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Super-Resolution Fluorescence Microscopy by Single-Molecule Switching

Super-Resolution Fluorescence Microscopy by Single-Molecule Switching
Biological research has been greatly impacted by the invention of fluorescent microscopy (Thorley, Pike and Rappoport, 2014). Although the impact on describing biological processes is revolutionary, diffraction of light is a limiting factor that blurs objects smaller than 250 nm in the x and y direction, and 500 nm in the z direction. Cellular structures, including ribosomes and action fibers are smaller than this size. Overcoming diffraction barrier has been challenge for many years (Patterson et al., 2010). The new field of microscopy called super-resolution microscopy have been discovered and provided insights into biological processes by enhancing spatial resolution (Huang, Babcock and Zhuang, 2010).
Super-resolution microscopy offers a great understanding of cell function and the dynamic interactions between individual molecules. This revolutionary technique has overcome resolution limit of light microscopy which is 250 nm in the x and y direction, and >450–700 nm in the z direction (Galbraith and Galbraith, 2011). Enhancing spatial resolution by over an order of magnitude, super-resolution imaging defines biological processes on a molecular scale (Patterson et al., 2010).
Newly developed approaches such as stochastic optical reconstruction microscopy (STORM), photoactivation localization microscopy (PALM), and fluorescent photoactivation localization microscopy (FPALM) break this diffraction of light wave (Thorley, Pike and Rappoport, 2014). Photoswitchable molecules are being used for overcoming the diffraction barrier in this approach.
Other conceptual strategy uses nonlinear optical approaches to smaller the size of focal spot, therefore structural details of the object can be resolved. Examples of such strategy are stimulated emission depletion (STED) fluorescence microscopy and structured illumination microscopy (SSIM) (Patterson et al., 2010).
Super-resolution fluorescence microscopy by single-molecule switching requires PALM, FPALM, and STORM techniques, where photoactivable fluorophores are being used to determine the position of molecules with high precision (Huang, Babcock and Zhuang, 2010). These far-field imaging techniques use fluorophore and its photoconversion process to detect fluorescence. Fluorophores in PALM are imaged and activated in multiple cycles whereas in STORM, the process of imaging and activation is simultaneous (Shashkova and Leake, 2017).
Fluorescent Probes
Fluorescent probes can switch between a fluorescent and a dark state (Huang, Bates and Zhuang, 2009). Light is emitted at a certain wavelength in fluorescent state, however dark state does not emit the light (Huang, Babcock and Zhuang, 2010).
The main requirement of the fluorescent probes is able to be either photoactivated, photoswitched, or photoconverted by a light of a specific range of wavelength. These functions are called optical highlighting (Patterson et al., 2010).
Individual imaging, localization, and deactivation of molecules is obtained when they are activated within a diffraction limited area at different times (Huang, Bates and Zhuang, 2009).
Parallel localization is achieved through wide-field imaging where fluorophore locations reconstruct super-resolution images. STORM, PALM, and FPALM techniques use photoswitchable fluorescent dyes or proteins that are being activated by light (Huang, Bates and Zhuang, 2009).
Photoswitchable probes are used for high-precision localization of single molecules.
Accumulation of sufficient fluorophores that are switched on is necessary for super-resolution image (Shim et al., 2012).
Resolution of the image is determined by fluorescent probe density. For high resolution images, high levels of protein labels need to be expressed. However, this can cause artifacts overexpression (Shim et al., 2012).
Different applications of super-resolution imaging, for instance three-dimensional (3D) imaging or live-cell imaging, use variety of probes ranging from photoactivable fluorescent proteins to photoswitchable cyanine dyes (Patterson et al., 2010). Many factors influence whether proteins or dyes are being used. Dyes are usually more flexible in terms of use in molecular species. Proteins, nucleic acids, and oligosaccharides are tagged with dyes (Huang, Babcock and Zhuang, 2010).

Fluorescent probes used in PALM/FPALM are photoactivable or photoconvertible (Patterson et al., 2010), whereas STORM uses switchable organic fluorophores which are placed in specific buffers; they are combination of Cy3 and Cy5 synthetic fluorophores. They can be stochastically and reversibly switched (Godin et al., 2014). Probes have specific physical and chemical properties which are significant for single-molecule super-resolution microscopy. Number of photons detected per photoactivation affects the resolution (Huang, Bates and Zhuang, 2009). In order to determine the precise localization of molecules, probes should emit a large number of photons (Huang, Babcock and Zhuang, 2010).

3D Imaging

Super-resolution microscopy based on single-molecule switching have expanded to 3D imaging (Laine et al., 2016).
Position of individual molecules can be determined in all three dimensions (3D). Demonstration of 3D data provides an extra information about the complex system. This approach improves the scientific understanding of biological structures (von Diezmann et al., 2017), and is one of the advantages of super-resolution microscopy (MacDonald et al., 2015).
Position of activated fluorescent probes is being determined in STORM/PALM/FPALM approach of 3D imaging. Methods of particle localization such as astigmatism method for axial localization is used (Huang, Bates and Zhuang, 2009), where insertion of cylindrical lens in the light path produces astigmatism imaging (Galbraith and Galbraith, 2011).
Image of the single molecule becomes elliptical, and the axial position is determined from ellipticity whereas the lateral position is determined from the centroid of the image (Huang, Babcock and Zhuang, 2010).
Other implementations take advantage of multiple focal planes, point spread function (PSF), and interferometric detection (von Diezmann et al., 2017). Interferometry uses two opposing objectives and provides highest axial resolution (Huang, Babcock and Zhuang, 2010).
Two-focal plane uses the emission of light. This light emission is divided and imaged to two regions of the camera, however with different path lengths. Defocused shape of the single-molecule images is captured and Z coordinates are determined (Huang, Bates and Zhuang, 2009), however increased processing time of the image and collection of data are disadvantages of this imaging (MacDonald et al., 2015).
Engineering a PSF creates geometrically detectable emission patterns. Double-helical shape of a PSF is one of the alternatives used for 3D imaging (Laine et al., 2016).
Each method has its specific advantages and disadvantages such as effects of optical aberrations or fluorophore labeling density which require consideration for further application and development (von Diezmann et al., 2017).
3D imaging resolution is around of 20 nm laterally and 50-60 nm axially (Galbraith and Galbraith, 2011), however for fixed samples results of 10 nm have been demonstrated (Shim et al., 2012). For instance, various proteins are being identified and imaged using 3D single-molecule localization microscopy and 3D STORM in fixed brain tissue (Shashkova and Leake, 2017).

Live-cell Imaging

Live-cell imaging approach involves reconstruction of image from single-molecule localizations that are recovered from thousands of frames. Well-separated fluorescent emitters are detected in each frame (Godin et al., 2014).
Live-cell fluorescent imaging has specific requirements such as adequate time resolution for recording of cell dynamics, and appropriate cell labeling. Fluorophore localizations is collected over many activation cycles, therefore imaging speed is relatively low (Huang, Bates and Zhuang, 2009), due to slow switching of fluorescent proteins to dark states. Brighter and faster organic dyes could improve speed of imaging (Huang, Babcock and Zhuang, 2010). For instance, cyanine dyes and caged dyes could improve optical properties and photostability for live-cell imaging of nucleic acids (Schwechheimer et al., 2018).
Focal adhesion proteins have been described in live cells using Eos fluorescent protein (EosFP). EosFP was used to label focal adhesion protein – paxillin, with effective resolution of 60-70 nm by the Nyquist criterion and time resolution of 25-60 s per frame (Huang, Bates and Zhuang, 2009). Nyquist-Shannon criterion of obtaining position for at least two emitters within each element of resolution should be considered. This sampling theorem will increase the number of well-localized molecules (Thompson et al., 2010).
Proteins in live bacteria such as C. crescentus can be studied using yellow fluorescent protein (EYFP). EYFP can be genetically encoded for protein in living cell. Desired Nyquist criterion for 40 nm was achieved using live-cell PALM technique (Biteen and Moerner, 2010)
PALM has quickly become the prime technique in super-resolution live-cell imaging due to specificity of genetically encoded, fluorescently tagged molecules in cells. Such technique allows a study of intracellular biomolecules (Godin et al., 2014).

Super-resolution microscopy has a great impact on understanding the inner life of the cell (Galbraith and Galbraith, 2011). It provides an insight into biological structure and can define these structures with nanometric localization precision (Patterson et al., 2010).
Applications of super-resolution microscopy are numerous, and different fields have benefited from this invention such as microbiology and neurobiology (Huang, Babcock and Zhuang, 2010).
There are some advantages of super-resolution approaches which use probe-based super-resolution imaging; one being individual identification of molecules at high densities. Moreover, the dynamics and distribution of subcellular structures can be analyzed (Patterson et al., 2010). However, live-cell imaging requires rapid image collection without the change in temperature. Slow imaging speed is one of the limitations in PALM/STORM techniques (MacDonald et al., 2015).

Biteen, J. and Moerner, W. (2010). Single-Molecule and Superresolution Imaging in Live Bacteria Cells. Cold Spring Harb Perspect Biol 2010;2:a000448
Galbraith, C. G.,

Glomeruli and Glomerular Filtration Rate (GFR)

The glomeruli are microscopic filters made of a network of capillaries that filter the blood plasma, which occurs in the renal corpuscle of the nephron in the kidneys (figure 1). Glomerular filtration rate (GFR) is defined by amount of blood filtered by the glomerulus into the Bowman’s capsule (ml/per unit of time), the resulting fluid is called the glomerular filtrate. GFR is influenced by the interaction of several important pressures (Khan Academy, 2019).

Figure 1 – Structure of the renal corpuscle filtering unit; glomeruli made up of a tuft of small blood capillaries contained with the Bowman’s capsule between the afferent and efferent arterioles. It is connected to the proximal tubule which leads to segments of the renal tubule such as Loop of Henle, the distal convoluted tubule and the collecting duct which leads to the urethra. (Khan Academy, 2019).
GFR is determined by the sum of the filtration rates of all the functioning nephrons in the kidneys. As a rough guide, a healthy GFR can be estimated by calculating 140 – [your age] i.e. a healthy 20-year old’s GFR would be 120ml/min. Body weight and height are also considered in the measurement. A GFR of less than 60ml/min can suggest chronic kidney disease (University of Washington, 2019).
GFR can be measured by quantifying the renal clearance of the endogenous substance of creatine, a by-product of muscle metabolism. It is freely filtered by the glomerulus so can be compared to the serum creatine concentration using the following equation:

This however often overestimates GFR due to secretion of creatine by tubules (15%). Instead, an endogenous substance such as inulin (a carbohydrate that is purely filtered by the kidneys as it is neither reabsorbed or secreted) can be used to provide a more accurate measure of GFR. (Traynor, J).

Figure 2 – Equation used to calculate GFR concentration; whereby the urine concentration refers to the concentration of inulin in the sample, the urine flow refers to the volume of urine produced in a given period and the plasma concentration is the concentration of inulin in the blood (Khan academy, 2019).
However, inulin is expensive, must be given by intravenous injection and measurements from blood and urine samples must be taken frequently over several hours to obtain an accurate measure of kidney function and filtration (Traynor, J).
The glomerulus is positioned between the afferent arteriole which delivers blood (1-1.1L per minute) to the glomerulus and the efferent arteriole which carries blood away. The efferent arteriole is significantly thinner than the afferent arteriole (figure 3) which maintains a high glomerular capillary pressure required for filtration (University of Washington, 2019).

