RESOLFT

Last updated April 17, 2024

RESOLFT, an acronym for REversible Saturable OpticaLFluorescence Transitions, denotes a group of optical fluorescence microscopy techniques with very high resolution. Using standard far field visible light optics a resolution far below the diffraction limit down to molecular scales can be obtained.

With conventional microscopy techniques, it is not possible to distinguish features that are located at distances less than about half the wavelength used (i.e. about 200 nm for visible light). This diffraction limit is based on the wave nature of light. In conventional microscopes the limit is determined by the used wavelength and the numerical aperture of the optical system. The RESOLFT concept surmounts this limit by temporarily switching the molecules to a state in which they cannot send a (fluorescence-) signal upon illumination. This concept is different from for example electron microscopy where instead the used wavelength is much smaller.

Working principle

RESOLFT microscopy is an optical microscopy with very high resolution that can image details in samples that cannot be imaged with conventional or confocal microscopy. Within RESOLFT the principles of STED microscopy ^[1]^[2] and GSD microscopy are generalized. Structures that are normally too close to each other to be distinguished are read out sequentially.

Within this framework all methods can be explained that operate on molecules that have at least two distinguishable states, where reversible switching between the two states is possible, and where at least one such transition can be optically induced.

In most cases fluorescent markers are used, where one state (A) which is bright, that is, generates a fluorescence signal, and the other state (B) is dark, and gives no signal. One transition between them can be induced by light (e.g. A→B, bright to dark).

The sample is illuminated inhomogeneously with the illumination intensity at one position being very small (zero under ideal conditions). Only at this place are the molecules never in the dark state B (if A is the pre-existing state) and remain fully in the bright state A. The area where molecules are mostly in the bright state can be made very small (smaller than the conventional diffraction limit) by increasing the transition light intensity (see below). Any signal detected is thus known to come only from molecules in the small area around the illumination intensity minimum. A high resolution image can be constructed by scanning the sample, i.e., shifting the illumination profile across the surface.^[3]

The transition back from B to A can be either spontaneous or driven by light of another wavelength. The molecules have to be switchable several times in order to be present in state A or B at different times during scanning the sample. The method also works if the bright and the dark state are reversed, one then obtains a negative image.

Resolution below the diffraction-limit

In RESOLFT the area where molecules reside in state A (bright state) can be made arbitrarily small despite the diffraction-limit.

One has to illuminate the sample inhomogeneously so that an isolated zero intensity point is created. This can be achieved e.g. by interference.
At low intensities (lower than the blue line in the image) most marker molecules are in the bright state, if the intensity is above, most markers are in the dark state.

Upon weak illumination we see that the area where molecules remain in state A is still quite large because the illumination is so low that most molecules reside in state A. The shape of the illumination profile does not need to be altered. Increasing the illumination brightness already results in a smaller area where the intensity is below the amount for efficient switching to the dark state. Consequently, also the area where molecules can reside in state A is diminished. The (fluorescence) signal during a following readout originates from a very small spot and one can obtain very sharp images.

In the RESOLFT concept, the resolution can be approximated by $\Delta d\simeq {\frac {\lambda }{\pi n{\sqrt {\frac {I}{Isat}}}}}$ , whereby $I_{sat}$ is the characteristic intensity required for saturating the transition (half of the molecules remain in state A and half in state B), and $I$ denotes the intensity applied. If the minima are produced by focusing optics with a numerical aperture ${\mbox{NA}}=n\sin \alpha$ , the minimal distance at which two identical objects can be discerned is $\Delta d={\frac {\lambda }{2n\cdot \sin \alpha \cdot {\sqrt {1+{\frac {I}{Isat}}}}}}$ which can be regarded as an extension of Abbe’s equation. The diffraction-unlimited nature of the RESOLFT family of concepts is reflected by the fact that the minimal resolvable distance $\Delta d$ can be continuously decreased by increasing $\varsigma ={\frac {I}{Isat}}$ . Hence the quest for nanoscale resolution comes down to maximizing this quantity. This is possible by increasing $I$ or by lowering $I_{sat}$ .

Variants

Different processes are used when switching the molecular states. However, all have in common that at least two distinguishable states are used. Typically the fluorescence property used marks the distinction of the states, however this is not essential, as absorption or scattering properties could also be exploited.^[4]

STED Microscopy

(Main article STED microscopy)

Within the STED microscopy (STimulated Emission Depletion microscopy)^[1]^[2] a fluorescent dye molecule is driven between its electronic ground state and its excited state while sending out fluorescence photons. This is the standard operation mode in fluorescence microscopy and depicts state A. In state B the dye is permanently kept in its electronic ground state through stimulated emission. If the dye can fluoresce in state A and not in state B, the RESOLFT concept applies.

