One of the most promising and exciting application of PCFPs and RSFPs is their use in nanoscopy. Indeed, thanks to techniques newly developed, those proteins can be used as efficient fluorescent probes, in a way that allows to defeat the resolution limitation imposed by diffraction.
1 - The resolution is limited by diffraction in far-field visible microscopy
The resolution of a microscope refers to the resolving power of the lenses that compose this microscope. The resolving power is the ability of an apparatus to measure the angular distance between two points (i.e. distinguish two points) of an object, and is limited by the phenomenon of diffraction. In far-field microscopy, the waves emitted by a light source in a microscope do not focus in an infinitely small point but interfere together near the image plane to form a so- called Fraunhofer diffraction pattern. As a consequence, this pattern at the focal plane will resemble a disc surrounded by concentric blurred rings called first, second... order maxima (Figure I.3.2) and separated by minima called first, second... zeros; this pattern is called the Airy disc. The Airy disc is characterized by an intensity distribution function called the point- spread function (PSF) of the microscope. The point spread function is thus the instrumental response of the imaging of a point object and the image is a convolution of the object and the PSF. The degree of spreading (blurring) is a measure for the quality of an imaging system.
Figure I.3.2 - The parasitic phenomenon of diffraction causes the formation of an Airy disc (left).
The corresponding point-spread function (right)
a - The Abbe's relation
The physicist Ernst Abbe (Figure I.3.3) found (Abbe 1873) that in far-field microscopy, using visible light, the smallest distance between two objects that can be distinguished defines the resolving power (r) of the apparatus. He described this value as being: r~/2n
Where is the wavelength that is used and n is the refractive index of the medium in which the light rays travel. For example, with n=1 (air) and=500 nm, we find that the best resolution equals r~250 nm.
Figure I.3.3 - Photograph of Ernst Karl Abbe and John William Strutt Rayleigh
b - The Rayleigh criterion
In reality, two objects start to be undistinguishable when their Airy discs are separated by an Airy radius. Below this distance, the objects will be unresolved. This expression that approaches very much the Abbe’s relation is known as the Rayleigh criterion (Figure I.3.4) and is expressed by:
𝑟 = 0.61 𝜆
𝑁𝐴 or 𝑟 =1.22 𝜆
2 𝑁𝐴 = 1.22 𝜆 2 𝑛 ∙ 𝑠𝑖𝑛(𝛼)
Equation I.1
Where NA and are the numerical aperture and the half aperture of the microscope, respectively. For example, with NA=1.2 and =500 nm, we find a resolution of 𝑟 ≈ 254 nm.
Figure I.3.4 - The Rayleigh criterion. When the distance between two objects is too close for the microscope resolution, they cannot be distinguished
This relation shows that the resolution of a microscope depends on two parameters: the wavelength (the resolution is worse when the wavelength increases) and NA (the resolution is better when the numerical aperture increases). The optical resolution is a complex topic.
Since state of the art air objectives in visible microscopy allow a numerical aperture of 0.95, the best resolution that can be achieved with such microscopes is r=/1.5. With oil immersion objectives, numerical aperture values up to 1.65 can be reached so that the best resolutions with such microscopes are about r=/2.7. Overall, it is admitted that for conventional optical
microscopy the resolution is limited to values around /2 or 250 nm for visible light (James 1976) and is never better than /3. The observation of a real specimen made of many punctual objects gives rise to a high number of Airy patterns whose PSF can be resolved or not (Figure I.3.5). This is a major problem in fluorescence microscopy because the smallest cellular structures cannot be detailed and there are always doubts concerning the co-localizations of elements of interest.
Figure I.3.5 - Resolution in far-field microscopy. The observed elements give rise to Airy patterns that can only be resolved if the objects are separated enough - Reproduced from Olympus Microscopy
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2 - Defeating the resolution limit, the quest for nanoscopy
The Abbe-Rayleigh relation stood as the golden rule in microscopy for almost 130 years, up until recent advances allowed breaking it for UV-Vis fluorescence microscopy, which have allowed the development of diffraction-unlimited resolution techniques.
a - Localization of fluorescent objects with high accuracy
Watt Webb (Figure I.3.7) at Cornell University, USA, discovered that fitting the PSF and calculating their centroid, an improved localization of individual fluorescent particles, and thus, their precise tracking (Thompson et al. 2002) could be obtained compared to conventional microscopy. This fitting procedure is made by simple gaussian curves instead of Airy functions because fluorescent images are rarely precise enough to distinguish between those alternatives.
