How TIRF works on a Microscope

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TIRF is a widely used tool for effectively creating a super resolution instrument out of a standard widefield microscope. The cool part of TIRF is that it bypasses the limits of axial resolution on the scope not by altering the optics per-se, but by altering what gets excited. In a normal widefield fluorescent microscope, photons are emitted from an arc lamp, LED, or other source that are of numerous polarization, phase and angular states. In effect you  shower the specimen by flooding light through the objective, exciting any fluorescent molecule in the path of the light. The problem is that what gets excited may be inside, below or out of the objective’s focal plane. Any image you collect from such a setup represents both the “signal” (molecules in focus) and “noise” (molecules that aren’t in focus) in the field of view. More importantly, It’s common to have out of focus noise overpower in-focus signal, thereby reducing or restricting what information can be obtained from the microscope. My fancy cartoon below shows an example of a widefield system and it’s illumination path.

Typical fluorescent excitation

Of course, one simple solution, but an expensive one, to this problem is to add a pinhole-based confocal to the optical system. While a confocal does reduce or eliminate the out of focus haze in the image, it doesn’t provide any increase in Z accuracy. This axial resolution limit is still based on the objective’s performance.

TIRF avoids this limit by restricting the excitation field. In order to accomplish this, TIRF uses a laser, coupled to the microscope and most commonly delivered through the objective, to deliver excitation energy into the specimen. The trick is not that the laser excites the fluorescent molecules, the trick is that the laser, if set at a proper angle (critical angle) will bounce off of the bottom of a specimen container. When the laser bounces back there is a small electromagnetic field created, just on the specimen-side of the coverslip. This EM field is the same frequency as the bounced laser light, allowing it to excite fluorescent molecules. The field has an exponential decay as it extends from the coverslip, so it’s only strong enough to excite molecules that are very close to the interface of the coverslip and the specimen (usually the excitation field in TIRF is ~100nm of depth).

TIRF Cartoon example. Note the steep approach angle for the beam, which can only be used with high NA lenses (~1.49)

It’s hard to believe that light could be reflected back into a medium simply because of a refractive index change, but I was able to visually capture this effect on a large scale when installing a customer’s system at UC Davis. The image below is of a fluid filled jar, with some bits of glass tube floating in it. The cool part is the laser beam. You can see the beam emitting from the objective and passing through the fluid. When the beam reaches the air (lower refractive index),  the refractive index change is at or beyond the critical angle the light can follow, so the light is reflected back into the previous, higher refractive index medium (the fluid). This is the fundamental effect that makes TIRF possible.

Laser emits from lens, but is trapped in fluid and reflected back down.

The limitations to this technique are that you need a laser, you need an optic that can reach the critical angle, and you need to have something in your research that can sit on the coverslip, i.e. this won’t work for anything farther than ~100nm from the coverslip. If everything lines up, you get an image representing roughly a 10x improvement in Z resolution over a confocal microscope, qualifying this as a “super resolution” technique.

If you’d like to learn more about the theory of TIRF microscopy I highly recommend the MicroscopyU explanation, which does a better job of explaining the physics than my overview here.

***Thanks to Professor Jawdat Al-Bassam of UC Davis for allowing me to demonstrate this effect on his instrument!

-Austin

Update on Andor Neo Camera

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I had an opportunity to demonstrate the Neo camera on site with a customer yesterday at UC Davis. We captured some great images of GFP tagged pathways on PKC cells. We were able to image the cells at exposures down to 1ms @ 2×2 binning using Metamorph. We were also able to capture extreme fields of view while scanning Z at full resolution and 1×1 bin, using 3-10ms exposures. The combination of the capture resolution and short exposures really change how you use the scope. I think the use of oculars on a fluorescent microscope might be sunsetting with the advent of these sensors. I’ll post some data from this demo once it’s completed.

 

Neo camera at 100fps

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Andor’s Neo camera has been out for about a year now. I first wrote about using it back in February. The camera immediately showed the major difference in field of view between sCmos sensors and traditional interlines quite well, but the speed, binning, sub-array and bias clamp controls weren’t available. Over the past few firmware/SDK updates, Andor has steadily addressed these limitations. I had a chance to field test the latest combo of firmware and driver SDK. Basically I found a camera that runs at 100fps with roughly four times the field of view of a typical Sony285 type sensor. In my last post I noted that people would wait until end user software platforms could take advantage of the sCmos architecture. I think the wait might be over.

I’ll continue to report on this as I have an opportunity to put it through it’s paces. I’m curious to see how people respond to it’s performance…

-Austin

Hands on with Imaris Bitplane

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During my company sales meeting I had the opportunity to receive training on Imaris Bitplane, which we are now able to sell in our dealership territory. I’ve come across this software package off an on over many years, and have always wanted to get under the hood and check it out. Below is an example of what the software can do. In this case the program identified neuronal processes, as well as tagged nodes for 3-D analysis.

The software fits the needs of a very specific market.  I would suggest looking at this software only if you already own or are buying a confocal of some type, or have a very good familiarity with deconvolving images. You would also have a need to visualize 3-D interactions or structures which occur on a larger scale than that of your axial resolution limit, and/’or have a need to measure volumetric objects either in a single data stack, or in a timelapse.

Software like this has an advantage over standard imaging software in that it is specifically targeted to do one thing and do it well. With general-use imaging products, the developers and support engineers are spread thin writing code to do everything from image analysis in 2-D, to device driver updates, to archive connections, boutique analysis protocols (i.e. FRET), image processing filters and so forth. The advantage of a specifically targeted product is that the engineers can concentrate on making the key jobs of the software work as well as possible, and because it has a targeted application, the interface can be built around a known user workflow. In this case Bitplane does extremely well with measuring and visualizing anything in 3-D, and has a very powerful 2-D/3-D tracking capability.

As with all new technologies I’m exposed to, this tool looks great -- but I haven’t learned it’s limits by working on real world data. In an effort to discover where this software fits, I’d like end-users to provide some data sets with requests for analysis from the data, so I can both learn and test this software. The upside for users is that they get to find out if this tool might help them, simply by uploading a stack. You can also get a cool 3-D movie like the one above to show off at the next lab meeting :-)

If you’re interested in finding out what this tool can do, and you have some data handy, please place a comment below, and I’ll email you my FTP upload information. I’m quite curious to see how this software stands up to some real world challenges.

-Austin

SOPA and the future of the internet

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I don’t normally post on political subjects here, but I believe, along with Google and many others in the online community, that the progressing “SOPA” bill carries a major potential for government censorship of speech. A line by line analysis of the bill can be found here ,with other perspectives here and here. I encourage US voters to research this bill (and it’s sister bill “PIPA”), and contact your legislator if you feel led to do so. My goal here is only to inform that this bill is moving forward and that at minimum we as voters should be informed of things like this, before they become law.

-Austin