With LSM 880 with Airyscan, photobleaching of the samples was drastically diminished. We could monitor the dynamics of ER and lysosomes live more than 3 hours in the same region in a cell, with no trade-off of the resolution of the imaging.
This fact changes our experimental design for live cell imaging: Now we can monitor cellular responses with relatively slow kinetics from beginning to end. For example, one that will peak several hours after stimulation. This is indeed what we seek for!
EGFP-tagged endoplasmic reticulum (ER) protein (green), mScarlet-tagged lysosomal protein (red)
I am interested in the interplay between lipid droplets and lysosomes within the cell. ZEISS LSM 880 with Airyscan provides the excellent resolving power and fast acquisition speeds necessary to monitor the interactions between these small structures.
Airyscan was able to reveal the inner structure of the organelles without having to compromise in regards to the selection of fluorescent dyes or the duration of image acquisition. We also did not have to deal with complex image processing.
It has been difficult to study the inner structures of organelles in living cells using conventional fluorescence microscopy. These images, taken with ZEISS Airyscan, show superresolution fluorescence imaging of cytoskeleton (actin filaments) and mitochondria in living COS7 cells.
Actin: SiR-actin (Spirochrome)
Mitochondria: MItoTracker Green FM (Thermofisher)
There are a number of super resolution techniques available today, but many require fixed samples, special fluorophores, or slow rates of image acquisition. We've been hoping for one that would perform well for live cell imaging. This means the microscope would acquire images quickly, use low light doses to prevent damage to live cells or fluorophores, work with traditional fluorophores, and be easy to use. We also wanted a system that is sensitive enough to pick up genetically encoded fluorophores expressed at low levels…”
For us, one of the best selling points, was the Airyscan Fast mode. It was just incredible how quickly we were able to image live cells. That is where ZEISS really set itself apart.
"Neurons expressing genetically encoded glutamate sensor, iGluSnFR, collected with ZEISS Airyscan Fast mode. Rapid acquisition of images using Fast allowed resolution of the fine details of dendritic spine heads with comparable image quality to Airyscan SR in much shorter time."
"We need high speed imaging for Z-stacks of our living cells and FRAP experiments."
"FRAP experiments with Airyscan Fast were successful despite low endogenous levels of fluorescence at the plasma membrane. This was tested because it is the most difficult of all our FRAP applications."
FRAP time series of fluorescent glutmatate sensor targeted to the plasma membrane of E18.5 Rat Hippocampal neurons @ 15DIV. Marvin, J.S., Borghuis, B.G., Tian, L., Cichon, J., Harnett, M.T., Akerboom, J., Gordus, A., Renninger, S.L., Chen, T.W., Bargmann, C.I. and Orger, M.B., 2013. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nature methods, 10(2), p.162.
"HEK 293T cells transfected with mitochondrial OMM targeted msGFP and ER lumen targeted mRuby3. The system was able to clearly image ER tubules, which is usually challenging in this cell type. In most cells, the ER exists as such a fine network of tubules that traditional light microscopy cannot resolve individual tubules and instead an ER sheet is observed. In fact, many people who study the ER, use a specific cell line, COS-7 cells, because the COS-7 ER network is more spread out at the cell periphery which allows researchers to image the ER tubules. Finally, we were able to clearly image ER tubules in HEK cells."
Airyscan allows me to more easily see the scolopidia of Johnston's organ to assess for morphological defects.
Johnston's organ is very small and can be difficult to get high resolution images using a normal confocal. Airyscan allows me to more easily see the scolopidia of Johnston's organ to assess for morphological defects.