Figure 3 – Filtration rate is determined by three main pressures, one of which promotes filtration (PGC) and two that oppose filtration (πGC and PBS). The overall pressure in the capillaries (PGC) also known as hydrostatic pressure is very high due to the increased diameter of the afferent arteriole. Osmotic pressure (πGC) counteracts this force and occurs due to the higher concentration plasma proteins (e.g. albumin) in the capillaries that cannot be filtered causing water to move back into the capillaries. Together are known as Starling’s forces. Hydrostatic pressure is greater than osmotic pressure, to allow glomerular filtration to occur. The overall net glomerular filtration pressure (NFP) is the sum of PGC minus the hydrostatic pressure in the Bowman’s space (PBS) and the osmotic pressure from the blood proteins (πGC). NFP in this example is 60 – (15 29) = 16mmHg. (University of Washington, 2019)
GFR is directly proportional to the NFP. The hydrostatic pressure must be precisely regulated to maintain efficient filtration. High PGC can be maintained through an intrinsic process in the kidney called renal autoregulation (figure 4), regardless of natural changes in blood pressure (between 80 and 160mmHg) (University of Washington, 2019).

Figure 4 – An autoregulation graph describes the relationship between arterial pressure and GFR. The plateau shows the autoregulation range, which is the mean arterial pressures at which autoregulation remains effective. This is achieved through two primary mechanisms; myogenic reflux and juxtaglomerular apparatus. Beyond the plateau, there are upper and lower limits of GFR which are of clinical importance (University of Washington, 2019).
Myogenic reflux is a sympathetic response. Baroreceptors in the afferent arterioles detect an increase in blood pressure. This stimulates opening of calcium channels, causing action potential and contraction of the smooth muscle, resulting in vasoconstriction. The volume of blood entering the glomerular capillaries decreases, reducing PGC and reducing GFR. The opposite occurs when a decrease in blood pressure is detected, to increase GFR (University of Washington, 2019).
The juxtaglomerular apparatus (figure 5) activates the angiotensin-aldosterone system in response to a detecting a decrease in blood pressure.

Figure 5 – Juxtaglomerular apparatus – juxtaglomerular cells line the afferent and efferent arterioles meet the macula densa cells in the distal convoluted tubule. The macula densa cells monitor the filtrate that it receives which was originally filtered in the Bowman’s capsule. Macula densa cells detect sodium composition of the filtrate (Histology, Yale. 2018).
In response to elevated sodium levels indicating low blood pressure, the macula densa cells stimulate juxtaglomerular cells to secrete the enzyme renin. This activates angiotensinogen (plasma protein in the blood) which is converted to angiotensin I. This is activated by ACE (angiotensin-converting enzyme) and converted to angiotensin II. This is a vasoconstrictor, causing constriction of the afferent arteriole, reducing blood flow and reducing GFR. Blood pressure is increased as it stimulates the thirst centre in the brain, increasing water retention. It also increases cardiac output by increasing stroke volume and heart rate (Histology, Yale. 2018).
ACE inhibitors are used to treat hypertension and lower blood pressure, by preventing the production of angiotensin II. This allows vasodilation of vessels, so they relax and widen, whilst also reducing water retention and blood volume (British Heart Foundation, 2019).
Conversely, the hormone atrial natriuretic peptide (ANP) can increase GFR. An increase in plasma volume activates secretion of the hormone from the heart which causes vasodilation of the afferent arteriole, an increase in sodium excretion (natriuresis) and increase in fluid excretion (diuresis) (University of Washington, 2019). They also decrease the release of renin, preventing the renin-angiotensin system (R, Klabunde. 2019).
Khan Academy. (2019). Renal physiology: Glomerular filtration. [online] Available at: [Accessed 4 Jan. 2019].
University of Washington. (2019). Regulation of GFR. [online] Available at: [Accessed 4 Jan. 2019].
Traynor, J. (2006). How to measure renal function in clinical practice. BMJ, 333(7577).
Histology, Yale (2019). Juxtaglomerular Apparatus. [online] Available at: [Accessed 16 Feb. 2019].
British Heart Foundation. (2019). Watch: What are ACE inhibitors and what do they do in your body?. [online] Available at: [Accessed 16 Feb. 2019].
Klabunde, R. (2019). CV Physiology | Atrial and Brain Natriuretic Peptides. [online] Available at: Pressure/BP017 [Accessed 16 Feb. 2019].