directly
([hidden email]).



Thanks so much,



Bryon



*******************************



Bryon Grove, Ph.D.

Associate Professor

Department of Basic Sciences

Director, Edward C Carlson Imaging and Image Analysis Core Facility

UND School of Medicine and Health Sciences

501 N Columbia Rd Stop 9037

Grand Forks, ND, 58202-9037

Phone: [phone]

Fax: [phone]

[Zac Arrac Atelaz]

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Bryon:
 
I have personally worked on the Olympus TIRF, the first versión with everything manual, and the new versión with the automated SIS system, and I have also have the chance to see the http://www.tirftechnologies.com/ system insert working with an Olympus IX81 microscope.
 
The manual TIRF from Olympus was a single user system as many manual adjustments were needed. Here is a great BUT, the new automated system is really impressive, you can adjust the depth from which you are having signal coming out. So the electromagnetic space adjacent to your coverglass is greatly controlled. All the laser alignment is made only once by the installation engineers and basically remains like that for life, they work now with led lasers, so maintenance would not be an issue, and expected life for leds is quite long.
 
Tirf tech have a very small system which is interesting, you might want to take a look to that one also.
 
I have not Heard from other TIRF systems
 
Best regards and happiness
 
Gabriel OH
 

> Date: Fri, 16 Aug 2013 11:49:38 -0500
> From: [hidden email]
> Subject: Feedback on TIRF systems
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
>
>
> We are currently looking at TIRF/widefield fluorescence microscopy
> systems from Olympus, Zeiss and DeltaVision and are seeking comments
> from users of these systems. We would greatly appreciate any feedback
> that users could provide. Since this is not directly a confocal microscopy
> question, I would be happy to receive comments directly
> ([hidden email]).
>
>
>
> Thanks so much,
>
>
>
> Bryon
>
>
>
> *******************************
>
>
>
> Bryon Grove, Ph.D.
>
> Associate Professor
>
> Department of Basic Sciences
>
> Director, Edward C Carlson Imaging and Image Analysis Core Facility
>
> UND School of Medicine and Health Sciences
>
> 501 N Columbia Rd Stop 9037
>
> Grand Forks, ND, 58202-9037
>
> Phone: [phone]
>
> Fax: [phone]

     

[Unruh, Jay]

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Hi all,

If you intend to work on interfaces other than glass/water, be very skeptical of claims to determine the TIRF field penetration depth.  This is highly refractive index dependent and will depend strongly on cell type/adhesion strength, basement membrane composition, etc...  I have seen scenarios where adherent cells will couple the light out of the coverslip into the solution.  Cell walls will also do funny things with the evanescent wave.

Nevertheless, the ability to calibrate the incidence angle can be useful for repeatability.  Whether it is worth the cost is another consideration.  Just don't assume that you can set the exact same TIRF angle for every sample type and get the same excitation depth.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zac Arrac Atelaz
Sent: Monday, August 19, 2013 11:40 AM
To: [hidden email]
Subject: Re: Feedback on TIRF systems

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Bryon:
 
I have personally worked on the Olympus TIRF, the first versión with everything manual, and the new versión with the automated SIS system, and I have also have the chance to see the http://www.tirftechnologies.com/ system insert working with an Olympus IX81 microscope.
 
The manual TIRF from Olympus was a single user system as many manual adjustments were needed. Here is a great BUT, the new automated system is really impressive, you can adjust the depth from which you are having signal coming out. So the electromagnetic space adjacent to your coverglass is greatly controlled. All the laser alignment is made only once by the installation engineers and basically remains like that for life, they work now with led lasers, so maintenance would not be an issue, and expected life for leds is quite long.
 
Tirf tech have a very small system which is interesting, you might want to take a look to that one also.
 
I have not Heard from other TIRF systems
 
Best regards and happiness
 
Gabriel OH
 

> Date: Fri, 16 Aug 2013 11:49:38 -0500
> From: [hidden email]
> Subject: Feedback on TIRF systems
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
>
>
> We are currently looking at TIRF/widefield fluorescence microscopy
> systems from Olympus, Zeiss and DeltaVision and are seeking comments
> from users of these systems. We would greatly appreciate any feedback
> that users could provide. Since this is not directly a confocal
> microscopy question, I would be happy to receive comments directly
> ([hidden email]).
>
>
>
> Thanks so much,
>
>
>
> Bryon
>
>
>
> *******************************
>
>
>
> Bryon Grove, Ph.D.
>
> Associate Professor
>
> Department of Basic Sciences
>
> Director, Edward C Carlson Imaging and Image Analysis Core Facility
>
> UND School of Medicine and Health Sciences
>
> 501 N Columbia Rd Stop 9037
>
> Grand Forks, ND, 58202-9037
>
> Phone: [phone]
>
> Fax: [phone]

