@article {69959, title = {Resolution in super-resolution microscopy - facts, artifacts, technological advancements and biological applications}, journal = {J Cell Sci}, volume = {138}, year = {2025}, month = {2025 May 27}, abstract = {

Super-resolution microscopy (SRM) has undeniable potential for scientific discovery, yet still presents many challenges that hinder its widespread adoption, including technical trade-offs between resolution, speed and photodamage, as well as limitations in imaging live samples and larger, more complex biological structures. Furthermore, SRM often requires specialized expertise and complex instrumentation, which can deter biologists from fully embracing the technology. In this Perspective, a follow-up to our recent Q\&A article, we aim to demystify these challenges by addressing common questions and misconceptions surrounding SRM. Experts offer practical insights into how biologists can maximize the benefits of SRM while navigating issues such as photobleaching, image artifacts and the limitations of existing techniques. We also highlight recent developments in SRM that continue to push the boundaries of resolution. Our goal is to equip researchers with the crucial knowledge they need to harness the full potential of SRM.

}, keywords = {Animals, Artifacts, Humans, Microscopy, Microscopy, Fluorescence}, issn = {1477-9137}, doi = {10.1242/jcs.263567}, url = {https://doi.org/10.1242/jcs.263567}, author = {Prakash, Kirti and Baddeley, David and Eggeling, Christian and Fiolka, Reto and Heintzmann, Rainer and Manley, Suliana and Radenovic, Aleksandra and Shroff, Hari and Smith, Carlas and Schermelleh, Lothar} } @article {69445, title = {Building momentum through networks: Bioimaging across the Americas}, journal = {Journal of Microscopy}, volume = {n/a}, year = {2024}, month = {2024 May 15}, abstract = {

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

}, keywords = {bioimaging networks, BioImaging North America, community-building, imaging scientists, Latin America Bioimaging}, doi = {https://doi.org/10.1111/jmi.13318}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/jmi.13318}, author = {De Niz, Mariana and Escobedo Garc{\'\i}a, Rodrigo and Ter{\'a}n Ramirez, Celina and Pakowski, Ysa and Abonza, Yuriney and Bialy, Nikki and Orr, Vanessa L. and Olivera, Andres and Abonza, Victor and Alleva, Karina and Allodi, Silvana and Almeida, Michael F. and Becerril Cuevas, Alexis Ricardo and Bonnet, Frederic and Burgos Solorio, Armando and Chew, Teng-Leong and Chiabrando, Gustavo and Cimini, Beth and Cleret-Buhot, Aur{\'e}lie and Contreras Jim{\'e}nez, Gast{\'o}n and Daza, Laura and De S{\'a}, Vanessa and De Val, Natalia and Delgado-{\'A}lvarez, Diego L. and Eliceiri, Kevin and Fiolka, Reto and Grecco, Hernan and Hanein, Dorit and Hern{\'a}ndez Herrera, Pa{\'u}l and Hockberger, Phil and Hernandez, Haydee O. and Hernandez Guadarrama, Yael and Itano, Michelle and Jacobs, Caron A. and Jim{\'e}nez-Garc{\'\i}a, Luis F. and Jim{\'e}nez Sabinina, Vilma and Kamaid, Andres and Keppler, Antje and Kumar, Abhishek and Lacoste, Judith and Lovy, Alenka and Luby-Phelps, Kate and Mahadevan-Jansen, Anita and Malacrida, Leonel and Mehta, Shalin B. and Miller, Caroline and Miranda, Kildare and Moore, Joshua A. and North, Alison and O{\textquoteright}Toole, Peter and Olivares Urbano, Mariana and Pietrasanta, L{\'\i}a I. and Portugal, Rodrigo V. and Rossi, Andr{\'e}s H. and Sanchez Contreras, Jonathan and Strambio-De-Castilla, Caterina and Soldevila, Gloria and Vale, Bruno and Vazquez, Diana and Wood, Chris and Brown, Claire M. and Guerrero, Adan} } @article {69517, title = {Resolution in super-resolution microscopy - definition, trade-offs and perspectives.}, journal = {Nat Rev Mol Cell Biol}, year = {2024}, month = {2024 Jul 01}, abstract = {

Super-resolution microscopy (SRM) is gaining popularity in biosciences; however, claims about optical resolution are contested and often misleading. In this Viewpoint, experts share their views on resolution and common trade-offs, such as labelling and post-processing, aiming to clarify them for biologists and facilitate deeper understanding and best use of SRM.

