Super-resolution microscopy ZMB

Temporal resolution

Refers to how quickly images can be acquired, especially important for dynamic live-cell imaging.

Multicolor

The ability to image multiple fluorescent markers simultaneously without significant spectral overlap.

Image Fidelity

Refers to the overall quality and accuracy of the image, including contrast, noise, and ability to faithfully represent the sample’s structure.

Live-cell friendliness

Indicates how suitable the technique is for imaging live samples without inducing significant phototoxicity or photobleaching.

Thick sample compatibility

Assesses how well the technique performs with thick or dense samples, where light scattering and absorption can interfere with imaging.

Z resolution

Refers to the axial (Z) resolution, or how well the technique can distinguish points at different depths within a 3D sample.

XY resolution

Measures the lateral (XY) resolution or how finely the technique can distinguish two closely spaced points on the same plane.

Versatility

Refers to how broadly the technique can be applied across different types of samples and experimental conditions. In the context of SRM, versatility indicates the ability to image various biological samples (e.g., live or fixed cells, tissue sections) and to adapt to different experimental goals (e.g., protein localization, dynamic processes).

SIM

ExM

STED

SMLM

PR-SRM

Availability

The accessibility of the technique at the ZMB.

FF-SRM

Our evaluations are based on a scoring system detailed in The Journal of Cell Science, helping you choose the best technique for your research needs.

Info

Temporal resolution

Refers to how quickly images can be acquired, especially important for dynamic live-cell imaging.

Multicolor

The ability to image multiple fluorescent markers simultaneously without significant spectral overlap.

Image Fidelity

Refers to the overall quality and accuracy of the image, including contrast, noise, and ability to faithfully represent the sample’s structure.

Live-cell friendliness

Indicates how suitable the technique is for imaging live samples without inducing significant phototoxicity or photobleaching.

Thick sample compatibility

Assesses how well the technique performs with thick or dense samples, where light scattering and absorption can interfere with imaging.

Z resolution

Refers to the axial (Z) resolution, or how well the technique can distinguish points at different depths within a 3D sample.

XY resolution

Measures the lateral (XY) resolution or how finely the technique can distinguish two closely spaced points on the same plane.

Versatility

Refers to how broadly the technique can be applied across different types of samples and experimental conditions. In the context of SRM, versatility indicates the ability to image various biological samples (e.g., live or fixed cells, tissue sections) and to adapt to different experimental goals (e.g., protein localization, dynamic processes).

SIM

ExM

STED

SMLM

PR-SRM

Availability

The accessibility of the technique at the ZMB.

FF-SRM

Our evaluations are based on a scoring system detailed in The Journal of Cell Science, helping you choose the best technique for your research needs.

Versatility

Fluctuation-based methods are purely analytical approaches that are compatible with most microscopes and do not depend on hardware modifications. They are thus easy to implement, and free software versions exist, but these require knowledge to properly tune the processing (Dedecker et al., 2012; Gustafsson et al., 2016). Moreover, the need to acquire a fast sequence of images (typically hundreds) can make multidimensional protocols complex.

Thick Sample Compatibility

SMLM is sensitive to light diffusion and spherical aberrations when imaging structures that are more than a few microns above the coverslip. More complex setups using light-sheet illumination schemes or adaptive optics allow to reach deeper in cells and tissue, but they are not yet broadly available (Liu et al., 2018).

Image Fidelity

Intensity in the generated images weakly relates to the local stoichiometry of fluorophores. While images will represent the structure (with some degree of unwanted defects), care needs to be taken when employing further analysis routines that take pixel brightness into account (Culley et al., 2018b).

Z Resolution

Minor (∼25%) axial resolution improvement has been demonstrated (York et al., 2013), and further improvement necessitates additional deconvolution of the collected data.

Temporal Resolution

STED microscopy allows the user to directly visualize the object of interest in super resolution without the need for offline computation or post-processing of multiple images. It is therefore suitable for the imaging of fast cellular events, such as organelle dynamics and protein trafficking (Bottanelli et al., 2016).

Availability

Abberior INFINITY STED

Leica SP8 3X STED

Temporal Resolution

The necessity to acquire thousands of images for a single reconstruction is a strong impediment to fast acquisition in SMLM. Higher laser intensities and fast cameras can be used (Lin et al., 2015). Furthermore, the use of artificial intelligence shows promise in the ability to infer structure from a limited number of acquired images (Ouyang et al., 2018).

Versatility

ExM has been successfully applied to a wide variety of samples, including cells (Chen et al., 2015), tissue sections (Zhao et al., 2017) and model organisms (Drosophila and zebrafish embryo) (Cahoon et al., 2017; Freifeld et al., 2017), as well as whole intact organs (Gao et al., 2019; Ku et al., 2016). ExM has also been used to observe RNA (Chen et al., 2016) and lipids (Karagiannis et al., 2019 preprint). Each new application of ExM needs careful and specific optimization.

XY Resolution

The resolution improvement considerably depends on the capacity of the algorithm used to detect fluctuations in fluorophores. It is thus difficult to predict the resolution that will be achieved prior to actual imaging, but an enhancement of two- to three-fold can typically be expected (Culley et al., 2018a).