     

[John Oreopoulos]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I very much concur with Jay's statements. I have also noticed that overly confluent cells usually lead to very poor TIRF images, probably for the same reason that he mentioned. In addition to the uncertainty in the penetration depth caused by the sample index of refraction, there are additional scattering within the objective lens that can cause the penetration depth to deviate from single exponential behavior. This has been documented here:

Mattheyses, A.L. and D. Axelrod, Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence. Journal of Biomedical Optics, 2006. 11(1).

and confirmed here:

Oreopoulos, J. and C.M. Yip, Combined scanning probe and total internal reflection fluorescence microscopy. Methods, 2008. 46(1): p. 2-10.

I don't think any of the commercial systems account for this effect as it would vary for each objective lens. Bottom line is, you should calibrate your penetration depth yourself on any commercial system if you really care about its exact value. Most people are usually satisfied with eliminating the out-of-focus light near the cell surface, however, in which case the penetration depth value doesn't matter so much. All of the commercial systems I'm aware of are capable of meeting that requirement.

Sincerely,

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-08-20, at 2:32 PM, Unruh, Jay wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> If you intend to work on interfaces other than glass/water, be very skeptical of claims to determine the TIRF field penetration depth.  This is highly refractive index dependent and will depend strongly on cell type/adhesion strength, basement membrane composition, etc...  I have seen scenarios where adherent cells will couple the light out of the coverslip into the solution.  Cell walls will also do funny things with the evanescent wave.
>
> Nevertheless, the ability to calibrate the incidence angle can be useful for repeatability.  Whether it is worth the cost is another consideration.  Just don't assume that you can set the exact same TIRF angle for every sample type and get the same excitation depth.
>
> Jay
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zac Arrac Atelaz
> Sent: Monday, August 19, 2013 11:40 AM
> To: [hidden email]
> Subject: Re: Feedback on TIRF systems
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Bryon:
>
> I have personally worked on the Olympus TIRF, the first versión with everything manual, and the new versión with the automated SIS system, and I have also have the chance to see the http://www.tirftechnologies.com/ system insert working with an Olympus IX81 microscope.
>
> The manual TIRF from Olympus was a single user system as many manual adjustments were needed. Here is a great BUT, the new automated system is really impressive, you can adjust the depth from which you are having signal coming out. So the electromagnetic space adjacent to your coverglass is greatly controlled. All the laser alignment is made only once by the installation engineers and basically remains like that for life, they work now with led lasers, so maintenance would not be an issue, and expected life for leds is quite long.
>
> Tirf tech have a very small system which is interesting, you might want to take a look to that one also.
>
> I have not Heard from other TIRF systems
>
> Best regards and happiness
>
> Gabriel OH
>
>> Date: Fri, 16 Aug 2013 11:49:38 -0500
>> From: [hidden email]
>> Subject: Feedback on TIRF systems
>> To: [hidden email]
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>>
>>
>>
>> We are currently looking at TIRF/widefield fluorescence microscopy
>> systems from Olympus, Zeiss and DeltaVision and are seeking comments
>> from users of these systems. We would greatly appreciate any feedback
>> that users could provide. Since this is not directly a confocal
>> microscopy question, I would be happy to receive comments directly
>> ([hidden email]).
>>
>>
>>
>> Thanks so much,
>>
>>
>>
>> Bryon
>>
>>
>>
>> *******************************
>>
>>
>>
>> Bryon Grove, Ph.D.
>>
>> Associate Professor
>>
>> Department of Basic Sciences
>>
>> Director, Edward C Carlson Imaging and Image Analysis Core Facility
>>
>> UND School of Medicine and Health Sciences
>>
>> 501 N Columbia Rd Stop 9037
>>
>> Grand Forks, ND, 58202-9037
>>
>> Phone: [phone]
>>
>> Fax: [phone]
>    

[Kate Luby-Phelps]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Since the evanescent field decays exponentially as you get away from the refractive index
interface, penetration depth is actually somewhat arbitrary in any case. If you have a very
high concentration of fluorophore and a sensitive camera you can end up collecting signal
deeper than what you calculate from the equation. Direct measurement is best, as already
pointed out.

