Design Stories – fluorcyte

Design Stories

FluorEx Lambda

The FluorEx graphic is based in part on the shape of the greek letter lambda

In the physical sciences, we use the lower case Greek letter lambda (λ) as a symbol to  describe the color of light, or wavelength. This letter's shape serves as a sort of base for the FluorEx graphic - representing light.

To explain what the colors in the designs mean, we need to learn a little bit about what fluorescence actually is.

We can think of light as a bunch of little particles called photons. With lasers, we can get a bunch of photons with a similar color  to travel in a general direction. And we can harness these photons to do some pretty cool stuff.

One of the things that has been revolutionary for scientists is the phenomenon of fluorescence. Certain molecules - or fluorophores - can absorb certain colors of photons. These molecules effectively catch some of these photons and throw them back into their environment while taking some of their energy in the process. This thrown photon will have less energy than the original caught photon. And when photons change energy, we can see this as a change in color. These re-emitted photons with a different color are called fluorescent photons or fluorescent light.

But what does that look like? Something like this:

A 488-nm laser beam exciting fluorescence in a solution containing a green-emitting fluorophore, Alexa488.

[Image courtesy of Dr. Bryan Millis, Vanderbilt Biophotonics Center - Vanderbilt University]

Notice there's a blue-ish-looking laser traveling into a vial of water with a little bit of a green fluorescent molecule dissolved in it. This particular molecule absorbs blue-ish colored photons and re-emits them as green photons. The physical/chemical process of molecules taking some of the light it's given and changing it's color is what we call fluorescence.

Explanation of the chemophysical basis behind the FluorEx graphic.

The concept of fluorescence excitation and emission is what serves as the color inspiration of the FluorEx graphics. Each graphic is based off of a different fluorescent molecule based on what wavelengths it absorbs or emits. The photons we throw at the fluorescent molecules (or the excitation light) interact with our sample molecules and can generate a different color of light. Some the thrown photons will reflect off of the sample surface - which we refer to as 'specular reflection'. This is partially the same reason you can see a weak reflection of yourself in a transparent window.

 

For the FluorEx logo, I used the concept of specular reflection and fluorescence to generate a abstracted shape of the letter lambda - to represent light.

Different molecules will have different excitation and emission colors. The range of colors here serves as the basis for a bunch of different fluorescent molecules that we use in the lab regularly to do research (more on that below).

Range of FluorEx graphics designed to represent different fluorescent molecules.

Spectral

So hopefully I've convinced you that fluorescence is cool. But now there's some important details that we need to keep in mind when using these fluorescent molecules - particularly in how we measure or image them.

Turns out, fluorescent molecules can absorb more than one color of light. And likewise, they usually emit more than one color of light. This is important in how we build instruments to look at these proteins. And the way we get an idea of what colors fluorophores absorb and emit is by measuring and  representing this information on a graph called an Excitation-Emission (or Ex-Em) profile.

Example excitation-emission spectrum of EGFP, from FBbase.org

The Ex/Em profile of green fluorescent protein (or GFP) is shown above. The x-axis of the graph represents wavelength, or color) of the light being absorbed or emitted. 700nm light is roughly the color red, 400nm light  is roughly the color violet, and the colors of the rainbow (remember ROYGBIV) fall sequentially in between. With GFP, we can see that it absorbs (or is excited by) blueish light, most strongly around 488nm. And it's emitted photons are mostly green dominantly around 515nm. When we want to image a particular fluorophore on a microscope, this information lets us pick what lasers / lights and optical filters we need to choose to image most effectively.

These Ex/Em profiles for different fluorophores serve as the basis for the Spectral graphic t-shirts. But I typically take it a step further and use the Ex/Em curve of 2 fluorophores. One of our more popular shirts is the Ex/Em profile of GFP and a protein called mCherry, which absorbs orange light (~560nm) and emits dominantly in red (~ 625nm). These two proteins are often used in combination because they are different colors that can be easily separated with filters based purely on color.

EGFP and mCherry excitation-emission spectra

When we're trying to figure out what fluorophores to use, there's often much more than color to consider (e.g. brightness, efficiency, dimerization tendancies, fluorescent lifetime). It's a lot to think about. So we use a bunch of tools, like those on FPbase.org to help guide us to picking the right fluorophores for our applications. They even and help a bit in  sourcing the chunks of DNA to necessary to do that.

What's crazy is that FPbase.org  is totally free, open source, community driven, and continually growing. Coordinating such an effort is hard work. And because of that, a significant portion of the profit made from the spectral line of products offered on fluorcyte.com goes directly to helping maintain FPbase.org.

The Bigger Picture

So who cares? Fluorescence looks cool. But why is this important?

Human iPSC-derived neural cell co-cultures. Red (NeuN) and Grey (GFAP).

It turns out that there are a handful of fluorescent molecules that occur naturally in living organisms - there's even a bunch of them in humans! Weirdly, a bunch of different cyanobacteria and jellyfish already make special proteins that are brightly fluorescent. This is really important because the fact that fluorescent proteins exist in nature means that there is DNA that code for them - which we can get from these organisms.

So in the lab, we can take the DNA that codes for these fluorescent proteins from these different organisms and stick them into cells that we want to learn about. This makes the cells we want to study fluorescent.

We can make these fluorescent proteins only show up in certain cells in the body where specific genes we might want to study are active. Having tools like this let us figure out what genes are important to controlling all different facets of our life.

We can also make these fluorescent proteins attach to other non-fluorescent proteins we want to learn about. With this approach, we can look at where these non-fluorescent proteins travel to within cells or body - giving us clues on what those proteins do.

We can get different colored fluorescent proteins to look at multiple different things in cells at once. And slowly, we can begin to build up an understanding of what all these genes we have do in each of the cell in the body. We can figure out if they have any impact on health or disease to help develop new drugs and therapies.

We can also modify these proteins to make them different colors, or react to different processes in cells, watching them live and interact with other cells in real time. And when using genetically encoded proteins doesn’t work, we can use fluorescent small molecules targeted to certain features of cells that we want to learn about in normal and diseased states.

Fluorescence has revolutionized the way we study living things - and will continue to do so for many years to come. Scientists and engineers today are constantly developing better, brighter, more responsive fluorophores and instruments to measure and image them more effectively than ever. These developments constantly lead to new discoveries in biology. They're used widely to study tons of facets of biology and physiology, like brain function, heart biomechanics, and even the ways the immune system works (or sometimes doesn’t work….). This knowledge helps us develop new drugs and therapies for treating diseases improving outcomes from surgery.