XY Resolution

The final ‘resolution’ of the image depends on the final size of the expended sample, as well as on the microscopy strategy used to acquire the images. ExM protocols typically lead to a 4.5-fold expansion in all dimensions (an expected resolution of ∼70 nm), but others attain a 10-fold expansion (an expected resolution of 25–30 nm) (Truckenbrodt et al., 2018). Samples can also be expanded sequentially (an expected resolution of ∼25 nm) (Chang et al., 2017). ExM is also compatible with other super-resolution modalities, including STED (Gao et al., 2018), SMLM (Shi et al., 2019 preprint) and SIM (Cahoon et al., 2017; Halpern et al., 2017).

Live-Cell Friendliness

SIM is commonly used for live-cell imaging (Burnette et al., 2014; Carisey et al., 2018; Fiolka et al., 2012). In addition, improvement in reconstruction algorithms are enabling the imaging of biological samples using low laser power over hour-long time lapses (Huang et al., 2018).

Availability

ZEISS ELYRA 7

Leica GSD

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Fluctuation-based super resolution microscopy

When excited with continuous light, the emitted light of every fluorophore randomly varies over time. This is due to transitions between the non-fluorescent states of the fluorophore (van de Linde and Sauer, 2014) or their interactions with the surrounding environment (Bagshaw and Cherny, 2006). After capturing these oscillations over a sequence of tens to hundreds of images, algorithms such as super-resolution radial fluctuations (SRRF) (Gustafsson et al., 2016) use the temporal correlations in these oscillations to predict the presence and location of fluorophores at improved resolution. The accuracy and resolution will considerably improve when samples are decorated with highly fluctuating probes, such as reversibly photoswitchable fluorescent proteins (Zhang et al., 2016) or organic dyes in a photoswitch-inducing buffer (van de Linde et al., 2011).

Gustafsson, Nils, et al. "Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations." Nature communications 7.1 (2016): 12471.

Multicolor

The technique does not rely on specific fluorophores and can be used for multicolor imaging. It has the same multicolor capacity as laser scanning confocal or spinning disc microscopy.

Live-Cell Friendliness

Pixel reassignment allows single-shot super-resolution imaging at low-illumination. Reassignment implies additional magnification, which requires the use of sensitive detection systems.

Expansion microscopy (ExM) is a sample preparation technique that physically increases the size of the specimen (Chen et al., 2015). Multiple variations of the ExM protocol have been described, but they all share a common workflow. After fixation, the sample is embedded and cross-linked to a swellable gel that is then expanded using water (Wassie et al., 2019). The resulting enlarged specimen can then be imaged using classical microscopy techniques (Chen et al., 2015; Chozinski et al., 2016; Ku et al., 2016; Tillberg et al., 2016).

Chen, Fei, Paul W. Tillberg, and Edward S. Boyden. "Expansion microscopy." Science 347.6221 (2015): 543-548.

Expansion microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Image Fidelity

The complexity of the required post-acquisition analysis (localization and image reconstruction) and the high precision attained by SMLM makes it prone to artifacts. Care must be taken to ensure proper quenching of fluorophores and to limit the density of their blinking.

Image Fidelity

The pure-optical resolution enhancement does not generate additional image defects, such as those commonly occurring in super-resolution techniques that require data processing to generate a final image. However, the additional use of deconvolution can lead to image artefacts.

Versatility

STED microscopy requires dedicated fluorophores which are compatible with the depletion laser line. STED does not need specific buffers, but special care should be taken during sample preparation (e.g. fixation) to ensure maximal preservation of cellular structures at the STED resolution (Blom and Widengren, 2017).

Z Resolution

In 3D STED, it is possible to also thin the emission spot along the Z-axis using a second phase mask, which provides up to a four-fold improvement in Z resolution when compared to point scanning confocal (Klar et al., 2000). For instance, one study achieved 90 nm in the Z dimension, while maintaining 35 nm in the XY dimensions (Osseforth et al., 2014).

Temporal Resolution

The necessity to acquire thousands of images for a single reconstruction is a strong impediment to fast acquisition in SMLM. Higher laser intensities and fast cameras can be used (Lin et al., 2015). Furthermore, the use of artificial intelligence shows promise in the ability to infer structure from a limited number of acquired images (Ouyang et al., 2018).

Thick Sample Compatibility

ExM can be used on thick samples, such as small model organisms or whole mouse organs (Cahoon et al., 2017; Freifeld et al., 2017; Gao et al., 2019; Ku et al., 2016). However, it is important to note that post-expansion, samples will be at least 100-fold more voluminous and, therefore, they can be challenging to image at high magnification using classical microscopes. Light-sheet microscopy is especially well suited for imaging such samples (Gao et al., 2019).

Image Fidelity

When using linear SIM, the fluorescence intensities in reconstructed images are not directly transposed from the intensities of the raw images (Heintzmann and Huser, 2017). The intensity of the reconstructed image typically correlates well with the brightness of the original structures, but SIM can attenuate constant signals and is thus not suited to image and quantify diffuse (cytoplasmic) staining.

Temporal Resolution

Owing to the mostly optical nature of the resolution improvement process, high-speed imaging is possible and expected to be similar to that of laser scanning confocal or spinning disc microscopes.

Versatility

A lateral resolution improvement of 1.4-fold can be achieved purely through optics without the need for an additional analytical step. This means researchers can observe the resolution-enhanced sample in real-time. MSIM implementations (York et al., 2012), however, require the digital analysis of the images being collected.

Multicolor

ExM is compatible with standard dyes and fluorescent proteins and is therefore well adapted to multicolor experiments.

