SPIFFI Turns Every Microscope Frame Into a Super-Resolution Movie of Living Cells

Living cells refuse to pose. Mitochondria pinch apart and fuse back together in seconds. Microtubules slide, overlap, and rearrange. Ordinary light microscopes smear those events once details fall below roughly 200 nanometers, the natural limit set by how light waves spread. Older super-resolution methods beat that blur by stacking hundreds or thousands of frames and combining the tiny brightness changes that appear over time.

The approach works when samples have been chemically fixed in place. On living cells the same structures keep moving, so the stacked frames turn into a smear. Wei Guo, first author and a PhD student at EPFL’s Laboratory of Nanoscale Biology, noted that earlier techniques used time to buy spatial sharpness, a trade that fails when the subject will not wait.

SPIFFI, also known as spatial polarization-induced fluorescence fluctuation imaging, is a game changer. The method was developed by a team from EPFL’s School of Engineering, including Guo, Lely Feletti, and Aleksandra Radenovic, and published in Nature Methods. Now, you may assume that fluorescent tags, which biologists currently employ to investigate their samples, are simple, but the light they release is polarized in different directions depending on how the molecules are arranged.

The brilliance of SPIFFI is that it takes that light and splits it into four separate polarization directions at the same time, and it also records all four images in real time. This means that you can compare those four pictures side by side and obtain a lot more spatial detail than you would with a single typical image. In fact, your resolution roughly doubles, allowing you to see structures as small as 160 to 170 nanometers across. The best part is that every frame in a series is sharpened as it progresses, allowing you to record true super-resolution films of living cells rather than simply constructing a high-resolution still from a slew of individual frames. Then, with a little extra processing that doesn’t require nearly as many frames as previous methods, you can reduce it to approximately 80 nanometers, which is simply fantastic.

According to Aleksandra Radenovic, the SPIFFI studies demonstrate that it is possible to gain a real look at fast-moving processes inside cells while also doing multidimensional imaging at considerably faster rates than conventional microscopes. It’s resulted in some very remarkable outcomes so far, such as being able to show mitochondria’s outer membranes as really neat, hollow tubes rather than just blobs on a screen. Also, the team was able to zoom in on fission and fusion events as they occurred, as well as gain a better understanding of how microtubules travel without the motion blur that usually distorts the picture. As an added bonus, the approach provides useful information about how the fluorescent molecules are orientated, which has some very fascinating implications for understanding protein structure.

The good news is that this additional piece of equipment can simply be added to your existing fluorescence microscope, eliminating the need to replace it. The team is now working on reducing the size of the entire setup and pairing it with 3D scanning, so that researchers can get a true view of all those nanoscale dances in both space and time, which will be pretty cool.
[Source]