8 www.olympusmicro.com
This method reveals that if enough fluorescent photons are integrated from an individual fluorescent spot, its PSF fitting allows a greater resolution by an order of magnitude!
The authors found that while conventional diffraction-limited imaging of a fluorescent object only allows a resolution of ~250 nm FWHM, the precise localization of its center can be estimated with an accuracy given by the FWHM divided by the signal to noise 𝑁𝑝ℎ (with 𝑁𝑝ℎ the number of photons integrated). Basically, if the total number of integrated photons for a single spot is 104, the localization of a fluorescent object can be:
𝐹𝑊𝐻𝑀 𝑁𝑝ℎ
= 250
104≅ ±1.3 𝑛𝑚
Equation I.2
This discovery gave rise to the development of the technique called FIONA, standing for Fluorescence Imaging with One-Nanometer Accuracy (Selvin et al. 2007). This technique allows the precise localization of single fluorescent molecules in the x,y plane with only using a sample fixed on a coverslip and excited by a total internal reflection fluorescence (TIRF) microscope (Yildiz et al. 2003; Yildiz & Selvin 2005).
b - Stimulated emission depletion (STED)
The first high resolution fluorescence microscopy technique was developed by Stefan Hell (Figure I.3.7) and colleagues at Göttingen, Germany (where Ernst Abbe obtained his doctorate in 1861) and is called STED (STimulated Emission Depletion).
The principle of STED (Hell & Wichmann 1994; Klar et al. 2001) is based on PSF engineering, increasing the fluorescence microscopy resolution by depleting the signal coming from molecules located in a ring-shaped light pulse. In practice, two synchronized laser pulses separated by a very short delay are sent on a fluorescent sample. The first pulse uses the excitation wavelength. A second doughnut-shaped and red-shifted pulse is applied at the
circumference, immediately after the first one, during the excitation lifetime. The excited state comes back to the ground state without fluorescence emission by stimulated emission. The consequence is that the resulting PSF that is really observed is much narrower than what would have been expected following the conventional diffraction-limited rule (Figure I.3.6).
Figure I.3.6 - The principle of STED microscopy. Left: a depletion wavelength is chosen so that a good stimulation emission is achieved without re-excitation of the fluorophore [reproduced from (Kellner
2007)]; Middle: by stimulating molecules at the periphery of the excitation spot with a longer wavelength, a doughnut-shaped STED pulse acts like a carving knife on the excitation PSF's tails so
that the effective PSF is shrunk. Right: example of resolution improvement between confocal fluorescence microscopy and STED microscopy on neurofilaments (source: MPI-BPC Göttingen website9)
The resolution is thus greatly improved (about /20 instead of /2) and it is possible to reconstruct details of the smallest structures inside cells at unprecedented resolutions of 15- 30 nm (150-300 Å), approaching resolutions of standard scanning electron microscopes (SEM).
The STED technique is based on the principle of RESOLFT (REversible Saturable Optically Linear Fluorescence Transitions)(Hell 2003; 2007). This principle claims that the Abbe- Rayleigh formula (Equation I.1) can be developed and approximated by :
𝑟 = 𝜆
2𝑁𝐴 ∙ 1 + 𝐼 𝐼𝑠𝑎𝑡
Equation I.3
9 http://www.mpibpc.mpg.de
Where I is the intensity of the depletion excitation light and Isat is the intensity needed to saturate the transition (half of the molecules excited are stimulated to return to S0). In theory, thus, one can improve the optical resolution by increasing the intensity I, and as the intensity of the STED pulse is increased, the detected (engineered) PSF shrinks further.
Figure I.3.7 - Photographs of Watt W. Webb, Stefan W. Hell, Eric Betzig and Xiaowei Zhuang
c - Photoactivated localization microscopy (PALM)
Very recently, the use of Kaede-like PAFPs in microscopy found an impressive application with the development of the PhotoActivated Localization Microscopy by Eric Betzig (Figure I.3.7) and colleagues at the Howard Hughes Medical Institute, USA (Betzig et al. 2006).
