Several of the inherent limitations in photoactivated localization microscopy (PALM), such as the often high level of background signal contamination and long image acquisition times were overcome by altering the imaging mode in single-molecule superresolution imaging. By recording non-triggered, spontaneous off-on-off cycles without synchronization of the excitation illumination with the EMCCD camera system, a technique termed PALM with independently running acquisition (PALMIRA) was developed. This methodology is useful for photoswitchable fluorescent proteins and synthetic fluorophores, and accelerates images acquisition over 100-fold, reducing recording time to 2-5 minutes. Additionally, due to the large reduction in background fluorescence, PALMIRA enables imaging from within the interior of intact cells without requiring total internal reflection to narrow the depth of the region being imaged.
Egner, A., Geisler, C., von Middendorff, C., Bock, H., Wenzel, D., Medda, R., Andresen, M., Stiel, A. C., Jakobs, S., Eggeling, C., Schonle, A. and Hell, S. W.
Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophysical Journal 93: 3285-3290 (2007). One of the original research reports introducing the PALMIRA technique where the authors demonstrate fast recording speeds from the interior of intact cells. Presented are images of the microtubule network with a resolution of approximately 40 nanometers.
Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching. Applied Physics A 88: 223-226 (2007). Another report on the technique of PALMIRA from Dr. Stefan Hell's research group. The authors demonstrate the ability to reduce acquisition times to approximately 2.5 minutes, which is two orders of magnitude lower than many previous implementations.
Bock, H., Geisler, C., Wurm, C. A., von Middendorff, C., Jakobs, S., Schonle, A., Egner, A., Hell, S. W. and Eggeling, C.
Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Applied Physics B 88: 161-165 (2007). Using fast acquisition techniques, the authors demonstrate two-color superresolution imaging based on photoswitching a fluorescent protein (rsFastLime) and a synthetic carbocyanine (Cy5). The position of individual emitters was determined with an accuracy of approximately 20 nanometers.
Foelling, J., Belov, V., Riedel, D., Schoenle, A., Egner, A., Eggeling, C., Bossi, M. and Hell, S. W.
Fluorescence nanoscopy with optical sectioning by two-photon induced molecular switching using continuous-wave lasers. ChemPhysChem 9: 321-326 (2008). A novel rhodamine amide fluorophore is introduced that can be photoswitched from a dark to a fluorescent state with single or two-photon excitation. The authors demonstrate two-photon induced non-linear photoswitching with continuous-wave illumination and PALMIRA detection.
Stiel, A. C., Andresen, M., Bock, H., Hilbert, M., Schilde, J., Schonle, A., Eggeling, C., Egner, A., Hell, S. W. and Jakobs, S.
Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophysical Journal 95: 2989-2997 (2008). The authors engineer mCherry fluorescent protein variants, termed rsCherry and rsCherryRev, which display antagonistic (reversed) switching modes for single-molecule superresolution microscopy. PALMIRA was also used to demonstrate time-lapse live-cell imaging with the new fluorescent proteins.
Foelling, J., Belov, V., Kunetsky, R., Medda, R., Schoenle, A., Egner, A., Eggeling, C., Bossi, M. and Hell, S. W.
Photochromic rhodamines provide nanoscopy with optical sectioning. Angewandte Chemie International Edition 46: 6266-6270 (2007). After developing a new photochromic rhodamine derivative that isomerizes to form a bright fluorescent species upon activation with ultraviolet light, the authors use PALMIRA to achieve single-molecule superresolution images.
Testa, I., Schonle, A., von Middendorff, C., Geisler, C., Medda, R., Wurm, C. A., Stiel, A. C., Jakobs, S., Bossi, M., Eggeling, C., Hell, S. W. and Egner, A.
Nanoscale separation of molecular species based on their rotational mobility. Optics Express 16: 21093-21104 (2008). In order to observe rotational mobility, the authors combine single-molecule superresolution techniques with serialized recording of the switchable emitters matched to polarization-sensitive fluorescence detection.
3D nanoscopy: Bringing biological nanostructures into sharp focus. Angewandte Chemie International Edition 46: 8330-8332 (2007). A brief review article highlighting superresolution imaging using photochromic rhodamines and other photoswitchable fluorophores with single-molecule techniques. The authors point out the use of PALMIRA to achieve fast imaging rates and to alleviate problems with PALM and related techniques.
Juette, M. F., Gould, T. J., Lessard, M. D., Mlodzianoski, M. J., Nagpure, B. S., Bennett, B. T., Hess, S. T. and Bewersdorf, J.
Three-dimensional sub-100 nm resolution fluorescence microscopy. Nature Methods 5: 527-529 (2008). The authors introduce a three-dimensional single-molecule superresolution technique based on biplane imaging that combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM). This technique will also be useful with PALMIRA imaging strategies.
A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants. Journal of the American Chemical Society 129: 13970-13977 (2007). Using variants of the photoswitchable fluorescent protein Dronpa, the authors demonstrate application of fast PALM imaging, and introduce a new approach to achieve faster single-molecule superresolution by using simultaneous two-color stroboscopic illumination in a PALMIRA-related technique.