Microscopy Introduction PDF

 1. Introduction to Microscopy (Completed) 1. Introduction to Microscopy...

 1. Introduction to Microscopy (Completed) 1. Introduction to Microscopy Why Microscopy? (Completed) Why Microscopy? You might be most familiar with microscopy in a biological setting, in which it is commonly used to capture images of the structure of cells. However, the @eld of microscopy is not limited to this, and can allow us to determine the structure and even composition of many diAerent objects and materials. Of speci@c interest to us in chemistry is that microscopy allows us to actually see crystal and molecular structures, which is not possible with the naked eye (which has an average resolution limit of 0.1 mm). DiAerent parts of a sample will interact with diAerent stimuli in unique ways, which we can exploit to capture our images. Some examples of this can be seen below. This topic will focus on optical microscopy, which covers techniques that use light to generate an image through conventional means (i.e. limited by Abbe's diAraction limit). Later topics will look at how we can use diAerent methods (e.g. using electrons) to image other types of systems. Grade 91 Steel Slide show. Select the next button to progress. Optical microscopy can be used to see how the martensitic structure of steel varies when prepared in diAerent ways.   There is incomplete content above. You must complete this before you can proceed through the course. Brief History of Microscopy (Completed) Brief History of Microscopy 400 BCE — First known reference to “burning-glasses”, an early form of lens using water 100 CE — Clear glass is invented in Alexandria (Roman Empire) 1300s — Appearance of eyeglasses in Europe 1610 — Galileo Galilei discovers that he can change the focus of his telescopes to magnify close objects 1620 — First complete compound microscope built by Cornelis Drebbel 1660 — Antonie van Leeuwenhoek uses self-built microscopes of magni@cation of up to 270x 1665 — Robert Hooke publishes @rst drawing of a cell (and coins the term) 1931 — Ernst Ruska and Max Knoll build the @rst prototype electron microscope 1981 — Gerd Binnig and Heinrich Rohrer invent the scanning tunneling microscope There is incomplete content above. You must complete this before you can proceed through the course. Basic Principle of (Most) Microscopy (Completed) Basic Principle of (Most) Microscopy Most microscopy techniques (and in particular optical ones), use a series of lenses to refract (de`ect) light to give the impression of a much larger object. This can be seen in the diagram below. Essentially, the objective lens creates an inverted image just in front of the objective lens. When observing the sample through the eye- piece, the objective lens creates a virtual image beyond the actual object (follow the dashed lines) which gives an eAective magni@cation of the subject. Image source: Keyence There is incomplete content above. You must complete this before you can proceed through the course. Quite a Few Techniques! Quite a Few Techniques! Throughout this part of the module, we will be looking at quite a number of diAerent techniques, which might make you feel a little overwhelmed. Don't worry! You will only need to know the basic principles for each technique, such as "How is an image captured?" and "What interactions between light/electrons/probe and sample are occurring?". Additionally, it will be useful for you to know what the main advantages of a technique are ("What problem is a technique trying to solve?"), but also its disadvantages ("Why might we not want to use it?"). Finally, you will be expected to do some basic calculations, so keep an eye out for equations in ; you will need to know what these mean and what their terms represent. There is incomplete content above. You must complete this before you can proceed through the course.   2. Bright-Field and Polarized Light Microscopy (Completed) 2. Bright-Field and Polarized Light Microscopy There are a number of techniques which use light in a conventional way; that is to say, the light that reaches the detector is the same as was used to illuminate the sample. They each have unique properties which provide a diDerent contrast to an image. Contrast (Completed) Contrast Contrast in an image is, quite simply, the diDerence in the intensity (or color) between a feature and its surroundings and the background. This contrast is very important, as it will provide us information about the diDerent ways in which the sample is interacting with the light, as we will see. Keep this in mind in future topics as well, as contrast will be achieved by this changing interaction with the probe (light in this case) and sample. Here is an example of two sets of micrographs with poor contrast (left) and good contrast (right). You should hopefully be able to see that in the images with good contrast, the edges of the features are much easier to distinguish from the background. Image source: Olympus There is incomplete content above. You must complete this before you can