GSD microscopy

(Main article GSD microscopy)

GSD microscopy (Ground State Depletion microscopy) also uses fluorescent markers. In state A, the molecule can freely be driven between the ground and the first excited state and fluorescence can be sent out. In the dark state B the ground state of the molecule is depopulated, a transition to a long lived excited state takes place from which fluorescence is not emitted. As long as the molecule is in the dark state, it's not available for cycling between ground and excited state, fluorescence is hence turned off.

SPEM and SSIM

SPEM (Saturated Pattern Excitation Microscopy)^[5] and SSIM (Saturated Structured Illumination Microscopy)^[6] are exploiting the RESOLFT concept using saturated excitation to produce "negative" images, i.e. fluorescence occurs from everywhere except at a very small region around the geometrical focus of the microscope. Also non point-like patterns are used for illumination. Mathematical image reconstruction is necessary to obtain positive images again.

RESOLFT with switchable proteins

Some fluorescent proteins can be switched on and off by light of an appropriate wavelength. They can be used in a RESOLFT-type microscope.^[7] During illumination with light, these proteins change their conformation. In the process they gain or lose their ability to emit fluorescence. The fluorescing state corresponds to state A, the non-fluorescing to state B and the RESOLFT concept applies again. The reversible transition (e.g. from B back to A) takes place either spontaneously or again driven by light. Inducing conformational changes in proteins can be achieved already at much lower switching light intensities as compared to stimulated emission or ground state depletion (some W/cm²). In combination with 4Pi microscopy images with isotropic resolution below 40 nm have been taken of living cells at low light levels.^[8]

RESOLFT with switchable organic dyes

Just as with proteins, also some organic dyes can change their structure upon illumination.^[9]^[10] The ability to fluoresce of such organic dyes can be turned on and off through visible light. Again the applied light intensities can be quite low (some 100 W/cm²).

Related Research Articles

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.

Angular resolution describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object, thereby making it a major determinant of image resolution. It is used in optics applied to light waves, in antenna theory applied to radio waves, and in acoustics applied to sound waves. The colloquial use of the term "resolution" sometimes causes confusion; when an optical system is said to have a high resolution or high angular resolution, it means that the perceived distance, or actual angular distance, between resolved neighboring objects is small. The value that quantifies this property, θ, which is given by the Rayleigh criterion, is low for a system with a high resolution. The closely related term spatial resolution refers to the precision of a measurement with respect to space, which is directly connected to angular resolution in imaging instruments. The Rayleigh criterion shows that the minimum angular spread that can be resolved by an image forming system is limited by diffraction to the ratio of the wavelength of the waves to the aperture width. For this reason, high resolution imaging systems such as astronomical telescopes, long distance telephoto camera lenses and radio telescopes have large apertures.

$Diffraction-limited system Optical system with resolution performance at the instruments theoretical limit$

In optics, any optical instrument or system – a microscope, telescope, or camera – has a principal limit to its resolution due to the physics of diffraction. An optical instrument is said to be diffraction-limited if it has reached this limit of resolution performance. Other factors may affect an optical system's performance, such as lens imperfections or aberrations, but these are caused by errors in the manufacture or calculation of a lens, whereas the diffraction limit is the maximum resolution possible for a theoretically perfect, or ideal, optical system.

$Airy disk Diffraction pattern in optics$

In optics, the Airy disk and Airy pattern are descriptions of the best-focused spot of light that a perfect lens with a circular aperture can make, limited by the diffraction of light. The Airy disk is of importance in physics, optics, and astronomy.

<span class="mw-page-title-main">Point spread function</span> Response in an optical imaging system

The point spread function (PSF) describes the response of a focused optical imaging system to a point source or point object. A more general term for the PSF is the system's impulse response; the PSF is the impulse response or impulse response function (IRF) of a focused optical imaging system. The PSF in many contexts can be thought of as the extended blob in an image that represents a single point object, that is considered as a spatial impulse. In functional terms, it is the spatial domain version of the optical transfer function (OTF) of an imaging system. It is a useful concept in Fourier optics, astronomical imaging, medical imaging, electron microscopy and other imaging techniques such as 3D microscopy and fluorescence microscopy.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis provides kinetic parameters of the physical processes underlying the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.

A superlens, or super lens, is a lens which uses metamaterials to go beyond the diffraction limit. The diffraction limit is a feature of conventional lenses and microscopes that limits the fineness of their resolution depending on the illumination wavelength and the numerical aperture (NA) of the objective lens. Many lens designs have been proposed that go beyond the diffraction limit in some way, but constraints and obstacles face each of them.

Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves. In SNOM, the excitation laser light is focused through an aperture with a diameter smaller than the excitation wavelength, resulting in an evanescent field on the far side of the aperture. When the sample is scanned at a small distance below the aperture, the optical resolution of transmitted or reflected light is limited only by the diameter of the aperture. In particular, lateral resolution of 6 nm and vertical resolution of 2–5 nm have been demonstrated.

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

Ground state depletion microscopy is an implementation of the RESOLFT concept. The method was proposed in 1995 and experimentally demonstrated in 2007. It is the second concept to overcome the diffraction barrier in far-field optical microscopy published by Stefan Hell. Using nitrogen-vacancy centers in diamonds a resolution of up to 7.8 nm was achieved in 2009. This is far below the diffraction limit (~200 nm).

$Talbot effect Near-field diffraction effect$

The Talbot effect is a diffraction effect first observed in 1836 by Henry Fox Talbot. When a plane wave is incident upon a periodic diffraction grating, the image of the grating is repeated at regular distances away from the grating plane. The regular distance is called the Talbot length, and the repeated images are called self images or Talbot images. Furthermore, at half the Talbot length, a self-image also occurs, but phase-shifted by half a period. At smaller regular fractions of the Talbot length, sub-images can also be observed. At one quarter of the Talbot length, the self-image is halved in size, and appears with half the period of the grating. At one eighth of the Talbot length, the period and size of the images is halved again, and so forth creating a fractal pattern of sub images with ever-decreasing size, often referred to as a Talbot carpet. Talbot cavities are used for coherent beam combination of laser sets.

Vertico spatially modulated illumination (Vertico-SMI) is the fastest light microscope for the 3D analysis of complete cells in the nanometer range. It is based on two technologies developed in 1996, SMI and SPDM. The effective optical resolution of this optical nanoscope has reached the vicinity of 5 nm in 2D and 40 nm in 3D, greatly surpassing the λ/2 resolution limit applying to standard microscopy using transmission or reflection of natural light according to the Abbe resolution limit That limit had been determined by Ernst Abbe in 1873 and governs the achievable resolution limit of microscopes using conventional techniques.

Speckle, speckle pattern, or speckle noise is a granular noise texture degrading the quality as a consequence of interference among wavefronts in coherent imaging systems, such as radar, synthetic aperture radar (SAR), medical ultrasound and optical coherence tomography. Speckle is not external noise; rather, it is an inherent fluctuation in diffuse reflections, because the scatterers are not identical for each cell, and the coherent illumination wave is highly sensitive to small variations in phase changes.

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.

Wide-field multiphoton microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 µm). The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy. Wide-field multiphoton microscopes are not yet commercially available, but working prototypes exist in several optics laboratories.

Super-resolution photoacoustic imaging is a set of techniques used to enhance spatial resolution in photoacoustic imaging. Specifically, these techniques primarily break the optical diffraction limit of the photoacoustic imaging system. It can be achieved in a variety of mechanisms, such as blind structured illumination, multi-speckle illumination, or photo-imprint photoacoustic microscopy in Figure 1.