One other point regarding choice of systems: I would strongly advise choosing a microscope
stand with laser autofocus. Otherwise, small temperature fluctuations, etc. will make your
timelapse data unusable. Laser autofocus is offered by most if not all the major microscope
vendors. I only have experience with Nikon perfect focus, which was a game changer for us
after having two systems from two other vendors before hardware autofocus became
available.

Finally, a question for the list - does anyone know if there is a vendor that sells a system
with some sort of scrambler to get rid of interference fringes?


Kate

[John Oreopoulos]

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Kate, that's a good point too. I've had this kind of argument with other people before as well. A TIRF penetration depth of 100 nm does not mean you see only 100 nm deep into the sample (which is how many people interpret the meaning of this number). It simply means the intensity of the evanescent field decays to an intensity of 1/e over that distance. You're right that very bright, high fluorophore concentration objects/vesicles can appear in the image beyond the 100 nm depth. That is to say, it's not so straightforward to translate pixel intensity into object height above the glass coverslip because the intensity is related to the distance from the coverslip AND the fluorophore concentration (and a few other things)... Now, having said all that, there have been several papers in the past that did you show you could determine object heights if you have a good way of capturing images of the same thing at multiple penetration depths. For example:

Olveczky, B.P., N. Periasamy, and A.S. Verkman, Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy. Biophysical Journal, 1997. 73(5): p. 2836-2847.

But even there, there are some assumptions made in those kinds of methods. Related to this topic, some people might find of interest this early view paper:

Chiu, C.-L. and E. Gratton, Axial super resolution topography of focal adhesion by confocal microscopy. Microscopy Research and Technique, 2013
http://onlinelibrary.wiley.com/doi/10.1002/jemt.22267/abstract

This method seems much more robust to me and simpler.


John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2013-08-21, at 8:22 AM, Kate Luby-Phelps wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Since the evanescent field decays exponentially as you get away from the refractive index
> interface, penetration depth is actually somewhat arbitrary in any case. If you have a very
> high concentration of fluorophore and a sensitive camera you can end up collecting signal
> deeper than what you calculate from the equation. Direct measurement is best, as already
> pointed out.
>
> One other point regarding choice of systems: I would strongly advise choosing a microscope
> stand with laser autofocus. Otherwise, small temperature fluctuations, etc. will make your
> timelapse data unusable. Laser autofocus is offered by most if not all the major microscope
> vendors. I only have experience with Nikon perfect focus, which was a game changer for us
> after having two systems from two other vendors before hardware autofocus became
> available.
>
> Finally, a question for the list - does anyone know if there is a vendor that sells a system
> with some sort of scrambler to get rid of interference fringes?
>
>
> Kate

[Nic Cade]

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I wrote a brief review of our TILL iMic TIRF system back in January (below).

We are very happy with the system now, and the image quality and illumination
homogeneity are excellent.
Two of the main advantages are
- TIRF angle calibration is fully automated and reproducible, which is obviously
vital if you need consistent illumination conditions
- 360 degree TIRF illumination using fast scanning galvos, which completely
eliminates interference fringes / speckling

Cheers
Nic

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We have recently bought a Till iMic TIRF system as we needed a customised
solution for 3 channel imaging, which none of the other companies were able to
provide.

Despite a few teething problems with the optics, overall we are now very
happy with the performance. A few specific comments:

Software - fully automated control of filters, TIRF angle, acquisition parameters
etc. Different experimental protocols for e.g. timelapse, z-stack, multichannel,
multi-point, all of which can be modified or combine.

TIRF - precise and reproducible control of angle, automatic angle calibration,
elimination of interference fringes with the 360 illumination module. Very easy
to switch between TIRF and epi. After putting in some additional correction
optics, there is about 25% drop in illumination uniformity across the fov (100x).

Autofocus -  the back reflected laser signal is used to monitor and correct for
focus drift. It can be a bit temperamental if you are using very low laser power,
but otherwise generally works well and was stable in an overnight test.

As it seems they are still developing the system to some extent, the engineers
were happy to work with us to get the best design and performance. The only
real issues we have at the moment are
- in conventional 1-point TIRF (i.e without 360 module) there is a high degree
of laser speckle. TILL are coming back to replace some dichroics, which they
believe will eliminate this.
- only 3 different filter cubes can be used at any one time.  If necessary you
can 'hot-swap' the cube holder for a different set without turning the system
off.

Best
Nic