Super resolution microscopy methods were developed over the last few decades in order to overcome the optical diffraction limit by Drs Betzig, Hell, and Moerner, who were awarded the 2014 Nobel Prize for their work. [1][2][3] The optical diffraction limit, also called the Abbe diffraction limit, 4 limits the size of structures that can be resolved using light microscopy to a few hundred nanometers which is near to or larger than many subcellular structures, such as proteins and vesicles, which are of interest to researchers, as shown in Fig. 1. 5 While electron microscopy can also observe length scales down to several nanometers it is incompatible with living systems and thus cannot provide information on biological processes that are of interest to researchers. The diffraction limit, given in the xy plane by eqn (1) 5,6 states that the smallest resolvable distance (d x,y ) is approximately equal to half of the excitation wavelength (l) at most numerical apertures (NA); eqn (2) states that the smallest resolvable distance in the z plane (d z ) is equal to double the wavelength divided by NA 2 . [4][5][6][7][8][9][10]
Super resolution microscopy is divided into two categories: single molecule localization-based super resolution such as STORM and PALM (photo-activated localization microscopy)developed separately by Moerner 3 and Betzig, 2 among others, and patterned excitation super resolution such as STED and (reversible saturable optical uorescence transitions) RESOLFT developed by Hell. 1 Localization-based super resolution techniques use uorophores or probes that are capable of switching from dark state to an on state and back, which limits the number of uorophores available for use. Typically used uorophores are small molecule uorophores such as Cy5 and Cy3, and photoswitchable uorescent proteins such as green uorescent protein (GFP), although these uorophores have several disadvantages; 8,9,[11][12][13][14][15] specic imaging buffers are needed for use with small molecule uorophores to induce photoswitching, however these buffers are toxic to cells. 16,17 Both small molecule uorophores and photoswitchable uorescent proteins oen have a lower quantum yield and rapid photobleaching. In addition, the number of photons detected positively impacts the localization precision -the more photons collected, the more precisely the uorophore can be locatedmaking a high quantum yield desirable. 7,[18][19][20][21] Recently the library of uorescent probes for super resolution microscopy has expanded to include nanomaterials such as carbon dots, quantum dots, and dye doped nanoparticles of varying compositions. Though they are signicantly larger than the previously established super resolution uorophoressingle molecule dyes are approximately 0.5 nm and uorescent proteins, specically green uorescent protein (GFP), are approximately 4.2 nm in length and 2.4 nm in diameter and the various nanomaterial probes discussed in this review range in size from approximately 5 nm up to approximately 100 nmthey have been successfully used for super resolution microscopy of both cell surfaces and subcellular structures. 16,[22][23][24] These nanomaterial probes show higher quantum yields and better photostability than the previously established probes; GFP for example has a quantum yield ranging from 58-79% depending on the type of GFP being used and the commonly used uorescent proteins have quantum yields ranging from 7% to 85%, and commonly used single molecule probes have Fig. 1 The minimum spatial resolution of many techniques currently used to study biological structures is quite large, with even some super resolution techniques unable to resolve structures smaller than the mitochondria. The super resolution techniques stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED) are shown to be able to resolve structures down to tens of nanometers, making them able to image proteins and other small structures that cannot be resolved using typical fluorescence microscopy. Used with permission. 5 Table 1 A table providing information on several fluorescent proteins commonly used for super resolution microscopy. l ex is the excitation wavelength, l em is the emission wavelength, 3 abs is the extinction coefficient, contrast is the fold increase in fluorescence at the emission wavelength after photoactivation, h fl (%) is the quantum yield, and N is the number of photons detected per single molecule per imaging cycle. NA and ND are not applicable and not determined, respectively. Used with permission 5 Fluorescent protein Activating pight Quenching pight Pre/post colors l ex (nm) l em (nm) been found to have quantum yields ranging from 23% to 93%, shown in Tables 1 and2 respectively. Nanoparticles such as quantum dots have quantum yields ranging from 10-85% depending on composition and excitation wavelength and while they have lower quantum yields in the visible light range than uorescent proteins and single molecule uorophores they have higher quantum yields in the NIR light range, however carbon dots typically have lower quantum yields around 5% although doping the carbon dots with atoms such as nitrogen(N), sulfur (S), phosphorous (P), and uorene (F) can increase the quantum yield and recently carbon dots have been developed by Arab et al. that have shown a quantum yield of 85.69%. The quantum yield for dye doped nanoparticles would depend on the dye imbedded in the nanoparticle and the structure of the nanoparticle (mesoporous silica, core-shell, etc.) and no denitive range was found. 5,17,23,[25][26][27][28][29] In addition, these nanomaterials oen exhibit surface chemistry allows them to be modied for the targeting of specic structures, as demonstrated by Dong et al. using Biotin-BSA, Neutravidin, and biotinylated antibodies to modify the surface of Ag@SiO 2 nanoparticles to target the surface of cells. 17,23,[30][31][32] This review will discuss the recent use of carbon dots and quantum dots, as well as dye doped polymer-, protein-, and silica-based nanoparticles, which include metal-silica core-shell nanoparticles, in super resolution uorescence microscopy.