Availability

ZEISS ELYRA 7

Leica GSD

Thick Sample Compatibility

The resolution and quality of the data reconstructed will depend on the density of fluorophores captured in each image. As such, optical sectioning techniques, such as total internal reflection fluorescence (TIRF), confocal or light-sheet considerably improve the data generated and enable these methods to super-resolve samples with a thickness of tens of micrometers.

Multicolor

The technique does not rely on specific fluorophores and can be used for multicolor imaging. It has the same multicolor capacity as laser scanning confocal or spinning disc microscopy.

Image Fidelity

When using linear SIM, the fluorescence intensities in reconstructed images are not directly transposed from the intensities of the raw images (Heintzmann and Huser, 2017). The intensity of the reconstructed image typically correlates well with the brightness of the original structures, but SIM can attenuate constant signals and is thus not suited to image and quantify diffuse (cytoplasmic) staining.

Live-Cell Friendliness

ExM is only compatible with fixed samples.

Live-Cell Friendliness

SIM is commonly used for live-cell imaging (Burnette et al., 2014; Carisey et al., 2018; Fiolka et al., 2012). In addition, improvement in reconstruction algorithms are enabling the imaging of biological samples using low laser power over hour-long time lapses (Huang et al., 2018).

Live-Cell Friendliness

Owing to the requirement for high-intensity illumination by the STED beam, this technique remains a challenge for live-cell imaging. From the various configurations available, depletion using a 775 nm laser line is the most live-cell friendly and has been shown to work for a panel of fluorescent proteins and probes (D'Este et al., 2015). Gentler approaches called RESOLFT, based on photoswitchable probes, allow resolution enhancement with a lower light dose (Grotjohann et al., 2011; Masullo et al., 2018).

Multicolor

ExM is compatible with standard dyes and fluorescent proteins and is therefore well adapted to multicolor experiments.

Thick Sample Compatibility

The SIM design is often based on widefield microscopy. In this case, SIM performance can be strongly affected by the sample thickness, as well as by the presence of out of focus light. ‘Grazing incidence’ illumination (Guo et al., 2018) or TIRF (Kner et al., 2009; Li et al., 2015) can be used to image objects that are close to the coverslip with a better signal. In addition, lattice light-sheet (Chen et al., 2014) or slit-confocal (Schropp et al., 2017) arrangements can be combined with SIM to image deeper into cells.

Temporal Resolution

ExM is only compatible with fixed samples.

Image Fidelity

STED microscopy does not require post-processing of the images, which strongly limits the risk for artifact generation. Image quality and signal-to-noise ratio can however be further improved by photon reassignment using computational deconvolution.

Z Resolution

In 3D STED, it is possible to also thin the emission spot along the Z-axis using a second phase mask, which provides up to a four-fold improvement in Z resolution when compared to point scanning confocal (Klar et al., 2000). For instance, one study achieved 90 nm in the Z dimension, while maintaining 35 nm in the XY dimensions (Osseforth et al., 2014).

Multicolor

The number of fluorophores compatible with STED remains limited and many commonly used dyes are irreversibly bleached by the high intensity STED beam. In addition, in multicolor experiments, special care must be taken when selecting fluorophores to ensure that no overlap exists between their excitation spectrum and the depletion laser to improve the resolution of the other channels. Sequential scanning can be used as a workaround but acquisitions are limited to a single time point and focal plane.

XY Resolution

The 1.4-fold lateral-resolution improvement can be increased to two-fold through additional and careful deconvolution of the collected data.

XY Resolution

SIM typically improves the spatial resolution in all three dimensions by a factor of 2-4 (Gustafsson et al., 2008). Non-linear SIM approaches that bypass this resolution use fluorophore saturation, but are not yet commercially available (Li et al., 2015; Rego et al., 2012).

Versatility

ExM has been successfully applied to a wide variety of samples, including cells (Chen et al., 2015), tissue sections (Zhao et al., 2017) and model organisms (Drosophila and zebrafish embryo) (Cahoon et al., 2017; Freifeld et al., 2017), as well as whole intact organs (Gao et al., 2019; Ku et al., 2016). ExM has also been used to observe RNA (Chen et al., 2016) and lipids (Karagiannis et al., 2019 preprint). Each new application of ExM needs careful and specific optimization.

Live-Cell Friendliness

The time necessary to accumulate enough localizations and the high illumination intensities needed to visualize single molecules makes SMLM challenging to use on live samples (Tosheva et al., 2020; Wäldchen et al., 2015), although it has been performed (Huang et al., 2013; Jones et al., 2011).

XY Resolution

SMLM reconstructs images at 10 to 20 nm resolution. The final resolution achieved depends on the brightness of the fluorophores detected, their labeling density and the capacity to accurately detect individual fluorophores (Culley et al., 2018b). Importantly, at this scale, the size of the probe (antibody or fusion protein) can start to degrade the precision of the imaging (Magrassi et al., 2019).

In the structured illumination microscopy (SIM) technique, the sample is illuminated using a patterned light. For each focus plane, multiple images are taken using a different pattern and are then combined by a computer algorithm to reconstruct a super-resolved image (Schermelleh et al., 2019). The most common patterns are parallel lines, but hexagonal or lattice patterns can be used (Heintzmann and Huser, 2017). In the case of lattice pattern as on our ZEISS ELYRA 7, multiple shifted patterns are obtained using a grid : 9 images for a 2D image live-cell and 13 images per plane for a 3D volume.

