Figure 6. Angular magnification by a single convex lens. A When an object is directly observed by the eye eye lens, EL , a relatively small angle is subtended at the eye by the object. B When a simple convex lens such as e. Because the eye has its own convex lens, it also has the ability to focus the diverging light rays originating from this virtual image to once again form a real image on the retina R.
Since the angle subtended by the virtual image at the eye is now much larger, a larger image of the object will form at the retina. In the early days of microscopy, all instruments were 'bright field' transmission microscopes. In such microscopes, homogeneous and sufficiently intense illumination is achieved through the use of a condenser lens, which focuses light of the illumination source onto the sample.
This light subsequently passes through the sample and is ultimately collected by the compound lens system of the microscope. Variations in transparency of the sample create contrast in the resulting image. Even though modern-day instruments are often much more complex than the earliest compound microscopes, one can still grasp the essential aspects of image formation in microscopy by considering the most basic two lens system consisting of an objective lens and an ocular lens. The objective lens typically features a very short focal length, producing an enlarged and inverted real image inside the microscope behind, or most optimally at, the focal point of the ocular.
This results in a virtual image that is magnified significantly, such that it can subsequently be observed by the eye figure 7 A. Figure 7. Image formation in simple lens systems in simple compound microscopes. EL Eye lens, R Retina. B A compound microscope with a tube lens T features an infinity space, allowing for the introduction of additional optical elements without impact to the tube length.
To ensure that the objective forms an image at the focal point of the ocular lens, the relative distance between both lenses, the so-called tube length, needs to be fixed, simply because the lenses themselves have fixed focal lengths. Introduction of any additional optical element such as e.
Because of this, modern microscopes typically feature an additional lens inside the optical tube. This lens is aptly named the 'tube-lens' and modern objective lenses are designed such that the light rays between the objective and the tube lens are perfectly parallel, i.
The tube lens is responsible for creating a real image at the ocular lens focus. This allows the section between the objective and tube lens, the infinity space, to be of arbitrary length. As such, it can cater for the relatively straightforward introduction of additional optical elements figure 7 B. The imaging systems outlined here are still highly simplified. In reality, a single objective can contain more than ten individual lens elements, made from different materials, arranged into multiple groups and featuring specialized coatings.
This is necessary for correcting optical aberrations that unavoidably occur when light passes through lenses.
These image distortions can generally be divided into chromatic-, spherical-, coma-, stigmatic- and field curvature aberrations, which are treated in depth elsewhere [ 12 ]. Fluorescence is a luminescence phenomenon where certain molecules and minerals emit light upon absorption of photons from an 'excitation' light source. Excitation to an emissive state can only occur when the wavelength of an incident photon matches the energy difference between the electronic ground state and an excited electronic state of the dye, provided that this electronic transition is also be allowed by the laws of quantum mechanics [ 13 ].
For organic fluorophores in the condensed phase, the excited molecules return to their ground energy state very shortly after the excitation event, i. Emitted photons feature a longer wavelength compared to the corresponding excitation photons. This red shift of the fluorescence emission, the 'Stokes shift', is caused by the fact that excited molecules lose a small amount of the absorbed energy through non-radiative processes such as molecular vibrations or interactions with surrounding media, i.
The radiative energy transition will therefore be smaller and, in accordance with Planck's law, the wavelength of emission will be longer figure 8 [ 13 ]. It should be noted that not every photon absorbed by a fluorophore gets re-emitted as a fluorescence photon.
Super-resolution imaging (SR) is a class of techniques that enhance the resolution of an imaging system. In some SR techniques—termed optical SR— the. Super-resolution microscopy, in light microscopy, is a term that gathers several techniques, which allow images to be taken with a higher resolution than the one .
As the fluorophore interacts with its surroundings, a number of other de-excitation processes can compete with fluorescence emission [ 13 , 14 ]. Figure 8. A Jablonski diagram showing the energy transitions in fluorescence. Upon excitation, the molecule will be in a higher vibrational state. Prior to emission, the molecule will relax non radiatively, after which emission can take place. B A Franck—Condon energy diagram shows how transitions can occur to different vibrational levels, resulting in characteristic shapes for the excitation and emission spectra.
C Excitation and emission spectra typically resemble each other's mirror image because similar transitions occur with the same probability. Before the advent of super-resolution microscopy, most fluorescence imaging and microscopy could be expected to occur under conditions where the excitation rate from the ground state S 0 to the first excited state S 1 would be lower than the radiative decay rate from S 1 to S 0. These conditions are said to be non-saturating. With a significant fraction of fluorophores in the ground state, the probability that processes, other than normal fluorescence decay take place from S 1 , is relatively small [ 15 ].
Nonetheless, competing pathways resulting in the formation of metastable dark states such intersystem crossing ISC from S 1 to the triplet T 1 or formation of radical states or are possible. Depending on e.
Figure 9. Jablonski diagrams showing processes that compete with fluorescence emission. A At low excitation powers, the excited S 1 state might convert to a longer lived and dark triplet state T 1 or to various other dark states, as indicated by 'D'.
B At increasing excitation powers, transitions to higher excited states also become prevalent. Moreover, at increasing power levels, excitation from S 1 and T 1 into higher excited states S n and T n will also become more prevalent figure 9 B. What is important is that all these excited states can be precursors to permanent photobleaching, resulting in irreversible loss of the ability to emit fluorescence light [ 15 ]. For low to moderate excitation powers, and well-defined chemical environments, it's value can be considered constant.