Super resolution microscopy can be divided into two categories: super resolution microscopy using spatially patterned excitation, called stimulated emission depletion (STED), reversible saturable optically linear uorescence transition (RESOLFT), and super resolution microscopy using single molecule/particle localization, called stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), uorescence photoactivation localization microscopy (FPALM), and point accumulation for imaging in nanoscale topography (PAINT). 7,8,28,[33][34][35][36][37] Two other techniques, structured illumination microscopy (SIM), and saturated-structured illumination microscopy (SSIM) will also be briey discussed. 37 Patterned excitation-based super resolution microscopy uses two lasers, one with a point of zero intensity in the center giving it a doughnut shape, which is used to switch the peripheral uorophores into the dark state in order to reduce the effective point spread function (PSF) (Fig. 2). 7 The optical resolution of STED and other patterned excitation approaches is represented by the size of the effective PSF created by the doughnut-shaped STED laser; the effective PSF is the area in which the uorescent probes are allowed to emit uorescence which is detected by the detector. 37,38 STED can achieve spatial resolutions of 50-80 nm with temporal resolution of seconds. 36 Data acquisition is relatively fast, on the order of seconds compared to localization-based methods which can take much longer to acquire enough images to construct a single-color composite image, and aer acquisition the super resolution image is immediately available, requiring no post processing. 6,8 A wider variety of uorophores can be used for STED microscopy because, due to the relative simplicity of the uorophore control, uorophores are not limited to those that can undergo photoswitching, photoconversion, or photoactivation; 6 STED has been successfully used with uorescent labeled antibodies and uorescent proteins, as well as quantum dots, carbon dots, and nanoparticles, with carbon dots and quantum dots being the most frequently used nanomaterial probes. 5,7,16,30,39 Structured illumination microscopy (SIM), excites the uorophores using high intensity periodic line patterns to achieve sub-diffraction limit resolution; the result of this approach is The average width of the microtubules given by the FWHM of the peaks is consistent with previous measurements performed with TEM and STORM using antibody labeling. The scale bars are 10 mm. Used with permission. 28,56 that due to saturation the orescence intensity is not proportional to the excitation source power. SIM uses patterned light to illuminate the sample and measures an interference pattern created by overlaying two grids that either have different mesh sizes or anglescalled the Moire pattern. 7,16,37,40,41 SIM can achieve a lateral resolution of approximately 125 nm and an axial resolution of approximately 350 nm. 36 Saturated structured illumination microscopy (SSIM) uses a strong excitation source in a sinusoidal pattern which allows detection of subdiffraction limit spatial resolution. The spatial resolution of SSIM is limited by the level of uorescence saturation rather than by diffraction, as in SIM, and has demonstrated spatial resolution down to 50 nm. 7 Localization-based super resolution microscopy is based on the precise localization of individual photoswitchable uorescent probes in order to build a super resolution image point by point. STORM super resolution uses a system of two lasers, one to activate individual uorophores and one to image the uorophores and return them to the dark state; once the uorophores the area of focus have been imaged the images are processed by a computer program (such as ThunderSTORM) which calculates the center of each uorescent point and constructs the composite super resolution image. 6,13,37,[42][43][44] Fig. 3 (ref. 6) illustrates the imaging process of STORM microscopy. The uorescent probes used for STORM microscopy must be able to photoswitch, or switch between a dark state and an on state, which limits the number and types of uorescent probes available for use. STORM was rst used with the small molecule uorophores Cy5 and Cy3 in a dye-pair system and has since been used with other small molecule uorophores as well as uorescent proteins, carbon dots, quantum dots, and nanoparticles. 5,6,13,16,17,23 PAINT methods rely on the diffusion and transient binding of an affinity probe conjugated to a uorophore; when bound the uorophore produces a bright spot on the recorded camera frame. The relative brightness of the spot is determined by measuring the brightness of the bright spot compared to the background signal from the unbound uorophores and can be enhanced by coupling the PAINT set up to a total internal reection (TIR) set up. 45 The resolution of single molecule/particle localization methods is inuenced by the labeling density and localization precision of the uorophores and to date has achieved the highest spatial resolution among super resolution techniques, with spatial resolutions of 20-25 nm being regularly achieved and spatial resolutions as low as 5 nm are possible at the expense of lower temporal resolution (minutes rather than seconds). 6,9,36,46 A variation on localization-based methods, blink microscopy uses self-blinking probes to perform single-molecule or singleparticle localization. The process of image acquisition is similar to STORM and PALM methods, but the self-blinking of the probe requires only a single excitation source and does not require biologically incompatible buffers. 23,28,34,[47][48][49][50][51] Nanomaterial probes