Rego, E.H., Shao, L. (2015). Practical Structured Illumination Microscopy. In: Verveer, P. (eds) Advanced Fluorescence Microscopy. Methods in Molecular Biology, vol 1251. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2080-8_10

Structured illumination microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Z Resolution

The final ‘resolution’ of the image depends on the final size of the expended sample, as well as on the microscopy strategy used to acquire the images. ExM protocols typically lead to a 4.5-fold expansion in all dimensions (an expected resolution of ∼70 nm), but others attain a 10-fold expansion (an expected resolution of 25–30 nm) (Truckenbrodt et al., 2018). Samples can also be expanded sequentially (an expected resolution of ∼25 nm) (Chang et al., 2017). ExM is also compatible with other super-resolution modalities, including STED (Gao et al., 2018), SMLM (Shi et al., 2019 preprint) and SIM (Cahoon et al., 2017; Halpern et al., 2017).

XY Resolution

The resolution improvement considerably depends on the capacity of the algorithm used to detect fluctuations in fluorophores. It is thus difficult to predict the resolution that will be achieved prior to actual imaging, but an enhancement of two- to three-fold can typically be expected (Culley et al., 2018a).

XY Resolution

In practice, in biological systems, the best resolution achieved are ∼40 nm in living tissue and cells (Bottanelli et al., 2016; Willig et al., 2006), and 20 nm in fixed samples (Göttfert et al., 2013), and a typical ∼60 nm resolution can be obtained in core facility settings. Recently, 1 nm resolution was achieved by using a hybrid microscopy technique (MINFLUX) combining STED and single-molecule localization microscopy (SMLM) (Balzarotti et al., 2017; Gwosch et al., 2020).

Stimulated emission depletion (STED) microscopy is based on laser scanning confocal microscopy where a donut-shaped STED beam is overlaid on top of the excitation laser beam (Heine et al., 2017; Hell and Wichmann, 1994; Klar et al., 2000). In the zone of overlap between the two beams, the STED beam depletes the fluorophores before fluorescence takes place, thinning the emission area to a sub-diffraction sized spot. The donut-shaped STED beam is obtained using a vortex phase mask and the most recent systems are combining femtosecond lasers and detector time gating to improve the signal-to-noise ratio and significantly reduce the background (Hernández et al., 2015; Moffitt et al., 2011).

Betzig, Eric, Stefan W. Hell, and William E. Moerner. "How the optical microscope became a nanoscope." The Nobel Prize in Chemistry (2014).

Stimulated emission depletion microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Z Resolution

A common way of retrieving the Z coordinate of fluorophores is to deform the point-spread function into an ellipse using a cylindrical lens (Huang et al., 2008). This provides a typical Z localization precision of 20–30 nm (Diezmann et al., 2017).

Availability

Abberior INFINITY STED

Leica SP8 3X STED

Thick Sample Compatibility

SMLM is sensitive to light diffusion and spherical aberrations when imaging structures that are more than a few microns above the coverslip. More complex setups using light-sheet illumination schemes or adaptive optics allow to reach deeper in cells and tissue, but they are not yet broadly available (Liu et al., 2018).

Live-Cell Friendliness

The time necessary to accumulate enough localizations and the high illumination intensities needed to visualize single molecules makes SMLM challenging to use on live samples (Tosheva et al., 2020; Wäldchen et al., 2015), although it has been performed (Huang et al., 2013; Jones et al., 2011).

Image Fidelity

The pure-optical resolution enhancement does not generate additional image defects, such as those commonly occurring in super-resolution techniques that require data processing to generate a final image. However, the additional use of deconvolution can lead to image artefacts.

Live-Cell Friendliness

Fluctuation-based SRM is one of the least phototoxic methods in existence; enhanced resolution can be achieved using an illumination intensity that is similar to conventional fluorescence imaging (mW/cm2 magnitude). Imaging from minutes to hours without significant photobleaching or apparent light-induced cell stress has been demonstrated (Culley et al., 2018a).

Z Resolution

Resolution improvement in the Z-axis cannot yet be directly achieved by the algorithms, but specialized multiplane-imaging setups are being developed to tackle this limitation.

Versatility

Owing to its high spatial resolution, SMLM requires specific care during sample preparation to ensure optimal ultrastructural preservation (Jimenez et al., 2019; Whelan and Bell, 2015). In addition, as single molecules are recorded, the density of labeling must be high enough to delineate the final structure of interest (Patterson et al., 2010).

Availability

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Temporal Resolution

ExM is only compatible with fixed samples.

Fluctuation-based super resolution microscopy

When excited with continuous light, the emitted light of every fluorophore randomly varies over time. This is due to transitions between the non-fluorescent states of the fluorophore (van de Linde and Sauer, 2014) or their interactions with the surrounding environment (Bagshaw and Cherny, 2006). After capturing these oscillations over a sequence of tens to hundreds of images, algorithms such as super-resolution radial fluctuations (SRRF) (Gustafsson et al., 2016) use the temporal correlations in these oscillations to predict the presence and location of fluorophores at improved resolution. The accuracy and resolution will considerably improve when samples are decorated with highly fluctuating probes, such as reversibly photoswitchable fluorescent proteins (Zhang et al., 2016) or organic dyes in a photoswitch-inducing buffer (van de Linde et al., 2011).

Gustafsson, Nils, et al. "Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations." Nature communications 7.1 (2016): 12471.

