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Advice on increasing S/N for dual channel single molecule TIRF setup

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Brad Nolen

Advice on increasing S/N for dual channel single molecule TIRF setup

We have a Nikon TE2000 that we’re using for through the objective TIRF to try to image Alexa488 and Alexa 568 labeled proteins with single molecule sensitivity. We’re using a Chroma ZT488/561rpc TIRF dichroic and a ZET488/561m TIRF emission filter, and we are currently demoing various EMCCD 512x512 cameras. While we are able to image single molecules, the signal is very weak. One possible culprit is high background due to a small percentage of unblocked excitation light. We are wondering if anyone with single molecule experience can give us some input on the following:

1. Is it usual with through the objective SM TIRF setups to need additional cleanup filters to get acceptable S/N?

2. If so, what is the best way to do this? The folks who sell filters have recommended additional single bandpass cleanup filters. We anticipate needing fairly fast acquisition times (~30ms), so we are hesitant about using a filter wheel. A second option would be a beam splitter like the optiview-II or the Dualview, which would allow us to pass through the single bandpass emission filters without loss of temporal resolution, but which has the disadvantage of some signal loss.

Any advice or input would be greatly appreciated, even if it’s a just a link to a good technical reference that might help us make a decision on this.

Thanks

Brad Nolen

University of Oregon

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Charles Felts

Re: Advice on increasing S/N for dual channelsingle molecule TIRF setup

I would suggest posting this question on the confocal list serv.

Best,

Charles Felts

From: Brad Nolen [mailto:[hidden email]]
Sent: Wednesday, November 16, 2011 5:45 PM
To: [hidden email]
Subject: [micro-manager-general] Advice on increasing S/N for dual channelsingle molecule TIRF setup

1. Is it usual with through the objective SM TIRF setups to need additional cleanup filters to get acceptable S/N?

Any advice or input would be greatly appreciated, even if it’s a just a link to a good technical reference that might help us make a decision on this.

Thanks

Brad Nolen

University of Oregon

Holden Seamus John

Re: Advice on increasing S/N for dual channel single molecule TIRF setup

In reply to this post by Brad Nolen

Relevant:

Imaging Single Molecules Using Total Internal Reflection Fluorescence Microscopy (TIRFM)

Samara L. Reck-Peterson, Nathan D. Derr and Nico Stuurman

doi:10.1101/pdb.top73

Discusses dichroics and filters about halfway down.

On 16/11/11 23:44, Brad Nolen wrote:

We have a Nikon TE2000 that we’re using for through the objective TIRF to try to image Alexa488 and Alexa 568 labeled proteins with single molecule sensitivity. We’re using a Chroma ZT488/561rpc TIRF dichroic and a ZET488/561m TIRF emission filter, and we are currently demoing various EMCCD 512x512 cameras. While we are able to image single molecules, the signal is very weak. One possible culprit is high background due to a small percentage of unblocked excitation light. We are wondering if anyone with single molecule experience can give us some input on the following:

1. Is it usual with through the objective SM TIRF setups to need additional cleanup filters to get acceptable S/N?

2. If so, what is the best way to do this? The folks who sell filters have recommended additional single bandpass cleanup filters. We anticipate needing fairly fast acquisition times (~30ms), so we are hesitant about using a filter wheel. A second option would be a beam splitter like the optiview-II or the Dualview, which would allow us to pass through the single bandpass emission filters without loss of temporal resolution, but which has the disadvantage of some signal loss.

Any advice or input would be greatly appreciated, even if it’s a just a link to a good technical reference that might help us make a decision on this.

Thanks

Brad Nolen

University of Oregon
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security threats, fraudulent activity, and more. Splunk takes this 
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Nico Stuurman-4

Re: Advice on increasing S/N for dual channel single molecule TIRF setup

In reply to this post by Brad Nolen

Hi Brad,

We have a Nikon TE2000 that we’re using for through the objective TIRF to try to image Alexa488 and Alexa 568 labeled proteins with single molecule sensitivity. We’re using a Chroma ZT488/561rpc TIRF dichroic and a ZET488/561m TIRF emission filter, and we are currently demoing various EMCCD 512x512 cameras. While we are able to image single molecules, the signal is very weak. One possible culprit is high background due to a small percentage of unblocked excitation light. We are wondering if anyone with single molecule experience can give us some input on the following:
1. Is it usual with through the objective SM TIRF setups to need additional cleanup filters to get acceptable S/N?

Yes. We have two single molecule scopes (a TE2000, and a TI), and I spend significant time speccing filters for these systems. Since the most of the illumination power travels back into the microscope, through the objective type TIRF illumination requires emission filters that block the illuminating light extremely well.

On our systems we use quadruple mirrors (405, 488, 561, 640 reflection), have cleanup excitation filters (these were essential in our TE 2000, even though the lasers were spectrally pure, we rationalized with the idea that there are reflections in the illumination pathway resulting in light hitting the emission filters under strange angles making them perform less than optimally, excitation filter cuts out these weird angles), laser line blocking quadruple emission filter (we first used the Semrock notch until Chroma made a nicer and cheaper one). These three are all mounted in a Chroma cube. The cube itself is also important. Until a few years ago, the Nikon cubes caused the mirrors to bend resulting in astichmatism. Not sure where the Nikon cubes stand these days, but the Chroma cube with the thicker mirrors work well.

On top of this, we often need additional emission filters. These are sitting in a filter wheel. This works all very well, and we easily and routinely do single molecule imaging. I can send you the part numbers for our filters (but these are specced to work with 4 wavelengths).

2. If so, what is the best way to do this? The folks who sell filters have recommended additional single bandpass cleanup filters. We anticipate needing fairly fast acquisition times (~30ms), so we are hesitant about using a filter wheel. A second option would be a beam splitter like the optiview-II or the Dualview, which would allow us to pass through the single bandpass emission filters without loss of temporal resolution, but which has the disadvantage of some signal loss.

I never liked these much, in part since they incur significant light loss, and if you are not careful, loss of effective NA. Why not get a filter wheel? You can then install filters for the individual wavelengths as well as another of your ZET488/561m filters. You can use the latter for excitation switching, and - if you have a compatible device controlling your AOTF - you would be able to acquire multi-channel images at speeds only limited by the readout time of your camera.

Best,

Nico