proceed through the course. Bright-Field Microscopy (Completed) Bright-Field Microscopy This is the simplest of all optical microscopy techniques which simply involves focusing white light onto a sample (biologists will often use the term specimen), and using a set of lenses to direct any light that passes through the sample to the eye-piece. Image contrast is determined by what light is absorbed or scattered (these regions will appear darker), and sometimes samples are stained with dyes to improve contrast. It is a very easy-to-use technique and quite versatile. You will likely already have used a bright-Qeld microscope (see image), as they are very commonly used in primary and secondary school science lessons. There is incomplete content above. You must complete this before you can proceed through the course. In the image adjacent you can see the path taken by light in a bright-Qeld microscope. 1. Light is emitted by a light source (often white light) 2. A condenser lens focuses the light onto a (thin) specimen/sample 3. Transmitted light is collected and focused by the objective lens 4. Finally, the ocular lens focuses the light into the viewer In a microscope as seen above, this would happen Yipped by 180° (the light source is at the bottom). Source: Lumen Learning There is incomplete content above. You must complete this before you can proceed through the course. Example: Lithocholic Acid (Completed) Example: Lithocholic Acid In the image below, the chemical structure of lithocholic acid can be seen in (a). Bright-Qeld optical microscopy images of tubular spherulites can be seen in (b-c) and LCA rods in (d-f), in aqueous solution at pH 12.0 (b,d,e) and 7.5 (c,f), respectively. You can hopefully see the change in the morphology (shape/structure) of the self-assembly of lithocholic acid molecules at diDerent pH levels, demonstrating that a simple instrument such as a bright-Qeld microscope can provide valuable chemical information. Source: Royal Society of Chemistry There is incomplete content above. You must complete this before you can proceed through the course. Polarized Light (Completed) Polarized Light The next technique uses polarized light to provide a unique contrast, so let's quickly review what this means. Light is an example of an electromagnetic wave, which means it is composed of an oscillating electric and magnetic Qelds. In most cases, light sources will emit unpolarized light, which means it is composed of a mixture of waves with electric Qelds pointing randomly in every direction (see below). By using a special polarizing Qlter, it is possible to allow light with only speciQc electric Qeld directions to pass through. If the light passing through the Qlter is conQned to oscillating in a single plane, then this light will be referred to as linearly polarized light. This is in contrast to circularly or elliptically polarized light, which we will not be covering in this topic. Adjacent is an example of unpolarized light being Qltered into light with a single plane of electric Qeld oscillation. Source: Britannica There is incomplete content above. You must complete this before you can proceed through the course. Polarized Light Microscopy (Completed) Polarized Light Microscopy If a sample is birefringent, then a polarized light microscope can be used to determine diDerences in the structure of a sample that is overwise optically similar. Birefringence is the optical property of a material that has diDerent refractive indices depending on the direction and polarization of light. In other words, a birefringent material will have light of diDerent polarizations travel through it at diDerent speeds, which is exploited by the polarized light microscope. Below we can see a birefringent crystal which "splits" the A quartz crystal demonstrating the principle of birefringence. beam of light (black arrow) into two by refracting at a Note the doubling of the underlying gridlines as the light is diDerent angle depending on the polarization of the incident refracted through the crystal in diDerent ways. light. The dashed path will have a longer path through the crystal, so its eDective speed through the crystal will be slower compared to the shorter solid path. Additionally, the two beams will be out of phase with each other relative to the thickness of the sample. There is incomplete content above. You must complete this before you can proceed through the course. The setup of a polarized light microscope is similar to that of a conventional optical microscope, but with 2 polarizers that will Qlter the desired polarizations of light. A diagram of the setup can be seen below. These polarizers can be rotated to determine how diDerent parts of the sample refract the light. The Qrst polarizer is referred to simply as the polarizer, and is placed between the light source and the sample, allowing only one polarization of light to illuminate it. The second polarizer, known as the analyzer and placed after the objective, allows for only one polarization of light to be captured. Source: Humboldt-Universität