Basically, this technique uses a sample marked with green-to-red PAFPs. A short pulse at
~400 nm induces the irreversible photoconversion of a few, well separated individual molecules whose localization can determined with extreme precision with the method described above, p.61. Those molecules are finally bleached by this light and the photoconversion step can be renewed, photoconverting other elements in the sample. This iterative method ultimately allows the reconstruction of stochastically photoconverted individual elements with a very high accuracy, which produces a diffraction-unlimited fluorescence microscopic image (Figure I.3.8).
Figure I.3.8 - Principles of PALM and STORM sub-diffraction microscopy techniques allowed by PAFPs and RSFPs. Thumbnail images show the improvement of resolution allowed by both techniques
[inset images reproduced from (Betzig et al. 2006) and (Bates et al. 2007)]
d - Stochastic optical reconstruction microscopy (STORM)
The same stochastic approach has been used by Xiaowei Zhuang (Figure I.3.7) and colleagues at Harvard University, USA, to develop the STochastic Optical Reconstruction Microscopy technique the same year (Rust et al. 2006). This technique is strictly comparable to the PALM technique but was initially developed using photochromic dyes. First, all the RSFP molecules are turned \off", then a short pulse at 405 nm randomly turns \on" very few and separated objects that can be precisely imaged and bleached before the activation of other molecules to be recycled (Figure I.3.8). The fast trans to cis isomerization of Dronpa-like RSFPs permits a faster acquisition time although the iterative process and reconstruction is still a slow process.
PALM and STORM are thus two extremely similar techniques that use the photoactivation properties of PAFPs or RSFPs to individually image all the points within an object with a very high accuracy and then obtain an image superposing all the points in order to reconstruct a
diffraction-unlimited high-resolution image that reveals the smallest details of the studied sample (Figure I.3.9).
Figure I.3.9 - The idea behind the PALM/STORM super-resolution techniques. Two objects are too close each other to be resolved since the Rayleigh criterion is reached (a). The precise localization of only
one object by temporarily bleaching the other one (b) and then of only the second one by the reverse action (c) allow the positioning of both objects with a very high accuracy (d)
e - Fast and three-dimensional PALM/STORM microscopies
The main drawbacks of PALM/STORM methods compared to STED are that the iterative pulse/reconstruction steps make them time-consuming techniques and also that they only can be used in TIRF-based (surface bound) systems. The recent use of fast RSFPs such as Dronpa mutants (Flors et al. 2007) in PALM/STORM-like experiments now allows solving the first problem. The development of 3D-PALM and 3D-STORM techniques now makes possible the fast super-resolutive microscopy of objects in solution. If, as we said previously, the lateral resolution is limited by the Airy radius 𝑟𝑥,𝑦 ≈ 0.61 ∙ 𝜆 𝑁𝐴 in conventional visible microscopy (and a bit better in confocal microscopy: 𝑟𝑥,𝑦 ≈ 0.46 ∙ 𝜆 𝑁𝐴), the axial resolution is really worse: 𝑟𝑥,𝑧 ≈ 2 ∙ 𝜆 𝑁𝐴2 (and 𝑟𝑥,𝑧 ≈ 1.4 ∙ 𝜆 𝑁𝐴2 in confocal microscopy) because of an elongated PSF.
The use of 4Pi-microscopy (Hell et al. 1994) along with sample sectioning allowed axial resolutions to be reached <80 nm in STED (Punge et al. 2008). In parallel, the development of the PALM technique with the so-called biplane detection (Juette et al. 2008) recently allowed an axial resolution of 75 nm to be reached and the development of the STORM technique with
elliptical astigmatism permitted the enhancement of the axial resolution up to 50-60 nm (Huang et al. 2008). The 3D visible far-field nanoscopy with axial resolutions of /6 to /10 is thus a reality.
To summarize, super-resolution techniques represent a revolution in visible far-field microscopy by allowing us to have detailed information on the smallest cellular structures.
They also provide direct and precise information on objects that colocalize without requesting mathematical tricks or beads calibrations.