Making scientific images : Super-resolution microscopy (2)
In my previous post, I talked briefly about resolution and why it’s important in microscopy. When you want to take pictures of stuff that you can’t see with your eyes, you want it to have the best resolution possible. Light microscopy is limited by the diffraction of light, which usually means that at best, we can have a resolution of 250 nm. That means that two points can be seen as seperate entities if they are 250 nanometers apart from each other, or 0,000000250 meters. It seems like a lot, but sometimes we need a better resolution. Electron microscopy can achieve this, but at the cost of many advantages that light microscopy can bring. Mainly the ability to stain proteins and organelles very easily. How does that work ?
We use antibodies. Normally, antibodies are part of the immune system. They recognize what we call antigens, that can be harmful to the organism. Scientists can thus produce antibodies that will recognize and attach to their protein of interest. These antibodies will in turn be recognized by other antibodies that are coupled to a fluorescent molecule. Meaning that it will emit a colored light when excited by a laser.
What does it mean ? We can stain for specific proteins, and they will emit a fluorescent light that we can detect using a light microscope. So we can pinpoint where they are inside cells. These signals, as mentioned before, are limited by the diffraction of light. To enhance the resolution, people had to get crafty. And on October 8, 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, W.E. Moerner and Stefan Hell for "the development of super-resolved fluorescence microscopy”. They invented a way to bypass the limitations of light microscopy and bring it to the nanodimension.
Since then, several ways to achieve super-resolved light microscopy were developed, I’ll mention a few of them and how they work.
- Structured illumination microscopy (SIM) : Different pictures are taken of different phases of the sample and then assembled by a computer, enhancing the resolution.
Dronpa–Lifeact in a mammalian CHO cell imaged with NL-SIM. (A) On the left, a portion of the entire image is shown with conventional microscopy. The image is displayed using a nonlinear intensity scale (gamma = 0.65) to highlight the small filaments in the background over the thick and bright stress fibers. A subset of the data is enlarged and shown with (B) conventional TIRF microscopy, (C) linear SIM-TIRF, and (D) nonlinear SIM-TIRF. Lifeact marks the actin network, the structure of which is most clearly resolved with nonlinear SIM-TIRF.
Live 3D SIM imaging of mitochondria labeled with MitoTracker Green and the actin cytoskeleton labeled with tdTomato-LifeAct in a HeLa cell over 30 time points. Imaged with conventional microscopy (A) and 3D SIM (B).
In these images of bovine pulmonary artery endothelial cells, the mitochondria are stained with MitoTracker Red CMXRos (red), and F-actin is stained with BODIPY FL phallacidin (green). On the left is a standard wide-field fluorescence image that includes signal fluorescence from above and below focus. On the right is the same image following deconvolution using the Zeiss Apotome Structured Illumination System. (Jon Ekman - ITG, Beckman Institute)
Mitosis, the cellular division that produces two genetically identical daughter cells, is perhaps the most fundamental process in biology. Without it, multicellular life wouldn’t exist, a broken bone would never heal, tissues would disintegrate. Cancer — essentially mitosis gone rogue — also wouldn’t exist. Scientists have been watching mitosis through a microscope for about 150 years, but new views are fleshing out the less-detailed pictures of the past (bottom, right). After DNA replicates, the nuclear envelope surrounding it dissolves. Spindle fibers (gold above, red at right) align pairs of chromosomes (blue) and then separate the genetic material into two daughter cells (shown forming, above).
- STORM, PALM and FPALM : Stochastic optical reconstruction microscopy (STORM), photo activated localization microscopy (PALM) and fluorescence photo-activation localization microscopy (FPALM) are super-resolution imaging techniques that utilize sequential activation and time-resolved localization of photoswitchable fluorophores to create high resolution images. In short, fluorescent molecules are switched on at random intervals, and the signals form the final image.
Nuclear pore complexes, PALM superresolution microscopy : Xenopus laevis A6 cells (epithelial kidney cells). gp120, a nuclear pore complex protein arranged in an eightfold symmetry (labeled with Alexa 647 conjugated antibodies). Courtesy of A. Löschberger, M. Sauer, University of Würzburg, Germany.
Dual color PALM microscopy imaging of a kinase (red) and an adapter protein (green). Left: orinary resolution image, right PALM image with zoomed region of interest on the bottom.
Samples were prepared using a setup for automated immunostaining. (A) One color STORM image of mitochondria (Tom20). (B) Two-color STORM image of mitochondria (Tom20) and microtubules (alpha-tubulin). In both panels, the zoomed region shows the same region imaged using conventional epifluorescence microscopy (left) and STORM (right). Scale bars, 5 µm in top images, and 1 µm in bottom images (zoomed regions).
The tiny diameter and high density of actin filaments are visible in the sheetlike protrusions at this monkey kidney cell’s edge (color-coded by depth, red farthest away). Scientists are still trying to figure out precisely how these sheets form and connect to the cell’s interior so they can understand more about how cells travel. Credit: K. Xu, H.P. Babcock and X. Zhuang/Nature Methods 2012
Hippocampal neurons labeled for actin (orange) imaged by STORM and synapsin (blue) imaged by TIRF. (NeuroCyto)
Check out NeuroCyto Lab’s STORM gallery for more !
- STED microscopy : Stimulated emission depletion (STED) uses two lasers pulses, the excitation pulse for excitation of the fluorophores to their fluorescent state and the STED pulse for the de-excitation of fluorophores by means of stimulated emission. This allows to “bleach” around the center of the STED beam :
Confocal (left) and super resolution (right) microscopy image of tubulin stained with LIVE 580 tubulin (cabazitaxel) in living human fibroblasts.
STED image of triple immunostaining in HeLa cells: Green: NUP153-Alexa 532, red: Clathrin-TMR, white: Actin-Alexa 488.
You can check Leica’s and Zeiss’ galeries for more pictures. I’ll see you later on the other side of the lens.