References

1 2 Stefan W. Hell & Jan Wichmann (1994). "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy". Optics Letters. 19 (11): 780–2. Bibcode:1994OptL...19..780H. doi:10.1364/OL.19.000780. PMID 19844443.
1 2 Thomas A. Klar; Stefan W. Hell (1999). "Subdiffraction resolution in far-field fluorescence microscopy". Optics Letters. 24 (14): 954–956. Bibcode:1999OptL...24..954K. doi:10.1364/OL.24.000954. PMID 18073907.
↑ See also Confocal laser scanning microscopy.
↑ Stefan W. Hell (2004). "Strategy for far-field optical imaging and writing without diffraction limit". Physics Letters A. 326 (1–2): 140–145. Bibcode:2004PhLA..326..140H. doi:10.1016/j.physleta.2004.03.082.
↑ Rainer Heintzmann; Thomas M. Jovin; Christoph Cremer (2002). "Saturated patterned excitation microscopy a concept for optical resolution improvement". Journal of the Optical Society of America A. 19 (8): 1599–2109. Bibcode:2002JOSAA..19.1599H. doi:10.1364/JOSAA.19.001599. hdl: 11858/00-001M-0000-0029-2C24-9 . PMID 12152701.
↑ Mats G. L. Gustafsson (2005). "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution". Proceedings of the National Academy of Sciences of the United States of America. 102 (37): 13081–6. Bibcode:2005PNAS..10213081G. doi: 10.1073/pnas.0406877102 . PMC 1201569 . PMID 16141335.
↑ Michael Hofmann; Christian Eggeling; Stefan Jakobs; Stefan W. Hell (2005). "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins". Proceedings of the National Academy of Sciences of the United States of America. 102 (49): 17565–9. Bibcode:2005PNAS..10217565H. doi: 10.1073/pnas.0506010102 . PMC 1308899 . PMID 16314572.
↑ Ulrike Böhm; Stefan W. Hell; Roman Schmidt (2016). "4Pi-RESOLFT nanoscopy". Nature Communications. 7 (10504): 1–8. Bibcode:2016NatCo...710504B. doi:10.1038/ncomms10504. PMC 4740410 . PMID 26833381.
↑ Mariano Bossi; Jonas Fölling; Marcus Dyba; Volker Westphal; Stefan W. Hell (2006). "Breaking the diffraction resolution barrier in far-field microscopy by molecular optical bistability". New Journal of Physics. 8 (11): 275. Bibcode:2006NJPh....8..275B. doi: 10.1088/1367-2630/8/11/275 .
↑ Jiwoong Kwon; Jihee Hwang; Jaewan Park; Gi Rim Han; Kyu Young Han; Seong Keun Kim (2015). "RESOLFT nanoscopy with photoswitchable organic fluorophores". Scientific Reports. 5: 17804. Bibcode:2015NatSR...517804K. doi:10.1038/srep17804. PMC 4671063 . PMID 26639557.

This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.

[STED-sources-1-1] 1 2 Stefan W. Hell & Jan Wichmann (1994). "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy". Optics Letters. 19 (11): 780–2. Bibcode:1994OptL...19..780H. doi:10.1364/OL.19.000780. PMID 19844443.

[STED-sources-2-2] 1 2 Thomas A. Klar; Stefan W. Hell (1999). "Subdiffraction resolution in far-field fluorescence microscopy". Optics Letters. 24 (14): 954–956. Bibcode:1999OptL...24..954K. doi:10.1364/OL.24.000954. PMID 18073907.

[3] See also Confocal laser scanning microscopy.

[4] Stefan W. Hell (2004). "Strategy for far-field optical imaging and writing without diffraction limit". Physics Letters A. 326 (1–2): 140–145. Bibcode:2004PhLA..326..140H. doi:10.1016/j.physleta.2004.03.082.

[5] Rainer Heintzmann; Thomas M. Jovin; Christoph Cremer (2002). "Saturated patterned excitation microscopy a concept for optical resolution improvement". Journal of the Optical Society of America A. 19 (8): 1599–2109. Bibcode:2002JOSAA..19.1599H. doi:10.1364/JOSAA.19.001599. hdl: 11858/00-001M-0000-0029-2C24-9 . PMID 12152701.

[6] Mats G. L. Gustafsson (2005). "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution". Proceedings of the National Academy of Sciences of the United States of America. 102 (37): 13081–6. Bibcode:2005PNAS..10213081G. doi: 10.1073/pnas.0406877102 . PMC 1201569 . PMID 16141335.

[7] Michael Hofmann; Christian Eggeling; Stefan Jakobs; Stefan W. Hell (2005). "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins". Proceedings of the National Academy of Sciences of the United States of America. 102 (49): 17565–9. Bibcode:2005PNAS..10217565H. doi: 10.1073/pnas.0506010102 . PMC 1308899 . PMID 16314572.

[8] Ulrike Böhm; Stefan W. Hell; Roman Schmidt (2016). "4Pi-RESOLFT nanoscopy". Nature Communications. 7 (10504): 1–8. Bibcode:2016NatCo...710504B. doi:10.1038/ncomms10504. PMC 4740410 . PMID 26833381.

[9] Mariano Bossi; Jonas Fölling; Marcus Dyba; Volker Westphal; Stefan W. Hell (2006). "Breaking the diffraction resolution barrier in far-field microscopy by molecular optical bistability". New Journal of Physics. 8 (11): 275. Bibcode:2006NJPh....8..275B. doi: 10.1088/1367-2630/8/11/275 .

[10] Jiwoong Kwon; Jihee Hwang; Jaewan Park; Gi Rim Han; Kyu Young Han; Seong Keun Kim (2015). "RESOLFT nanoscopy with photoswitchable organic fluorophores". Scientific Reports. 5: 17804. Bibcode:2015NatSR...517804K. doi:10.1038/srep17804. PMC 4671063 . PMID 26639557.

[1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]