}, issn = {1471-0080}, doi = {10.1038/s41580-024-00755-7}, author = {Prakash, Kirti and Baddeley, David and Eggeling, Christian and Fiolka, Reto and Heintzmann, Rainer and Manley, Suliana and Radenovic, Aleksandra and Smith, Carlas and Shroff, Hari and Schermelleh, Lothar} } @article {67792, title = {Light sheet fluorescence microscopy.}, journal = {Nature Reviews Methods Primers}, volume = {1}, year = {2021}, month = {2021 Nov 03}, abstract = {

Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to excite only fluorophores within the focal volume. Light sheet microscopes (LSMs) have a true optical sectioning capability and, hence, provide axial resolution, restrict photobleaching and phototoxicity to a fraction of the sample and use cameras to record tens to thousands of images per second. LSMs are used for in-depth analyses of large, optically cleared samples and long-term three-dimensional (3D) observations of live biological specimens at high spatio-temporal resolution. The independently operated illumination and detection trains and the canonical implementations, selective/single plane illumination microscope (SPIM) and digital scanned laser microscope (DSLM), are the basis for many LSM designs. In this Primer, we discuss various applications of LSFM for imaging multicellular specimens, developing vertebrate and invertebrate embryos, brain and heart function, 3D cell culture models, single cells, tissue sections, plants, organismic interaction and entire cleared brains. Further, we describe the combination of LSFM with other imaging approaches to allow for super-resolution or increased penetration depth and the use of sophisticated spatio-temporal manipulations to allow for observations along multiple directions. Finally, we anticipate developments of the field in the near future.

}, doi = {10.1038/s43586-021-00069-4}, url = {https://www.nature.com/articles/s43586-021-00069-4}, author = {Stelzer, Ernst H. K. and Strobl, Frederic and Chang, Bo-Jui and Preusser, Friedrich and Preibisch, Stephan and McDole, Katie and Fiolka, Reto} } @article {47527, title = {A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.}, journal = {Nature Biotechnology}, volume = {34}, year = {2016}, month = {2016 May 30}, pages = {760-7}, abstract = {

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

}, issn = {1546-1696}, doi = {10.1038/nbt.3550}, author = {Chu, Jun and Oh, Younghee and Sens, Alex and Ataie, Niloufar and Dana, Hod and Macklin, John J and Laviv, Tal and Welf, Erik S and Dean, Kevin M and Zhang, Feijie and Kim, Benjamin B and Tang, Clement Tran and Hu, Michelle and Baird, Michelle A and Davidson, Michael W and Kay, Mark A and Fiolka, Reto and Yasuda, Ryohei and Kim, Douglas S and Ng, Ho-Leung and Lin, Michael Z} } @article {6279, title = {High speed phase distortion measurement and compensation for focusing in space and time.}, journal = {Proceedings of SPIE}, volume = {8589}, year = {2013}, month = {2013}, pages = {85890V}, abstract = {

Random scattering and aberrations severely limit the imaging depth in optical microscopy. We introduce a rapid, parallel wavefront compensation technique that efficiently compensates even highly complex phase distortions. Using coherence gated backscattered light as a feedback signal, we focus light deep inside highly scattering brain tissue. We demonstrate that the same wavefront optimization technique can also be used to compensate spectral phase distortions in ultrashort laser pulses using nonlinear iterative feedback. We can restore transform limited pulse durations at any selected target location and compensate for dispersion that has occurred in the optical train and within the sample.

}, doi = {10.1117/12.2001121}, author = {Fiolka, Reto and Cui, Meng} } @article {6480, title = {Three-dimensional live microscopy beyond the diffraction limit.}, journal = {Journal of Optics}, volume = {15}, year = {2013}, month = {2013 Sep}, pages = {094002}, abstract = {

In fluorescence microscopy it has become possible to fundamentally overcome the diffraction limited resolution in all three spatial dimensions. However, to have the most impact in biological sciences, new optical microscopy techniques need to be compatible with live cell imaging: image acquisition has to be fast enough to capture cellular dynamics at the new resolution limit while light exposure needs to be minimized to prevent photo-toxic effects. With increasing spatial resolution, these requirements become more difficult to meet, even more so when volumetric imaging is performed. In this review, techniques that have been successfully applied to three-dimensional, super-resolution live microscopy are presented and their relative strengths and weaknesses are discussed.

