Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution well below the diffraction limit. However, resolution in the double-digit nanometer range can be obtained only using high intensity depletion laser, at the cost of increased photo-damage, which significantly limits STED applications in live specimens. To minimize this, we use the separation by lifetime tuning (SPLIT) technique, in which phasor analysis is used to efficiently distinguish photons emitted from the center and from the periphery of the excitation spot of a STED microscope. Thus, it can be used to improve the resolution without increasing the STED beam intensity. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. The photons stream are successively analyzed using the SPLIT technique, demonstrating that the resolution improves without increasing the depletion laser intensity.
Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.