zu Berlin There is incomplete content above. You must complete this before you can proceed through the course. Michel-Lévy Chart (Completed) Michel-Lévy Chart Depending on the thickness of the sample, the color of the image will change if white light was used to illuminate the sample. This is due to the phase di:erence of the two light paths, and the relationship between the thickness of a uniform crystal and the color change is described by the Michel-Lévy Interference Color Chart, shown below. There is incomplete content above. You must complete this before you can proceed through the course. Example: Asbestos Testing (Completed) Example: Asbestos Testing Polarized light microscopy is commonly used in the analysis of minerals and other materials, such as in determining the presence of asbestos (which is birefringent) in bulk materials. Not only can the technique identify which Qbers are asbestos or not, but it can also discriminate between the major types of asbestos. Since using an optical is generally much cheaper and simpler than using more advanced instruments such as electron microscopes, the polarized light microscope is one of the main analytical tools in asbestos testing. There is incomplete content above. You must complete this before you can proceed through the course. Video: Polarized Light Microscope Video: Polarized Light Microscope Below is a short video that shows the basic working principle behind the polarized light microscope.    0:00 / 1:48  1x     4. Fluorescence and Confocal Microscopy (Completed) 4. Fluorescence and Confocal Microscopy The last four techniques have involved using white light to illuminate a sample, with the image formed by this same light reaching the detector (albeit with some parts scattered or absorbed). In the Bnal two techniques, we exploit the phenomenon of !uorescence to image only select parts of a sample we are interested in. Fluorescence (Completed) Fluorescence Fluorescence is the phenomenon in which a substance emits light after having absorbed light itself. The process in which this occurs is described below, but basically requires the absorption of a higher energy (shorter wavelength) photon which eventually leads to the emission of a lower energy (longer wavelength) photon. If a molecule is capable of Euorescence, it is known as a !uorophore. These will be useful to us as the absorption and emission wavelengths will be characteristic to a particular Euorophore used, and thus allows us to label a sample with various diGerent Euorescent tags. Take a look at the diagram, which shows a series of electronic (bold, S0-S3) and vibrational (non-bold, V0-V5) energy levels of an arbitrary molecule. When a molecule absorbs a photon of speci4c energy (↑, hν0), an electron is promoted to a higher excited energy level. The electron can then relax non-radiatively (⇝, without emitting a photon) to a lower energy excited state. Finally, the electron can relax electronically to a lower state (e.g. ground), emitting a lower energy photon (↓, hν1). There is incomplete content above. You must complete this before you can proceed through the course. Fluorescence Microscopy (Completed) Fluorescence Microscopy In order to use Euorescence to capture we need to use quite a diGerent setup than we have seen so far. While previously all the light we measured was transmitted through the sample, in Euorescence microscopy we do not want to measure anything but the light emitted by our Euorophores. The diagram below shows the usual setup for a Euorescent microscope, and the steps involved from light source to detector are as follows: 1. White light from the source passes through an excitation 4lter which is used to select a single wavelength of light to reach the sample. A laser of a speciBc wavelength may also be used. This allows us to specify which Euorophores we wish to excite. 2. The beam of light is then reEected by a dichroic mirror (this reEects certain wavelengths of light, while allowing others to just pass through) into an objective lens which focuses the light onto the sample. 3. Any Euorophores present which respond to this wavelength of light will then emit at a longer wavelength, and this light passes back through the objective lens and dichroic mirror (without being reEected). 4. An emission 4lter ensures that only the desired emission wavelength light is able to pass through and into our detector to generate an image. This second Blter allows us to be certain that the only light reaching the detector is due to Euorophores in the sample and is not stray light. Source: Tomas Majtner There is incomplete content above. You must complete this before you can proceed through the course. We now have a technique that allows for imaging of targeted molecules, which we can also bind to speciBc parts in a controlled way (more on this in a later topic). Not only that, we can genetically modify living cells to express proteins designed to Euoresce at a particular wavelength! Below is the structure of one commonly used Euorescent probe known as, perhaps