XY Resolution

In practice, in biological systems, the best resolution achieved are ∼40 nm in living tissue and cells (Bottanelli et al., 2016; Willig et al., 2006), and 20 nm in fixed samples (Göttfert et al., 2013), and a typical ∼60 nm resolution can be obtained in core facility settings. Recently, 1 nm resolution was achieved by using a hybrid microscopy technique (MINFLUX) combining STED and single-molecule localization microscopy (SMLM) (Balzarotti et al., 2017; Gwosch et al., 2020).

Image Fidelity

STED microscopy does not require post-processing of the images, which strongly limits the risk for artifact generation. Image quality and signal-to-noise ratio can however be further improved by photon reassignment using computational deconvolution.

XY Resolution

The 1.4-fold lateral-resolution improvement can be increased to two-fold through additional and careful deconvolution of the collected data.

Image Fidelity

Intensity in the generated images weakly relates to the local stoichiometry of fluorophores. While images will represent the structure (with some degree of unwanted defects), care needs to be taken when employing further analysis routines that take pixel brightness into account (Culley et al., 2018b).

Thick Sample Compatibility

The resolution and quality of the data reconstructed will depend on the density of fluorophores captured in each image. As such, optical sectioning techniques, such as total internal reflection fluorescence (TIRF), confocal or light-sheet considerably improve the data generated and enable these methods to super-resolve samples with a thickness of tens of micrometers.

Multicolor

Multicolor remains a challenge for most SMLM strategies – photoactivatable and/or convertible proteins rapidly occupy all available channels in PALM (Shroff et al., 2007), and the photophysics of organic fluorophores makes it difficult to identify those that are spectrally distinct and have good blinking properties (Dempsey et al., 2011; Lehmann et al., 2015). However, DNA-PAINT allows for virtually unlimited sequential imaging of distinct targets, and is easily used for imaging of three to four colors (Jimenez et al., 2019; Jungmann et al., 2014).

Stimulated emission depletion (STED) microscopy is based on laser scanning confocal microscopy where a donut-shaped STED beam is overlaid on top of the excitation laser beam (Heine et al., 2017; Hell and Wichmann, 1994; Klar et al., 2000). In the zone of overlap between the two beams, the STED beam depletes the fluorophores before fluorescence takes place, thinning the emission area to a sub-diffraction sized spot. The donut-shaped STED beam is obtained using a vortex phase mask and the most recent systems are combining femtosecond lasers and detector time gating to improve the signal-to-noise ratio and significantly reduce the background (Hernández et al., 2015; Moffitt et al., 2011).

Betzig, Eric, Stefan W. Hell, and William E. Moerner. "How the optical microscope became a nanoscope." The Nobel Prize in Chemistry (2014).

Stimulated emission depletion microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

During single-molecule localization microscopy (SMLM) experiments, the emission of individual fluorophores are recorded using a camera. The resulting images are composed of diffraction-limited spots (typically ∼200 nm in width) that are then fitted to precisely pinpoint the fluorophore position (typically within ∼10–15 nm) (Diezmann et al., 2017). In practice, tens of thousands of images of blinking fluorophores are acquired in rapid succession (Jimenez et al., 2019). A processing software is then used to fit the blinking events and create a super-resolved image (Baddeley and Bewersdorf, 2018). To detect single molecules from densely labeled samples, only a small fraction of the fluorophores can be emitting photons at any one time (Li and Vaughan, 2018). This can be achieved by using sparsely activated photoactivatable/photo-convertible fluorescent proteins in (fluorescence-) photo-activated localization microscopy (PALM) (Betzig et al., 2006; Hess et al., 2006). Organic dyes can also be induced to blink using specific buffers in (direct) stochastic optical reconstruction microscopy (dSTORM) (Heilemann et al., 2008; Rust et al., 2006) or ground-state depletion (GSD) microscopy (Fölling et al., 2008). Alternatively, blinking can be generated by the transient interaction between two short DNA sequences, one labeled and one unlabeled, in so-called DNA point-accumulation in nanoscale topography (DNA-PAINT) (Jungmann et al., 2014).

Rust, Michael J., Mark Bates, and Xiaowei Zhuang. "Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution." Nature methods 3.10 (2006): 793.

Single-molecule localization microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

In the structured illumination microscopy (SIM) technique, the sample is illuminated using a patterned light. For each focus plane, multiple images are taken using a different pattern and are then combined by a computer algorithm to reconstruct a super-resolved image (Schermelleh et al., 2019). The most common patterns are parallel lines, but hexagonal or lattice patterns can be used (Heintzmann and Huser, 2017). In the case of lattice pattern as on our ZEISS ELYRA 7, multiple shifted patterns are obtained using a grid : 9 images for a 2D image live-cell and 13 images per plane for a 3D volume.

Rego, E.H., Shao, L. (2015). Practical Structured Illumination Microscopy. In: Verveer, P. (eds) Advanced Fluorescence Microscopy. Methods in Molecular Biology, vol 1251. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2080-8_10

Structured illumination microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Versatility

Owing to its high spatial resolution, SMLM requires specific care during sample preparation to ensure optimal ultrastructural preservation (Jimenez et al., 2019; Whelan and Bell, 2015). In addition, as single molecules are recorded, the density of labeling must be high enough to delineate the final structure of interest (Patterson et al., 2010).

Temporal Resolution

Recent developments such as instant Lattice SIM allow acquisitions as fast as 255 fps, with the use of a sliding window over the varying patterns (Guo et al., 2018; Huang et al., 2018).

Thick Sample Compatibility

Both the intensity and the geometry of the excitation and depletion beams are differently affected while traveling across the biological sample, leading to difficulties when imaging deep into tissues. This can be partially improved by using multiphoton excitation lasers, which allow for a deeper penetration imaging capability.