Carbon-based nanomaterials, called carbon dots, are luminescent nanomaterials whose optical properties are dependent on their size and the excitation wavelength being used. Carbon dots can be synthesized through top-down or bottom-up methods using various starting materials such as citric acid, glucose, or amino acid. Characterization shows that they can contain signicant amounts of oxygen and nitrogen dye to incomplete carbonization of the starting materials; in some instances carbonization of the starting materials may not happen but nanodots are still formedanalysis shows that the primary components of these may be polymers, leading to the name polymer carbon dots (PCD). 52 Carbon dots have been shown to be nontoxic, biocompatible, stable in aqueous solution, resistant to photobleaching, allow many possibilities for surface functionalization, are cost effective for large scale production, and also have the potential to uoresce in multiple colors. 16,30,[53][54][55] Originally carbon dots were thought to not exhibit blinking behaviors, however some were later discovered to experience uorescence uctuations; this was attributed to energy traps and different surface oxidation states. 16 Reversible photoswitching of carbon dots was achieved by alternating the excitation wavelength and it was found that carbon dots exhibit long lived off states when in the presence of an electron acceptor, making carbon dots a suitable uorescent probe for STORM and STED microscopies. 16,30 Compared with typical small molecule super resolution probes carbon dots show increased brightness, greater photostability, and a lower duty cycle, however they are larger than single molecule dyes, with an approximate diameter of 5 nm. 16,30 Carbon dots can be effectively used in super resolution imaging of cells and can be made to either bind to the cell membrane or, due to their small size, be taken into a living cell in order to bind to and image specic organelles or structures. Lemenager et al. were able to use carbon dots to obtain STED images the lysosomes of both xed and living cells, shown in Fig. 4, and were able to show a >6Â increase in spatial resolution compared to confocal microscopy. 53 Additionally, blinking carbon dots were used by He et al. to perform STORM imaging of subcellular structures such as microtubules (Fig. 5). 56 STORM imaging of the cell membrane was also performed in order to study the G protein coupled receptors. 56 Khan et al. also had some success in using carbon dots for single molecule imaging of the nucleolus of living cells. 57
Quantum dots are inorganic metallic nanomaterials composed of semiconducting metals; they are among the earliest reported inorganic uorescent nanoparticles used for uorescence labeling and have undergone more development in size, availability, biocompatible surface, and suitability for use with existing microscopy systems. 22,30 The quantum dots used in biological studies have a core-shell structure, such as CdSe and ZnS, resulting in narrow emission and wide absorption spectra. Quantum dots are synthesized in organic solvent in coordination with compounds that coat it to make it hydrophobic, thus requiring additional surface modication to become usable for biological studies. 58 Their emission color is size dependent, however they are considerably larger than conventional dyes and many are toxic to biological systems. 30,59,60 Because the emission wavelength can be changed by adjusting the quantum dot size during synthesis and purication, they are ideal for use in multicolor imaging and super resolution applications. Quantum dots are used with both STED and STORM super resolution microscopies, as well as for other methods such as SOFI (super resolution optical uctuation imaging) and single particle tracking. 30,55,58,61 Besides toxicity, another signicant drawback to using quantum dots is that they have a high on-off duty cycle (blinking) that needs to be stabilized before they can be used for super resolution imaging, though this property can be used advantageously for blinking-based super resolution (BBS) microscopy and SOFI. 30,58,59 Quantum dots can be used effectively in the super resolution imaging of cells and internal cellular structures, however due to limitations such as blinking behavior and a spectral blue shi (blueing) complicate their use and require some minor modi-cations to either the quantum dots themselves (to reduce blinking) or to the super resolution technique being used; 30,58 an example of the latter is the use of a transmission grating as part of the equipment set up in order to be able to distinguish between neighboring quantum dots that show different degrees of blueing. 30 Recent use of quantum dots in super resolution microscopy include the use of quantum dots to image internal cellular structures using both STED and a localization-based super resolution microscopy called 3B by Yang et al., 62 images of which are shown in Fig. 6. Another recent use of quantum dots for multicolor 3D STORM imaging reported by Xu et al. 61 a representative microscopy the time the quantum dot spends in the off state must be greater than the time spent in the on state, however quantum dots spend a shorter time in the off state compared to other photoswitchable probes, making them difficult to use for single molecule localization super resolution microscopy; in order to address this problem Xu et al. developed a technique to induce quantum dot blueing that has made quantum dots more suitable for STORM imaging by causing a stochastic shi of the emission to shorter wavelengths and observing the emission through a band pass lter which allows uorescence from individual quantum dots to be measured (Fig. 7). 61
