Optomechanics Are Rad - Part II

In my last installment, I described the construction of a dip-coating apparatus for applying thin films onto the tips of optical fibers.  With that out of the way, the next step involved the construction of an optics system for measuring the fluorescence produced by a thin film on the tip of an optical fiber.  And so that's what I set about doing today.  Similarly to the dip-coater, I started with a clean breadboard.  Because I am measuring fluorescence with this system, I wanted to minimize any stray light, so I enclosed the breadboard within a black box.  The box is hinged in such a way that the top and front can be opened.

Inside the box, I put my detector - a handheld spectrometer (left) - and my light source - a blue laser (right).  Not shown is the laser's power supply.  This unit is somewhat bulky and, since it doesn't necessarily need to be inside the box, I left it out.  By the way, I apologize for the blurriness of these images; this is the product of a mediocre phone camera combined with shaky hands brought on by a bit too much morning coffee.

I decided that I would take all of my measurements by submerging the fiber tip into a fluid sample, into which I could then inject the analyte that I'm trying to detect and thus monitor any change in the fluorescence output in real time.  I also wanted to illuminate the fiber tip with the laser transversely - or perpendicular to the direction of the fiber - by slicing the laser's coherent beam directly across the tip of the fiber to maximize my excitation intensity.  In order for all this to work correctly, I decided to use a cuvette to hold the sample, therefore minimizing scattering as the beam enters the liquid sample.  So I put a cuvette holder into the box and secured it in place using a few breadboard mounts.  Then I aligned the laser so that the beam was directed through the center of the cuvette holder and bolted it into place.

With the cuvette holder and laser aligned and anchored onto the breadboard, I then needed some way of holding the fiber in place.  In order to make sure that each fiber could be accurately positioned into the path of the laser, I bolted two micro-manipulators onto the breadboard to act as the base of the fiber holder while also allowing me to adjust the X and Y position of the fiber.

Onto the micro-manipulators I then added a fiber holder.  The fiber holder seen in this image came more-or-less preassembled.  The lower section has a turnscrew that clamps the fiber in place and I added the tube seen in the upper section to help keep the fiber straight as it enters the cuvette.  Astute observers might notice that the fiber holder I've used is manufactured by Newport, whereas pretty much every other optomechanic component I've used is manufactured by ThorLabs.  What can I say?  The Newport fiber holder was all I had on hand.  It was a bit of a pain in the ass getting it secured to the ThorLabs components because the dimensions and threading are different between the two manufacturers, but I got things worked out nonetheless.  And once the fiber holder was in place, the whole thing was finished.

To test the system out, I popped an empty cuvette into the cuvette holder and threaded a fiber through the fiber holder and down into the cuvette.  I attached the other fiber terminus - the one with the SMA connector - to the spectrometer and turned on the laser.  Then I used the micromanipulators to position the fiber tip directly into the beam of the laser.  You can see in the picture below that once the laser was incident onto the fiber, the blue laser light was scattered everywhere and fiber tip seemed to glow bright blue.

If there were a thin layer of my novel fluorescence sensor material on the tip of the optical fiber, which there is not because this was just a spare fiber that I found lying around, it would emit a bright green fluorescence when excited by the laser.  The fluorescence emission would be guided through the fiber and into the spectrometer.  The spectrometer is connected via USB to a laptop computer equipped with software that shows the real-time fluorescence spectrum being sent through the fiber.  And there you have it.  Pretty easy to set up and use.  By using a coherent light source along with a fluorescent sensor material coated directly onto an optical fiber, I've negated the need for lenses or other optics.  Once you start adding lenses, alignment becomes a major issue, and the components have to be aligned and adjusted precisely.  On the other hand, this system required only minimal alignment and went from a clean breadboard to a fully functional optical measurement system with about 2 hours of build time.

Now you can hopefully see why these optomechanics systems are so fun.  Once I finished building, part of me wanted to break the whole thing down and start over.  Just like I did with my LEGOs when I was a kid.

Optomechanics Are Rad - Part I

There are a lot of fun things about my job.  And I'd like to devote a couple of posts to one of my favorite aspects of developing optical sensors.  One challenge that arises occasionally is when the optical or fluorescent analysis instruments that we have on hand in the lab are not sufficient for analysis of the optical sensor platforms that I've developed.  This is often the case when dealing with flat, planar substrates onto which a sensor architecture or layer has been applied or when we apply a sensing mechanism onto the tip of an optical fiber.  When this happens, I have to build a bench-top optics system using our inventory of detectors (PMTs and hand-held spectrometers), light sources (LEDs and lasers), and optomechanics components that we keep in the lab just for this type of situation.  And let me just say this:  building a bench-top optics platform is outrageously fun.  It's like playing with LEGOs.  But these LEGOs are bigger, studier, and able to achieve precise alignments.  They also cost thousands of dollars.

My research project is nearing a point at which we will begin applying our fluorescent sensing material onto optical fibers (I'll discuss the application of a sensing mechanism onto optical fibers another time).  This has left me with a couple of specific needs that must be addressed before the project can move forward:  1) the sensor material must be dip-coated onto the optical fiber tip and to do this, the rate of withdrawal of the fiber tip from the precursor solution must be kept constant at a particular rate, and 2) I have to have some way of accurately measuring the fluorescence emission from the layer coated onto the fiber tip.  In this post, I'm going to focus on the dip coating apparatus.  As it turns out, the optomechanics components that are used to build bench-top optics systems are also quite useful for constructing a home-built dip-coater.  Here's how I did it:

First, I started with a plain breadboard.

Next, I bolted one large post into the center of the breadboard.  The large post allowed me to align the entire system vertically.

Onto the post, I clamped a small system of rails.  The top and bottom of the rails are secured using filter/lens holders.  I used these because they have large holes cut out of their centers.  In between the two filter/lens holders is a modified filter/lens holder that has a small metal cylinder attached with epoxy.  The cylinder is just the right size to a hold a 4 ml vial - and this will hold the precursor fluid that is to be dip-coated onto the optical fibers.  While the top and bottom holders are clamped tightly to the rails, the vial holder in the center was left loose, allowing it to slide up and down the rails freely.

Above the rail system I added a small v-clamp.  The clamp will hold the optical fiber in place during the dip-coating process.

Last, I added a small actuator and the accompanying controller.  The actuator moves the vial holder up and down, like an elevator.

When I clamp an optical fiber into place, you can hopefully see how this apparatus works.  The fiber is submerged into the fluid to be dip-coated onto the exposed tip.  Then I just flip a switch on the actuator and the vial holder slowly descends, withdrawing the fiber tip from the liquid.  When the fiber tip has been completely removed from the fluid, it can be removed from the apparatus and the thin layer that has been deposited on the tip is allowed to crosslink and/or cure into a solid film.

Now this setup was pretty simple.  And because no light sources or detectors were involved, alignment of the entire apparatus was not a big deal.  I just needed the little elevator to withdraw the fiber from the fluid at a constant rate.  My next project - which I'll be working on over the next couple of days - will be to build a system for measuring the fluorescence of the thin film dip-coated onto the fiber tip.  This will be slightly more difficult, though using the fiber itself to gather the fluorescence emission does make things much easier.  But more on that to come.

MUG: It's Not Just For Coffee Anymore

Recently, I have been playing around with a special cell culture media that uses a fluorescent indicator to signal the presence of E. coli bacteria. The culture media is a nutrient broth called LST-MUG, and is often referred to by its proprietary name, Hach media. The nutrient broth is similar to other types of cell culture media, with a couple of notable exceptions. First, the media contains surfactants that inhibit the growth of non-coliform organisms. This basically means that everything but E. coli is killed in the broth, allowing only E. coli to grow in the media. Second, the media contains a molecule called 4-methylumbelliferyl-β-D-glucuronide, or MUG. MUG is the part of the media that is responsible for indicating the presence of E. coli.

Under normal conditions, MUG emits very weak fluorescence at a wavelength down around where blue and UV meet, somewhere around 400 nm, when exposed to UV light. When MUG is introduced to E. coli, it is cleaved (or cut) into two separate smaller molecules by an enzyme within the bacteria called β-glucuronidase. One of the products of this cleavage reaction (the glucoronide part) is basically inert and does nothing at all. But the other product of the reaction is 4-methylumbelliferone, a highly fluorescent molecule. When exposed to the same UV light as before, the fluorescence of 4-methylumbelliferone is easily observable as a pale blue glow. In this way, the MUG acts a fluorogenic reporter of the presence of E. coli bacteria.

This bacterial growth media with the fluorescent reporter is commercially available and widely used to detect the presence of E. coli. However, it has some pitfalls. One problem with the media is that the sample to be tested must be introduced to the broth and then incubated, which is best done in a laboratory environment by a trained technician. A more pressing issue with the broth is its response time, which is 16-24 hours. If you are testing a sample of, say, raw beef, you would take a random in-line sample during processing and inoculate the Hach media. Twenty four hours later, a fluorescent signal is observed in the media, indicating that the beef is contaminated with E. coli. But that particular beef product is already on a truck on its way to the grocery store.

One of the projects that we're focusing on at LU's Center for Nanotechnology & Biosensors is a method for making this process much more effective and drastically faster, so that E. coli can be detected efficiently and contaminated products can be kept off of store shelves.

There's Nothing Wrong With People Wanting to Experiment

Let's start with a fluorescent hydrazine derivative, which looks like this:
This fluorescent dye has had the hydrazine group added so that it will be reactive to ketones and aldehydes. When the fluorescent dye is exposed to an aldehyde or ketone, it undergoes a dehydration reaction, something along the lines of this:

Now, the reactive dye has specific optical properties. These properties include quantum yield, which is basically the amount of fluorescent emission produced per input light. This means that a certain concentration of dye will produce a predictable fluorescence intensity when excited with a known intensity of illumination light. Other optical properties of interest are the excitation and emission wavelengths. These are the wavelengths of light at which absorption by the dye is maximum and the fluorescent emission of the dye is maximum, respectively.

So here's what I'm wondering. If you react the fluorescent hydrazine with a ketone or aldehyde, do any of these properties change? When the two molecules are combined, does the quantum yield change, making the fluorescent emission more or less bright than it was initially? Will the emission wavelength change, causing a shift in the color of the fluorescence output?

I think both of these possibilities are quite likely, particularly the emission wavelength shift. The reason I believe this is likely is that the makeup of the fluorescent molecule has changed when it combines with the ketone or aldehyde, essentially creating a new molecule. So why wouldn't the optical properties be altered in some way? I've been scouring the internet to answer this question, but have found no definitive answers thus far. So I guess there's only one way to find out. Conduct the experiment myself. And that's what I think I'll do.