Multicolor

The number of fluorophores compatible with STED remains limited and many commonly used dyes are irreversibly bleached by the high intensity STED beam. In addition, in multicolor experiments, special care must be taken when selecting fluorophores to ensure that no overlap exists between their excitation spectrum and the depletion laser to improve the resolution of the other channels. Sequential scanning can be used as a workaround but acquisitions are limited to a single time point and focal plane.

Live-Cell Friendliness

Owing to the requirement for high-intensity illumination by the STED beam, this technique remains a challenge for live-cell imaging. From the various configurations available, depletion using a 775 nm laser line is the most live-cell friendly and has been shown to work for a panel of fluorescent proteins and probes (D'Este et al., 2015). Gentler approaches called RESOLFT, based on photoswitchable probes, allow resolution enhancement with a lower light dose (Grotjohann et al., 2011; Masullo et al., 2018).

Image Fidelity

The complexity of the required post-acquisition analysis (localization and image reconstruction) and the high precision attained by SMLM makes it prone to artifacts. Care must be taken to ensure proper quenching of fluorophores and to limit the density of their blinking.

Z Resolution

Resolution improvement in the Z-axis cannot yet be directly achieved by the algorithms, but specialized multiplane-imaging setups are being developed to tackle this limitation.

Multicolor

A temporal stream of a few hundred images needs to be collected to capture a single channel, generally taking one to five seconds.

Availability

ExM sample preparation only requires commercially available reagents and is therefore readily available; it is also relatively inexpensive to implement. We implemented the Pro-Exm Protocol at the ZMB (Asano et al., 2018).

XY Resolution

SIM typically improves the spatial resolution in all three dimensions by a factor of 2-4 (Gustafsson et al., 2008). Non-linear SIM approaches that bypass this resolution use fluorophore saturation, but are not yet commercially available (Li et al., 2015; Rego et al., 2012).

Live-Cell Friendliness

ExM is only compatible with fixed samples.

XY Resolution

The final ‘resolution’ of the image depends on the final size of the expended sample, as well as on the microscopy strategy used to acquire the images. ExM protocols typically lead to a 4.5-fold expansion in all dimensions (an expected resolution of ∼70 nm), but others attain a 10-fold expansion (an expected resolution of 25–30 nm) (Truckenbrodt et al., 2018). Samples can also be expanded sequentially (an expected resolution of ∼25 nm) (Chang et al., 2017). ExM is also compatible with other super-resolution modalities, including STED (Gao et al., 2018), SMLM (Shi et al., 2019 preprint) and SIM (Cahoon et al., 2017; Halpern et al., 2017).

Thick Sample Compatibility

The SIM design is often based on widefield microscopy. In this case, SIM performance can be strongly affected by the sample thickness, as well as by the presence of out of focus light. ‘Grazing incidence’ illumination (Guo et al., 2018) or TIRF (Kner et al., 2009; Li et al., 2015) can be used to image objects that are close to the coverslip with a better signal. In addition, lattice light-sheet (Chen et al., 2014) or slit-confocal (Schropp et al., 2017) arrangements can be combined with SIM to image deeper into cells.

Versatility

STED microscopy requires dedicated fluorophores which are compatible with the depletion laser line. STED does not need specific buffers, but special care should be taken during sample preparation (e.g. fixation) to ensure maximal preservation of cellular structures at the STED resolution (Blom and Widengren, 2017).

Availability

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Z Resolution

SIM typically improves the spatial resolution in all three dimensions by a factor of 2-4 (Gustafsson et al., 2008). Non-linear SIM approaches that bypass this resolution use fluorophore saturation, but are not yet commercially available (Li et al., 2015; Rego et al., 2012).

Multicolor

As SIM is compatible with most fluorophores, its setup can typically accommodate three to four different color channels (Jacquemet et al., 2019; Vietri et al., 2015).

Versatility

Fluctuation-based methods are purely analytical approaches that are compatible with most microscopes and do not depend on hardware modifications. They are thus easy to implement, and free software versions exist, but these require knowledge to properly tune the processing (Dedecker et al., 2012; Gustafsson et al., 2016). Moreover, the need to acquire a fast sequence of images (typically hundreds) can make multidimensional protocols complex.

Multicolor

As SIM is compatible with most fluorophores, its setup can typically accommodate three to four different color channels (Jacquemet et al., 2019; Vietri et al., 2015).

Thick Sample Compatibility

Reassignment systems are expected to generate images of similar fidelity to those collected in laser scanning confocal or spinning disc microscopy for thick samples.

Pixel reassignment super resolution microscopy

In Pixel reassignment super resolution microscopy, the fluorescence signal is not captured by a single-point detector, such as a photomultiplier tube, but by an array detector (Ströhl and Kaminski, 2016; Wu and Shroff, 2018). The signal detected in each element of the detector array is reassigned in space to achieve a smaller point-spread-function and thus higher resolution. After data processing, a 1.4-fold improvement in resolution can be achieved laterally with a minor axial improvement (Vangindertael et al., 2018). A number of variants exist for pixel reassignment, such as image scanning microscopy (ISM) (Müller and Enderlein, 2010), our Zeiss Airyscan (Huff, 2015), rescan confocal microscopy (RCM) (Luca et al., 2013) and multifocal structured illumination microscopy (MSIM) (York et al., 2012).