However, as will become apparent in the remainder of this manuscript, many super-resolution modalities, notably SIM and STED by definition will not operate under these conditions. These techniques specifically rely on illumination intensities that are such that fluorescence brightness no longer increases linearly to the excitation intensity and photobleaching can become a significant concern.
In fluorescence microscopy, fluorophores can be excited in any number of ways, ranging from voltaic arc lamps to LED's and lasers. In general, high intensity illumination is preferred to ultimately ensure generation of sufficient fluorescent photons. While many optical arrangements exist, so called 'epi'-fluorescence microscopy, where a single lens acts as both the condenser and objective, is by far the most frequently used implementation figure For practical reasons, this lens is most often placed underneath the sample, the so called 'inverted configuration'.
Figure Schematic representation of a typical fluorescence microscope and its essential components. Key is the combination of an excitation filter, dichroic mirror and emission filter, often termed a filter-cube. This combination ensures that excitation and emission light can be separated and the latter relayed to the observer. In contrast with a transmission microscope, most of the excitation light in an inverted epi-fluorescence microscope is not absorbed by the sample and simply passes through, never reaching the detector figure Moreover, due to back scattering some of the excitation light will nevertheless be collected by the objective lens.
mountkawibillde.ml Therefore, proper separation of excitation and emission light on their way to and from the sample, is highly important. A dedicated optical element, the dichroic mirror, is used to achieve this. A small fraction of back scattered excitation light might still be transmitted by the dichroic mirror but it is blocked by an emission filter, before it can reach the detector.
This way, emission and excitation light can be completely separated figure The ability to create filters that allow one or more precisely defined wavelength bands to pass, while efficiently blocking all other light, is central to all fluorescence microscopy studies of complex biological phenomena as it enables simultaneous observation of multiple, distinctly colored species.
Fluorescence microscopy offers many benefits over transmission microscopy in biological applications. Indeed, fluorescent labels attached to the structures of interest will be visible as bright point emitters against a vast dark background, like stars in the night sky, drastically improving contrast.
Indeed, careful tuning of the photophysical and bio functional properties of fluorescent labels has become an indispensable aspect of high resolution imaging of biological samples, as will become clear in the following sections. One of the most important innovations in fluorescence microscopy, particularly for life science applications, might well be the invention of the confocal microscope. Although patented in by Marvin Minsky of Harvard University, it would take around 20 years before it could be implemented practically [ 17 ].
All microscope arrangements discussed up to this point are 'wide-field' WF microscopes, where the entire sample volume is illuminated at the same time. By contrast, in a confocal microscope, light is focused into a relatively small volume within a three-dimensional sample.
The emission from this focal volume is collected by the objective lens, as it normally would. However, instead of recording it using an imaging device such as the human eye or a camera, it is relayed to a light sensitive point detector such as a photomultiplier tube PMT or an even more sensitive avalanche photon detector APD. Although technically distinct, both PMTs and APDs convert the incident photons into an electrical signal which can be amplified, by many orders of magnitude, resulting in enhanced contrast. More in-depth discussions on photon detectors for CLSM can be found elsewhere [ 14 ].
To properly observe a sample that is many times larger than the single illumination volume, either the sample or the illumination volume need to be moved across the sample in discrete steps.
The latter approach, called confocal laser scanning confocal microscopy CLSM , is by far the most common approach in biology applications. In CLSM, a set of movable mirrors is used to direct the illumination spot. A computer collects the detector signal throughout the scanning procedure and digitally reconstructs an image from the recorded information. The most important feature of a confocal microscope is the pinhole placed in front of the detector at a specific distance, the confocal plane. This pinhole will attenuate all light which does not originate from the focal volume, thus removing out of focus emission and greatly enhancing signal-to-noise ration and contrast figure Schematic representation of confocal detection.
Light originating from outside of the current focal position blue will be blocked by the pinhole whereas light red from the focal plane will be allowed to pass. Because emission is only observed from a relatively thin axial section of the sample, thicker samples can also be optically sectioned. By moving the illumination spot axially as well as laterally, three-dimensional structures such as biological tissues or even whole animals can be imaged.
Further developments, i. For these reasons CLSM became extremely popular in the biological and biomedical sciences. Over time, many variations on the basic confocal design were developed such as the spinning disk confocal microscope and non-linear confocal imaging methods such as 2-photon imaging. An exhaustive review of all these variations is beyond the scope of this review but excellent sources on these topics exist [ 14 ]. The resolving power or resolution of an optical imaging system, is defined as the smallest distance between two points for which both these points can still be distinguished.
To a certain extent, resolution can be improved through careful design of lenses and optics.
Moreover, because illumination occurs side-on, there is no out of focus emission as is typically the case in wide-field imaging figure 28 B [ 59 ]. This corresponds with the overlap of the Airy disk of one Airy functions with the first minima of the second Airy function. All Rights Reserved. Groeneweg, F. Journals Books Databases.
However, a physical limit will ultimately be reached, deeply rooted in the fundamental laws governing light diffraction. This implies that any optical microscope has a finite resolution and this physical limit is generally referred the 'diffraction limit'.