}, issn = {2040-8978}, doi = {10.1088/2040-8978/15/9/094002}, author = {Fiolka, Reto} } @article {5278, title = {Breaking the spatial resolution barrier via iterative sound-light interaction in deep tissue microscopy.}, journal = {Scientific Reports}, volume = {2}, year = {2012}, month = {2012 Oct}, pages = {748}, abstract = {

Optical microscopy has so far been restricted to superficial layers, leaving many important biological questions unanswered. Random scattering causes the ballistic focus, which is conventionally used for image formation, to decay exponentially with depth. Optical imaging beyond the ballistic regime has been demonstrated by hybrid techniques that combine light with the deeper penetration capability of sound waves. Deep inside highly scattering media, the sound focus dimensions restrict the imaging resolutions. Here we show that by iteratively focusing light into an ultrasound focus via phase conjugation, we can fundamentally overcome this resolution barrier in deep tissues and at the same time increase the focus to background ratio. We demonstrate fluorescence microscopy beyond the ballistic regime of light with a threefold improved resolution and a fivefold increase in contrast. This development opens up practical high resolution fluorescence imaging in deep tissues.

}, doi = {doi:10.1038/srep00748}, author = {Si, Ke and Fiolka, Reto and Cui, Meng} } @article {5102, title = {Complex wavefront corrections for deep tissue focusing using low coherence backscattered light.}, journal = {Optics Express}, volume = {20}, year = {2012}, month = {2012}, pages = {16532-43}, abstract = {

Aberrations and random scattering severely limit optical imaging in deep tissue. Adaptive optics can in principle drastically extend the penetration depth and improve the image quality. However, for random scattering media a large number of spatial modes need to be measured and controlled to restore a diffraction limited focus. Here, we present a parallel wavefront optimization method using backscattered light as a feedback. Spatial confinement of the feedback signal is realized with a confocal pinhole and coherence gating. We show in simulations and experiments that this approach enables focusing deep into tissue over up to six mean scattering path lengths. Experimentally the technique was tested on tissue phantoms and fixed brain slices.

}, doi = {10.1364/OE.20.016532}, author = {Fiolka, Reto and Si, Ke and Cui, Meng} } @article {5101, title = {Fluorescence imaging beyond the ballistic regime by ultrasound-pulse-guided digital phase conjugation.}, journal = {Nature Photonics}, volume = {6}, year = {2012}, month = {2012 Aug}, pages = {657}, abstract = {

Fluorescence imaging has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivalled single-molecule-level sensitivity have enabled breakthroughs in a number of research fields. For in vivo applications its major limitation is its superficial imaging depth, a result of random scattering in biological tissues causing exponential attenuation of the ballistic component of a light wave. Here, we present fluorescence imaging beyond the ballistic regime by combining single-cycle pulsed ultrasound modulation and digital optical phase conjugation. We demonstrate a near-isotropic three-dimensional localized sound{\textendash}light interaction zone. With the exceptionally high optical gain provided by the digital optical phase conjugation system, we can deliver sufficient optical power to a focus inside highly scattering media for not only fluorescence imaging but also a variety of linear and nonlinear spectroscopy measurements. This technology paves the way for many important applications in both fundamental biology research and clinical studies.

}, doi = {doi:10.1038/nphoton.2012.205}, author = {Si, Ke and Fiolka, Reto and Cui, Meng} } @article {5389, title = {Parallel wavefront measurements in ultrasound pulse guided digital phase conjugation.}, journal = {Optics Express}, volume = {20}, year = {2012}, month = {2012 Oct 22}, pages = {24827-34}, abstract = {

Ultrasound pulse guided digital phase conjugation has emerged to realize fluorescence imaging inside random scattering media. Its major limitation is the slow imaging speed, as a new wavefront needs to be measured for each voxel. Therefore 3D or even 2D imaging can be time consuming. For practical applications on biological systems, we need to accelerate the imaging process by orders of magnitude. Here we propose and experimentally demonstrate a parallel wavefront measurement scheme towards such a goal. Multiple focused ultrasound pulses of different carrier frequencies can be simultaneously launched inside a scattering medium. Heterodyne interferometry is used to measure all of the wavefronts originating from every sound focus in parallel. We use these wavefronts in sequence to rapidly excite fluorescence at all the voxels defined by the focused ultrasound pulses. In this report, we employed a commercially available sound transducer to generate two sound foci in parallel, doubled the wavefront measurement speed, and reduced the mechanical scanning steps of the sound transducer to half.

}, issn = {1094-4087}, doi = {http://dx.doi.org/10.1364/OE.20.024827}, author = {Fiolka, Reto and Si, Ke and Cui, Meng} } @article {4632, title = {Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Apr 3}, pages = {5311-5}, abstract = {

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.