For the purposes of this review the term "nanoparticle" will refer to any nanomaterial that is not a quantum dot or a carbon dot. The specic nanoparticles discussed here will be dye doped nanoparticles of varying compositions, including silica-based, additionally embedding dyes that weakly uoresce on their own may cause them to exhibit stronger uorescence, possibly due to suppression of non-radiative deexcitation events. 16 The addition of a metal core to the nanoparticle can further alter the behavior of the incorporated dye through metal enhanced uorescence. 17,23,25 Polymer-based nanoparticles, called polymer dots, are nanoparticles composed of mainly p-conjugated polymers and can be dye doped or composed of uorescent polymers such as CN-PPV or PFBT; a polymer dot can be composed of a single polymer or multiple polymers in specic amounts depending on the needs of the user and have been shown to be bright, photostable, biocompatible, and able to label subcellular structures. 10,60 The composition of the polymer dots can inuence the optical behavior, producing nanoparticles that show continuous uorescence, no uorescence, or photoblinking. 16,30 Recently dye doped polystyrene nanoparticles have been successfully used for STORM and STED microscopy, shown in Fig. 8. 16,63 Protein-based nanoparticles are composed of proteins such as bovine serum albumin (BSA) or transferrin as well as a variety of other sources including bacteria, viruses, amino acids, and plant or animal cells and are typically used in medicine for drug delivery. Because they are composed of proteins they are nontoxic, highly biocompatible, and can be easily modied for specic targeting; they also have symmetrical structures, uniform size distribution, and high reproducibility. 64 These nanoparticles have been used for both localization-based methods (STORM) and patterned excitation methods (STED). 16,65 Recently, transferrin-based protein nanoparticles developed by Lin et al. 16 have been shown to be able to be taken into cells and were successfully used for STED microscopy of HeLa cells, shown in Fig. 9, and BSA-based protein Silica-based nanoparticles, including mesoporous silica nanoparticles and core-shell nanoparticles, have been used for both STORM and STED super resolution microscopy. Silicabased nanoparticles are fairly easy to add dye to and using a core-shell design allows the microscopist to take advantage of metal enhanced uorescence (MEF) effects, which include increased brightness and photoblinking. 17,23,25,39,66,67 Mesoporous silica nanoparticles, which have numerous pores into which various types of molecules can be introduced, 67 have been used in STORM microscopy aer being taken into cells, shown in Fig. 11. 66 Silica nanoparticles with a metal core show increased brightness due to metal enhanced uorescence (MEF), which may also play a role in causing photoblinking behavior in dyes that do not otherwise exhibit this behavior, as observed by our group 23,25 and Lu et al. 17 when self-blinking nanoparticles were fabricated. The advantages of these selfblinking nanoparticles are that only a single light source is needed, rather than the two lasers normally needed for single particle localization-based super resolution methods, and that the imaging buffers normally required for these methods are not needed to cause the blinking behavior. 17
Super resolution microscopy is still a relatively new technology that is continuously being modied and improved upon; one such category of improvements is to the uorescent probes used with super resolution microscopy. The established uorophores are photoswitchable proteins and small molecule uorophores, the drawbacks of which are relatively low quantum yield and poor photostability; small molecule uorophores specically require the use of buffers which are toxic to living cells and many must be used in combination with another uorophore in order to obtain the desired behavior. 5,16,30 Recently, nanomaterials have been explored for use as uorescent probes for super resolution microscopy; although they are larger than the established uorophores they show higher quantum yield and resistance to photobleaching and have no need for toxic buffers or use in combination with other uorophores. Additionally, the structure of the nanomaterial probes can be modied to produce different optical properties, allowing the user to customize the probe to their specic needs. Nanoparticle probes have been developed that exhibit self-blinking behavior which eliminates the need for the two-laser system normally required for super resolution microscopy, which are used specically with localization-based super resolution. 16,17,23,25,30 These nanomaterials have already been successfully used for super resolution and have potential to become widely used in super resolution due to showing the desired optical properties and the potential to be customizable. Nanoparticles have been used to label the exterior of cells and the smaller nanoparticles have been shown to be able to be taken into the cells and imaged the labeling of subcellular structures such as mitochondria and microtubules is possible and because their surface chemistry allows for easy modication the nanoparticles can be modied in order to bind to specic cell types or organelles. The most signicant drawbacks of these nanomaterials are the size and the toxic materials used for quantum dots, both of which are currently being addressed.
Nanoscale Adv.© 2021 The Author(s). Published by the Royal Society of Chemistry
© 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv.