scholarly journals Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

2021 ◽  
Author(s):  
Takahiro K. Fujiwara ◽  
Shinji Takeuchi ◽  
Ziya Kalay ◽  
Yosuke Nagai ◽  
Taka A. Tsunoyama ◽  
...  

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the world′s highest time resolutions for single fluorescent-molecule imaging of 33 (100) µs (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640x640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research.

2009 ◽  
Vol 185 (7) ◽  
pp. 1135-1148 ◽  
Author(s):  
Jennifer C. Waters

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.


2020 ◽  
Vol 6 (16) ◽  
pp. eaay8271 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.


Photonics ◽  
2021 ◽  
Vol 8 (7) ◽  
pp. 273
Author(s):  
Lixin Liu ◽  
Meijie Qi ◽  
Yujie Liu ◽  
Xinzhu Xue ◽  
Danni Chen ◽  
...  

Fluorescence imaging is an important and efficient tool in cell biology and biomedical research. In order to observe the dynamics of biological macromolecules such as DNA, RNA and proteins in live cells, it is extremely necessary to surpass the Abbe diffraction limit in microscopic imaging. Single-molecule localization microscopy (SMLM) is a sort of super-resolution imaging technique that can obtain a large number of images of sparse fluorescent molecules by the use of photoswitchable fluorescent probes and single-molecule localization technology. The center positions of fluorescent molecules in the images are precisely located, and then the entire sample pattern is reconstructed with super resolution. In this paper, we present a single-molecule localization algorithm (SMLA) that is based on blind deconvolution and centroid localization (BDCL) method. Single-molecule localization and image reconstruction of 15,000/9990 frames of original images of tubulins are accomplished. In addition, this fluorophore localization algorithm is used to localize high particle-density images. The results show that our BDCL-SMLA method is a reasonable attempt and useful method for SMLM imaging when the imaging system is unknown.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anindita Dasgupta ◽  
Joran Deschamps ◽  
Ulf Matti ◽  
Uwe Hübner ◽  
Jan Becker ◽  
...  

Abstract3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.


2020 ◽  
Author(s):  
Anindita Dasgupta ◽  
Joran Deschamps ◽  
Ulf Matti ◽  
Uwe Hübner ◽  
Jan Becker ◽  
...  

Abstract3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for z localization in a method called supercritical angle localization microscopy (SALM). Here, we realize the full potential of SALM by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying new fitting routines to extract precise intensities of single emitters, resulting in a four-fold improved z-resolution compared to the state of the art. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.


Cell Reports ◽  
2020 ◽  
Vol 33 (2) ◽  
pp. 108248 ◽  
Author(s):  
Samantha Kent ◽  
Kyle Brown ◽  
Chou-hsun Yang ◽  
Njood Alsaihati ◽  
Christina Tian ◽  
...  

2001 ◽  
Vol 80 (5) ◽  
pp. 2396-2408 ◽  
Author(s):  
Gregory S. Harms ◽  
Laurent Cognet ◽  
Piet H.M. Lommerse ◽  
Gerhard A. Blab ◽  
Thomas Schmidt

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