unoriginally, green !uorescent protein (GFP). Next to this is a series of Euorescent micrographs of human cells taken before and during a mission of the crew on the International Space Station. Three diGerent Euorescent tags have been applied to diGerent parts of the cells (nucleus, vimentin, actin), each protein emitting at a diGerent wavelength, therefore allowing for these structures to be imaged separately. By combining all three images each labelled part of the cell can be clearly seen in relation to the others. Source: Zeiss Source: NASA There is incomplete content above. You must complete this before you can proceed through the course. We are not limited to biological samples either, as can be seen in this image of a dragon which has been created by using a scanning probe microscope. There is incomplete content above. You must complete this before you can proceed through the course. Confocal Microscopy (Completed) Confocal Microscopy By using a set of pinholes (one near the source and the other near the detector), Euorescence microscopy's resolution and contrast can be improved; this is referred to as confocal microscopy. It works by directly focussing the incoming beam onto a small area of the sample and blocking any light that isn't in the focal plane of the detector (hence the term confocal, which means to share a focal length). Let's take a look at how this works in a bit more detail (see Bgure below). 1. Light from a laser is focused through a pinhole, reEected by the dichroic mirror, and focused onto a small area of the sample. 2. Fluorescent molecules emit light in this small volume that has been illuminated by the laser; some will be emitted from the desired focal plane, while some will be emitted from above and below this focal plane. 3. The emitted light travels back up through the microscope and through the dichroic mirror. 4. Any light that is in the desired focal plane (solid red line) will pass through a second pinhole and pass into the detector. If the emitted light is not in the correct focal plane (i.e. above or below, dashed red line), it will be blocked by the pinhole. 5. The sample is scanned horizontally to capture the emission at every point (by repeating the steps above at every location) to create a 2D image. The result of the above process is that only in-focus light from a speciBc focal plane is captured, and a high-resolution and high-contrast image can be obtained. It is possible to also vary the vertical position, which allows for a 3D reconstruction of the sample by imaging its various focal planes. There is incomplete content above. You must complete this before you can proceed through the course. See below a comparison of the resolution and contrast achievable with confocal microscopy (d-f) compared to (wideBeld) Euorescence microscopy (a-c). Next to this is an example of a 3D reconstructed chick ciliary ganglion. Both examples courtesy of Olympus. Media player.  There is incomplete content above. You must complete this before you can proceed through the course. Video: Fluorescence and Confocal Microscopy Video: Fluorescence and Confocal Microscopy    0:00 / 2:41  1x   There is incomplete content above. You must complete this before you can proceed through the course.   5. Resolution Limit (Completed) 5. Resolution Limit Optical microscopy techniques o7er a large amount of :exibility in the type of sample they can image, and are relatively inexpensive as well. However, they have one major disadvantage: their resolution is di#raction limited. Di7raction (Completed) Di7raction When any wave goes through a conGned space (relative to its wavelength, e.g. an aperture for light), it will become di#racted; it will "bend" around the corners. This is why, for example, you can hear sounds coming from around a corner where you cannot see the source of the sound. The di7raction will become more pronounced the closer the aperture approaches the wavelength of the wave, as seen below and to the left. Di7raction is also not limited to light or sound either, and can be seen in other waves such as in water. There is incomplete content above. You must complete this before you can proceed through the course. If the aperture is circular, as is the case in a microscope, then the di7raction pattern forms what is known as an airy disc. Looking at an example of this below (left), we can see that a spot of light has a strong intensity signal in the center, which fades out to a series of concentric circles around it. On the right is a graph of intensity vs position along the central axis. Depending on the wavelength of light, the size of the central disc will change (smaller for shorter wavelengths). Even if we were able to make a perfect lens, the light going through the aperture would always be di7racted and e7ectively spread out and become "fuzzy", limiting the resolution we can achieve by simply using a set of lenses. The precise limit is described by Abbe's di#raction limit. There is incomplete content above. You must complete this before you can proceed through the course. Demo: Di7raction and Wave Interference (Completed) Demo: Di7raction and Wave Interference Below is another simulation in which you can play with various wave phenomena, such as wave interference and di7raction. While you can skip this, it might still be helpful to come back to it at some point to see how various properties a7ect the phenomena we have discussed so far. If the simulation does not load below, click here. Wave Interference Interference Slits Diffraction Waves There is incomplete content above. You must complete this before you can proceed through the course. Abbe Di7raction Limit (Completed) Abbe Di7raction Limit Ernst Abbe determined (for microscopes) the relationship between the minimum resolvable separation, , at any given wavelength of light, : is known as the numerical aperture, which is characteristic for the instrument used. It usually is between 1-1.4 for modern microscopes. Therefore, the resolution limit of optical microscopy will be approximately 250 nm. In the next topic we will look at how we can improve this by using some clever tricks, known as super-resolution microscopy. Ideal Situation Slide show. Select the next button to progress. In an ideal case, points of light would be deGned with perfectly sharp edges, as seen by two objects in green here.   There is incomplete content above. You must complete this before you can proceed through the course. Getting Around the Di7raction Limit? Getting Around the Di7raction Limit? Clearly, simply using a set of lenses and apertures will lead to di7raction-limited resolutions of our images. However, we can get around this in a number of ways, which will form the basis of the next three topics: Super-resolution microscopy Use some clever tricks while continuing to use light to probe the sample Electron microscopy E7ectively reduce the wavelength being used (so still di7raction-limited, but with a much greater resolution!) Scanning probe microscopy Capture an image by using a physical probe interacting with the surface at close proximity The Gnal topic will look at how we can use these same techniques to write on a surface There is incomplete content above. You must complete this before you can proceed through the course.   1. Introduction (Completed) 1. Introduction You might be wondering what exactly the term super-resolution is referring to, which is simply any optical microscopy technique which allows for imaging at a higher resolution than Abbe's di@raction limit. There are two main categories of super-resolution techniques: deterministic and stochastic. The majority of these techniques use Euorescence microscopy as their basis, using localized Euorophores to enable the capture of high-resolution images. In this topic we will be looking at so-called functional techniques; true sub-di@raction limit techniques require the use of evanescent waves (see Topic 4). Deterministic Techniques (Completed) Deterministic Techniques Deterministic techniques exploit the nonlinear response of Euorophores (i.e. emission is not linearly proportional to excitation) in order to enhance resolution. In other words, we can control and determine where and how di@erent parts of a sample will Euoresce, and hence have a greater control on the resolution that we can achieve. There are two main techniques we will be looking at in this topic, STED and GSD, which use the more general RESOLFT principles. Don't worry about the acronyms for now, we will be looking at them in more details in the next section. There is incomplete content above. You must complete this before you can proceed through the course. Stochastic Techniques (Completed) Stochastic Techniques Stochastic techniques use the complexity of the environment surrounding each Euorophore to cause changes in the temporal response of Euorophores, which can be used to reconstruct a higher resolution image. To put more simply, Euorophores will Euoresce at di@erent times allowing them to be di@erentiated more easily from one another. We will be looking at two (very similar) stochastic techniques known as PALM and STORM. There is incomplete content above. You must complete this before you can proceed through the course. Ways Around Di@raction Limit? (Completed) Ways Around Di@raction Limit? So what can we do to increase our resolution, without changing the wavelength of light we are using? Essentially, we need a way to di@erentiate between two separate Euorophores that are close enough that they are not resolved under normal Euorescence microscopy. As a reminder, Abbe's di@raction limit is given by. Let's take a look at some of the di@erent ways we can increase our resolution in the diagram below. Going from left to right: Do nothing Here we are di@raction-limited as before, and there is a limit to how close two Euorophores can get before we can no longer tell them apart. Change the shape of the airy disc (RESOLFT) By making the "fuzzy spot" emitted by Euorophores smaller, we can distinguish between them if they are closer than normal. Use a variety of Duorophores (not feasible) While it would be possible to di@erentiate between di@erent emission wavelengths close to each other quite easily, the sample preparation would make this incredibly di\cult to achieve. Measure emission at diGerent times (PALM/STORM) If two Euorophores are very close to each other, but Euoresce at di@erent points in time, we can measure their central position and reconstruct an image over time. There is incomplete content above. You must complete this before you can proceed through the course.   2. Deterministic Techniques (STED/GSD) (Completed) 2. Deterministic Techniques (STED/GSD) RESOLFT (Completed) RESOLFT The main principle of the deterministic techniques we will be looking at can be generalized under the principle of REversible Saturable (or Switchable) OpticaL Fluorescence Transitions (RESOLFT). This set of techniques relies on having Huorophores which have two distinct states: Bright state The Huorophore produces a Huorescent signal upon excitation Dark state No Huorescence is exhibited by the Huorophore after illumination by the excitation beam Importantly, it is necessary that the Huorophores are able to be controllably switched between these two states. If we now inhomogeneously (non-uniformly) illuminate parts of the sample with a depletion laser (which forces the exposed Huorophores into the dark state), we can leave behind a tiny area unilluminated (which remains in the bright state). By doing this we have reduced the e,ective excitation spot area, leading to a new resolution limit: λ d= 2NA√ 1 + I Isat where Isat is the intensity of light required to saturate the transition (half of states are in dark/bright state), and I is the intensity of the depletion laser. Essentially, as we increase the depletion beam intensity, we are reducing the minimum resolvable separation, d. Don't worry if the equation and Rgures don't make too much sense just yet; come back to it later once you've gone through the rest of the section. Here we can see an example of a photoswitchable Below we can see a number of diWerent possible excitation Huorophore. "proRles" (eWective spot sizes). On the left is the molecule in the bright ("on") state, while on If we use an intensity of I = 10 × Isat then we would have the right is its dark ("oW") state isomer. Switching between an eWective excitation proRle as shown by the blue curve the two is a reversible process. (labelled as 1). As we increase our depletion beam intensity, we continue narrowing this proRle until we reach the pink proRle (5) by using an intensity of. Image source: Zeiss Image source: Zeiss There is incomplete content above. You must complete this before you can proceed through the course. Stimulated Emission (Completed) Stimulated Emission Before we can look at our Rrst RESOLFT technique, we need to consider the process of stimulated emission, which is one of the three possible transitions between two electronic energy levels (as seen below). Absorption Slide show. Select the next button to progress. You are likely already familiar with absorption. Here, an incoming photon of the correct energy can be absorbed to cause an electron to be promoted from one electronic energy level to another.   There is incomplete content above. You must complete this before you can proceed through the course. Stimulated emission eWectively leads to the ampliRcation of the incident light if there are enough electrons in an excited state, and the outgoing light will be very coherent (all waves in phase). In fact, it is very likely you will have encountered one of the most common devices to exploit stimulated emission: a laser. There is one important fact to remember: Stimulated emission becomes much more likely than spontaneous emission if many photons are present. There is incomplete content above. You must complete this before you can proceed through the course. STED (Completed) STED We will be using the concept of stimulated emission to selectively cause a part of our sample to Huoresce, but at the wrong wavelength (i.e. not the one we are detecting). The rest of the excited part of the sample will Huoresce at the correct wavelength and be detected. As we saw earlier, the eWective size of this emission proRle will be much smaller, and thus we will have achieved a higher resolution. This is known as STimulated Emission Depletion (STED) microscopy. Let's take a closer look as to how we accomplish this. Adjacent is a diagram showing the general construction of a STED microscope. Unlike a normal Huorescence/confocal microscope, we have two diWerent laser pulses (i.e. non-continuous beams of light) coming into the instrument at slightly diWerent times: 1. Excitation Laser - This (higher energy) pulse is focused onto a point on the sample, causing the Huorophores to become excited. 2. Depletion Laser - This (lower energy) beam follows the excitation pulse (but before any Huorescence occurs) and is focused as a ring around the central spot of the excitation laser. Following this set of laser pulses some Huorescence will occur, which is then detected after passing through the usual set of lenses and dichroic mirrors (to Rlter out unwanted wavelengths of light). Image source: Zeiss There is incomplete content above. You must complete this before you can proceed through the course. But how does using a set of two laser pulses allow for higher resolution imaging to be possible? We can explain this by taking a look at the diagram below. On the left-hand side, we can see a couple of point spread functions (PSFs) for both the excitation (green spot) and the depletion (STED, red ring) laser pulses. The PSF of a system is essentially a description of how a point source is modiRed by an optical system; for example the PSF of an aperture will lead to the formation of an Airy disc. The larger the PSF, the larger the airy disc, so by reducing the size of the PSF we are essentially decreasing our eWective resolution limit. So, our unmodiRed excitation beam will have a wide PSF, which will lead to Huorescence in a wide area of the sample. However, we are exposing the same area with a depletion (STED) PSF in the shape of a ring (with a zero-intensity central spot) before the Huorophores have had a chance to Huoresce. Therefore, the only region to only have been exposed by the excitation beam will be at this very center point (see under "Saturated Depletion"), and the e,ective PSF of the excitation pulse is now much narrower. This then leads to us having a much greater control over the diWraction limit, allowing for higher resolution micrographs as seen on the right. Image source: Zeiss There is incomplete content above. You must complete this before you can proceed through the course. Depletion Ring (Completed) Depletion Ring You might be wondering just how we can create a ring of light surrounding the central spot. This can be relatively easily achieved by using a set of phase plates and using the superposition principle to cause light to destructively interfere where we choose. Excitation Laser Slide show. Select the next button to progress. Here we can see the excitation spot without any modiRcations, which has a 240 nm spot size.   There is incomplete content above. You must complete this before you can proceed through the course. Choice of STED Wavelength (Completed) Choice of STED Wavelength The Rnal bit we need to consider is what wavelength of light to use for our depletion (STED) laser. What we are looking for is for the regions exposed to this depletion laser to not Huoresce, in order for the smaller eWective excitation spot size to be the only region that Huoresces normally. We can exploit the principle of stimulated emission to force any excited molecule in the depletion region to emit a photon at a longer wavelength (which we will Rlter out!), instead of Huorescing normally. Let's take a look at the diagram below. In blue (peak to left) we have the excitation spectrum (i.e. absorbance spectrum) of our molecule, from which we choose the peak absorption wavelength for our excitation laser. The green spectrum (peak to the right) shows the Huorescence emission spectrum, which is the spectrum obtained from the molecule relaxing back down to its ground state, from which we would Rlter out any light not close to the emission maximum () from going into the detector. Our choice of STED wavelength must therefore be present in the emission spectrum, but at a greater wavelength than the Huorescence wavelengths we are not Rltering out. This choice is shown in orange (at around ), and constitutes a smaller energy relaxation from the excited to the ground state. By saturating the depletion region with light at this wavelength, we force stimulated emission at the longer wavelength, ensuring there are eWectively no Huorophores which will Huoresce at a shorter wavelength. There is incomplete content above. You must complete this before you can proceed through the course. Comparison with Confocal (Completed) Comparison with Confocal If we put all of this together, we have eWectively reduced the size of our airy disc, and hence can achieve a greater resolution image. This is constructed in the same way as a confocal micrograph, in which we scan across the sample, taking measurements at each pixel. Below is an example of how much of an improvement STED can be over confocal microscopy. Notice that we still retain the ability to tag diWerent parts of a sample with varied Huorophores. Source: Merck There is incomplete content above. You must complete this before you can proceed through the course. Video: STED Recap (Completed) Video: STED Recap    0:00 / 11:48  1x   There is incomplete content above. You must complete this before you can proceed through the course. GSD (Completed) GSD Another technique to use the RESOLFT principle is Ground State Depletion (GSD) microscopy. The method of operation is much the same as in STED, but instead of forcing a dark state through stimulated emission, the Huorescent marker is excited to a long- lived triplet dark state. The same type of "donut" depletion beam proRle is used, creating a very small eWective excitation area. A much less powerful laser is