Huff, Joseph, Annette Bergter, and Benedikt Luebbers. "Multiplex mode for the LSM 9 series with Airyscan 2: fast and gentle confocal super-resolution in large volumes." Nat. Methods 10 (2019): 1-4.

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Multicolor

A temporal stream of a few hundred images needs to be collected to capture a single channel, generally taking one to five seconds.

Thick Sample Compatibility

Reassignment systems are expected to generate images of similar fidelity to those collected in laser scanning confocal or spinning disc microscopy for thick samples.

Temporal Resolution

Recent developments such as instant Lattice SIM allow acquisitions as fast as 255 fps, with the use of a sliding window over the varying patterns (Guo et al., 2018; Huang et al., 2018).

During single-molecule localization microscopy (SMLM) experiments, the emission of individual fluorophores are recorded using a camera. The resulting images are composed of diffraction-limited spots (typically ∼200 nm in width) that are then fitted to precisely pinpoint the fluorophore position (typically within ∼10–15 nm) (Diezmann et al., 2017). In practice, tens of thousands of images of blinking fluorophores are acquired in rapid succession (Jimenez et al., 2019). A processing software is then used to fit the blinking events and create a super-resolved image (Baddeley and Bewersdorf, 2018). To detect single molecules from densely labeled samples, only a small fraction of the fluorophores can be emitting photons at any one time (Li and Vaughan, 2018). This can be achieved by using sparsely activated photoactivatable/photo-convertible fluorescent proteins in (fluorescence-) photo-activated localization microscopy (PALM) (Betzig et al., 2006; Hess et al., 2006). Organic dyes can also be induced to blink using specific buffers in (direct) stochastic optical reconstruction microscopy (dSTORM) (Heilemann et al., 2008; Rust et al., 2006) or ground-state depletion (GSD) microscopy (Fölling et al., 2008). Alternatively, blinking can be generated by the transient interaction between two short DNA sequences, one labeled and one unlabeled, in so-called DNA point-accumulation in nanoscale topography (DNA-PAINT) (Jungmann et al., 2014).

Rust, Michael J., Mark Bates, and Xiaowei Zhuang. "Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution." Nature methods 3.10 (2006): 793.

Single-molecule localization microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Z Resolution

Minor (∼25%) axial resolution improvement has been demonstrated (York et al., 2013), and further improvement necessitates additional deconvolution of the collected data.

XY Resolution

SMLM reconstructs images at 10 to 20 nm resolution. The final resolution achieved depends on the brightness of the fluorophores detected, their labeling density and the capacity to accurately detect individual fluorophores (Culley et al., 2018b). Importantly, at this scale, the size of the probe (antibody or fusion protein) can start to degrade the precision of the imaging (Magrassi et al., 2019).

Temporal Resolution

Provides excellent temporal resolution because it captures the entire field of view in a single exposure, making it ideal for fast imaging applications (Diaspro et al., 2005).

Image Fidelity

ExM leads to an isotropic expansion and has been shown to preserve the structural integrity of the various cellular structures imaged. In addition, using the nuclear pore as a reporter, ExM was found to have a uniform accuracy in the range of 20 nm (Pesce et al., 2019). Nevertheless, one concern that remains is whether all the structures of interest within a sample expand at the same rate or to the same extent. For instance, in bacteria, the expansion efficacy varies across species (Lim et al., 2019). Moreover, the digestion procedure typically attenuates the fluorescence of organic or protein-based fluorophores, and the expansion itself dilutes the fluorescent signal spatially (a 4-fold expansion results in 64 times less fluorescence per volume unit), requiring sensitive microscopes for ExM sample imaging.

Availability

ExM sample preparation only requires commercially available reagents and is therefore readily available; it is also relatively inexpensive to implement. We implemented the Pro-Exm Protocol at the ZMB (Asano et al., 2018).

Live-Cell Friendliness

Fluctuation-based SRM is one of the least phototoxic methods in existence; enhanced resolution can be achieved using an illumination intensity that is similar to conventional fluorescence imaging (mW/cm2 magnitude). Imaging from minutes to hours without significant photobleaching or apparent light-induced cell stress has been demonstrated (Culley et al., 2018a).

Live-Cell Friendliness

Pixel reassignment allows single-shot super-resolution imaging at low-illumination. Reassignment implies additional magnification, which requires the use of sensitive detection systems.

Thick Sample Compatibility

Both the intensity and the geometry of the excitation and depletion beams are differently affected while traveling across the biological sample, leading to difficulties when imaging deep into tissues. This can be partially improved by using multiphoton excitation lasers, which allow for a deeper penetration imaging capability.

Temporal Resolution

Owing to the mostly optical nature of the resolution improvement process, high-speed imaging is possible and expected to be similar to that of laser scanning confocal or spinning disc microscopes.

Versatility

SIM is generally easy to use, suitable for a wide variety of biological samples and is compatible with most fluorophores with the conditions that they are relatively resistant to photobleaching and non-blinking (Demmerle et al., 2017). SIM performances are affected by both the sample and the imaging conditions, and therefore dedicated training and optimizations are required to achieve good results.

Expansion microscopy (ExM) is a sample preparation technique that physically increases the size of the specimen (Chen et al., 2015). Multiple variations of the ExM protocol have been described, but they all share a common workflow. After fixation, the sample is embedded and cross-linked to a swellable gel that is then expanded using water (Wassie et al., 2019). The resulting enlarged specimen can then be imaged using classical microscopy techniques (Chen et al., 2015; Chozinski et al., 2016; Ku et al., 2016; Tillberg et al., 2016).

Chen, Fei, Paul W. Tillberg, and Edward S. Boyden. "Expansion microscopy." Science 347.6221 (2015): 543-548.

Expansion microscopy

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Z Resolution

A common way of retrieving the Z coordinate of fluorophores is to deform the point-spread function into an ellipse using a cylindrical lens (Huang et al., 2008). This provides a typical Z localization precision of 20–30 nm (Diezmann et al., 2017).

Image Fidelity

ExM leads to an isotropic expansion and has been shown to preserve the structural integrity of the various cellular structures imaged. In addition, using the nuclear pore as a reporter, ExM was found to have a uniform accuracy in the range of 20 nm (Pesce et al., 2019). Nevertheless, one concern that remains is whether all the structures of interest within a sample expand at the same rate or to the same extent. For instance, in bacteria, the expansion efficacy varies across species (Lim et al., 2019). Moreover, the digestion procedure typically attenuates the fluorescence of organic or protein-based fluorophores, and the expansion itself dilutes the fluorescent signal spatially (a 4-fold expansion results in 64 times less fluorescence per volume unit), requiring sensitive microscopes for ExM sample imaging.

Z Resolution

The final ‘resolution’ of the image depends on the final size of the expended sample, as well as on the microscopy strategy used to acquire the images. ExM protocols typically lead to a 4.5-fold expansion in all dimensions (an expected resolution of ∼70 nm), but others attain a 10-fold expansion (an expected resolution of 25–30 nm) (Truckenbrodt et al., 2018). Samples can also be expanded sequentially (an expected resolution of ∼25 nm) (Chang et al., 2017). ExM is also compatible with other super-resolution modalities, including STED (Gao et al., 2018), SMLM (Shi et al., 2019 preprint) and SIM (Cahoon et al., 2017; Halpern et al., 2017).

Temporal Resolution

STED microscopy allows the user to directly visualize the object of interest in super resolution without the need for offline computation or post-processing of multiple images. It is therefore suitable for the imaging of fast cellular events, such as organelle dynamics and protein trafficking (Bottanelli et al., 2016).

Z Resolution

SIM typically improves the spatial resolution in all three dimensions by a factor of 2-4 (Gustafsson et al., 2008). Non-linear SIM approaches that bypass this resolution use fluorophore saturation, but are not yet commercially available (Li et al., 2015; Rego et al., 2012).

Multicolor

Multicolor remains a challenge for most SMLM strategies – photoactivatable and/or convertible proteins rapidly occupy all available channels in PALM (Shroff et al., 2007), and the photophysics of organic fluorophores makes it difficult to identify those that are spectrally distinct and have good blinking properties (Dempsey et al., 2011; Lehmann et al., 2015). However, DNA-PAINT allows for virtually unlimited sequential imaging of distinct targets, and is easily used for imaging of three to four colors (Jimenez et al., 2019; Jungmann et al., 2014).

Thick Sample Compatibility

ExM can be used on thick samples, such as small model organisms or whole mouse organs (Cahoon et al., 2017; Freifeld et al., 2017; Gao et al., 2019; Ku et al., 2016). However, it is important to note that post-expansion, samples will be at least 100-fold more voluminous and, therefore, they can be challenging to image at high magnification using classical microscopes. Light-sheet microscopy is especially well suited for imaging such samples (Gao et al., 2019).

Versatility

SIM is generally easy to use, suitable for a wide variety of biological samples and is compatible with most fluorophores with the conditions that they are relatively resistant to photobleaching and non-blinking (Demmerle et al., 2017). SIM performances are affected by both the sample and the imaging conditions, and therefore dedicated training and optimizations are required to achieve good results.

Versatility

A lateral resolution improvement of 1.4-fold can be achieved purely through optics without the need for an additional analytical step. This means researchers can observe the resolution-enhanced sample in real-time. MSIM implementations (York et al., 2012), however, require the digital analysis of the images being collected.

Pixel reassignment super resolution microscopy

In Pixel reassignment super resolution microscopy, the fluorescence signal is not captured by a single-point detector, such as a photomultiplier tube, but by an array detector (Ströhl and Kaminski, 2016; Wu and Shroff, 2018). The signal detected in each element of the detector array is reassigned in space to achieve a smaller point-spread-function and thus higher resolution. After data processing, a 1.4-fold improvement in resolution can be achieved laterally with a minor axial improvement (Vangindertael et al., 2018). A number of variants exist for pixel reassignment, such as image scanning microscopy (ISM) (Müller and Enderlein, 2010), our Zeiss Airyscan (Huff, 2015), rescan confocal microscopy (RCM) (Luca et al., 2013) and multifocal structured illumination microscopy (MSIM) (York et al., 2012).

Huff, Joseph, Annette Bergter, and Benedikt Luebbers. "Multiplex mode for the LSM 9 series with Airyscan 2: fast and gentle confocal super-resolution in large volumes." Nat. Methods 10 (2019): 1-4.

Due to its purely analytical nature, Super-Resolution Radial Fluctuations (SRRF) enables super-resolution microscopy (SRM) imaging on standard modern microscopes using conventional fluorophores, without the need for specialized equipment. The free NanoJ plugin for Fiji provides the SRRF algorithm (available on our VMs), enabling SRRF imaging.

Temporal Resolution

Provides excellent temporal resolution because it captures the entire field of view in a single exposure, making it ideal for fast imaging applications (Diaspro et al., 2005).