}, issn = {1091-6490}, doi = {10.1073/pnas.1119262109}, author = {Fiolka, Reto and Shao, Lin and Rego, E Hesper and Davidson, Michael W and Gustafsson, Mats G L} } @article {4634, title = {Simplified approach to diffraction tomography in optical microscopy.}, journal = {Optics Express}, volume = {17}, year = {2009}, month = {2009 Jul 20}, pages = {12407-17}, abstract = {

We present a novel microscopy technique to measure the scattered wavefront emitted from an optically transparent microscopic object. The complex amplitude is decoded via phase stepping in a common-path interferometer, enabling high mechanical stability. We demonstrate theoretically and practically that the incoherent summation of multiple illumination directions into a single image increases the resolving power and facilitates image reconstruction in diffraction tomography. We propose a slice-by-slice object-scatter extraction algorithm entirely based in real space in combination with ordinary z-stepping. Thereby the computational complexity affiliated with tomographic methods is significantly reduced. Using the first order Born approximation for weakly scattering objects it is possible to obtain estimates of the scattering density from the exitwaves.

}, keywords = {Algorithms, Cheek, Computer-Assisted, Equipment Design, Fourier Analysis, Green Fluorescent Proteins, Humans, Image Processing, Imaging, Interferometry, Microscopy, Models, Optics and Photonics, Radiation, Scattering, Statistical, Three-Dimensional, Tomography}, issn = {1094-4087}, author = {Fiolka, Reto and Wicker, Kai and Heintzmann, Rainer and Stemmer, Andreas} } @article {4635, title = {Variable phase retarder made of a dielectric elastomer actuator.}, journal = {Optics Letters}, volume = {34}, year = {2009}, month = {2009 Mar 15}, pages = {803-5}, abstract = {

We present a polymeric optical phase retarder that is electrically tunable by a dielectric elastomer actuator. The soft material device affords a large tuning range (14pi at lambda=488 nm) combined with high accuracy in optical path length and low drift rate (8.3 nm/min). Furthermore, the phase retarder is not sensitive to polarization, introduces a wavefront distortion141 kW/cm2). We show the dynamics for periodic phase modulation and demonstrate a simple drive technique for fast phase stepping. The polymer-based device is inexpensive, easy to fabricate, and its design can be adapted to specific applications.

}, issn = {0146-9592}, author = {Beck, Markus and Fiolka, Reto and Stemmer, Andreas} } @article {4636, title = {Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator.}, journal = {Optics Letters}, volume = {33}, year = {2008}, month = {2008 Jul 15}, pages = {1629-31}, abstract = {

In wide-field fluorescence microscopy, illuminating the specimen with evanescent standing waves increases lateral resolution more than twofold. We report a versatile setup for standing-wave illumination in total internal reflection fluorescence microscopy. An adjustable diffraction grating written on a phase-only spatial light modulator controls the illumination field. Selecting appropriate diffraction orders and displaying a sheared (tilted) diffraction grating allows one to tune the penetration depth in very fine steps. The setup achieves 91 nm lateral resolution for green emission.

}, issn = {0146-9592}, author = {Fiolka, Reto and Beck, Markus and Stemmer, Andreas} } @article {4637, title = {Widefield fluorescence microscopy with extended resolution.}, journal = {Histochemistry and Cell Biology}, volume = {130}, year = {2008}, month = {2008 Nov}, pages = {807-17}, abstract = {

Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform K{\"o}hler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.

}, keywords = {Animals, Computer-Assisted, Confocal, Equipment Design, Fluorescence, Fluorescent Dyes, Humans, Image Enhancement, Image Processing, Imaging, Microscopy, Reproducibility of Results, Three-Dimensional}, issn = {0948-6143}, doi = {10.1007/s00418-008-0506-8}, author = {Stemmer, Andreas and Beck, Markus and Fiolka, Reto} } @article {4639, title = {Virtual slit scanning microscopy.}, journal = {Histochemistry and Cell Biology}, volume = {128}, year = {2007}, month = {2007 Dec}, pages = {499-505}, abstract = {

We present a novel slit scanning confocal microscope with a CCD camera image sensor and a virtual slit aperture for descanning that can be adjusted during post-processing. A very efficient data structure and mathematical criteria for aligning the virtual aperture guarantee the ease of use. We further introduce a method to reduce the anisotropic lateral resolution of slit scanning microscopes. System performance is evaluated against a spinning disk confocal microscope on identical specimens. The virtual slit scanning microscope works as the spinning disk type and outperforms on thick specimens.

}, keywords = {Confocal, Electron, Equipment Design, Hela Cells, Humans, Microscopy, Scanning, Sensitivity and Specificity, User-Computer Interface}, issn = {0948-6143}, doi = {10.1007/s00418-007-0342-2}, author = {Fiolka, Reto and Stemmer, Andreas and Belyaev, Yury} }