Wednesday, October 8. Today: Last Time: Changes in T & P Units Equilibrium calculations: some examples. Readings: Chang & Thoman:

Transcription

1 Wednesday, October 8 Last Time: Entropy of mixing Chemical potential and equilibrium The equilibrium constant Today: Changes in T & P Units Equilibrium calculations: some examples Readings: Chang & Thoman: Handouts: Reminders: Homework 5 on the website Take-home Exam 1 due at the beginning of class Friday

2 2014 Nobel Prize in Chemistry "for the development of super-resolved fluorescence microscopy Eric Betzig Jannelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA Stefan W. Hell Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, German Cancer Research Center, Heidelberg, Germany William E. Moerner Stanford University, Stanford, CA, USA ABBE S DIFFRACTION LIMIT (0.2 µm) ant hair mammalian cell bacterium mitochondrion virus protein small molecule 1 mm 100µm 10µm 1µm 100 nm 10 nm 1nm

3 2014 Nobel Prize in Chemistry intensity laser irradiates the fluorophores that define the structure of the studied object. D microscopy citing laser am This displays a broad focal region as determined by Abbe s diffraction limit. Another, high-intensity STED, red-shifted in relation to the first, has a zero intensity "for the development of super-resolved minimum in the focal region and its intensity grows in all directions from the focus. It rapidly brings fluorophores that have been excited by the first, from the vibrational ground state fluorescence microscopy of the first excited singlet state, S, down to a high vibrational energy state of the electronic STED microscope 1 ground state from which they rapidly move to the vibrational ground state (Fig. 3). Stimulated Emission Depletion 2 final image gets a 3 The resolution that is much Figure 2 better than 0.2 micrometre. The principle of STED microscopy STED microscope Regular optical microscope Quenching laser exactly where 2 the hits the The final image gets a sample, they can use that 3informaresolution that is much than 0.2 tion to render the image at abetter much micrometre. higher resolution. Exciting laser 1 laser s scan over the 2 The sample. Since scientists know Exciting laser Quenching laser In a STED microscope, an annular laser quenches all fluorescence except that in In a regular optical microscope, volume. the contours ofaa nanometre-sized mitochondrion can be distinguished, but the resolution can never get better than 0.2 micrometres. laser s scan over the 2 The sample. Since scientists know exactly where the hits the sample, they can use that information to render the image at a much higher resolution. a STED microscope, an 1 Inannular laser quenches all fluorescence except that in a nanometre-sized volume. Figure 3: (Hell et al., Current Opinion in Neurobiology 2004, 14: ). (a) Excitation from the electronic ground state S0 to S1 with green light and return to S0 with the emission of yellow light or by STED with yellow light. (b) Depletion of S 1 with STED. (c) Left: yellow fluorescence density in z- and x-directions in focal plane without STED. Middle: description of STED microscope (top) and (bottom) green excitation without STED, red low-intensity STED and ght from a The STED microscope collects light from a green excitation spot and red high-intensity STED and green excitation spot. Right: yellow of small volumes to create a large eate a large multitude fluorescence spot with high intensity STED. whole. In contrast, the second principle rewarded, single-molecule microscopy, entails the inciple superposition of several images. Eric Betzig By this arrangement, in conjunction with optimal pulse sequences for the excitation and STED py, entails theand W. E. Moerner (who always has been Figure 3. One of the first images taken by Stefan Hell using a s, light emission is turned off everywhere except in a small part of the diffraction-limited called by his initials, W. E.) have independently STED microscope. To the left, an E. coli bacterium imaged using Eric Betzig of each other contributed different fundamental conventional microscopy; to the right, the same bacterium imaged focal region. The latter region shrinks indefinitely with increasing intensity of the maximal using STED. The resolution of the STED image is three times insights in its development. The foundation was better. Image from Proc. Natl. Acad. Sci. USA 97: value, I0, of the STED. The width, Δmin, of the effectively fluorescing region is in the lateral has been Figure 3. Onesucceeded of the first images laid when W. E. Moerner in detecting a taken by Stefan Hell using a plane approximated by (Hell et al., 2004) STED microscope. single small fluorescent molecule. To the left, an E. coli bacterium imaged using independently Images from nobelprize.org conventional microscopy; to the right, the same bacterium imaged (3) t fundamental min W. E. using Moerner first detect a single fluorescent STED. Theto resolution of the STED image molecule is three times5: (Klar et al., 2000, PNAS, 2n97, sin ). ( 1 I 0 / I sat(a) ) Nonlinear decrease in fluorescence Figure In most chemical methods, for instance measuring absorption and fluorescence, scientists study oundation wasmillionsbetter. Image from Proc. Natl. Acad. Sci. USA 97: intensity with increasing STED intensity, ISTED. Illustration of fluorescence intensity spot in xof molecules simultaneously. The results of such experiments represent a kind of typical, but for in the absence (b) and presence (d) of STED. Measured fluorescence d in detectingaverage a molecule. Scientists have had to accept this since nothing else has been andpossible, z-directions a long time they dreamt of measuring single molecules, because the richer and more detailed the distribution in the knowledge, the greater the possibility to understand, for instance, how diseases develop. z-direction in the absence (black) and presence (red) of STED.

4 practical problems, for instance a lack of molecules with a sufficient amount of distinguishable optical properties. from each other greater than Abbe s diffraction limit of 0.2 micrometres. Hence the position of each In 1995 Eric Betzig published his theoretical ideas in the journal Optics Letters, and subsequently left glowing protein could be registered very precisely in the microscope. After a while, when their fluoacademia and joined his father s company. rescence died out, the scientists activated a new subgroup of proteins. Again, the pulse was so weak that only a fraction of the proteins began to glow, whereupon another image was registered. This procedure was then repeated over and over again. Lured back to microscopy by green fluorescent proteins 2014 Nobel Prize in Chemistry For many years Eric Betzig was entirely disconnected from the research community. But one day a longing for science sprang to life again, and returning to the scientific literature he came across When Betzig superimposed the images he ended up with a super-resolution image of the lysosome the green fluorescent protein for the first time. Realizing there was a protein that could make other proteins visible inside cells revived Betzig s thoughts of how to circumvent Abbe s diffraction limit. membrane. Its resolution was far better than Abbe s diffraction limit. An article published in Science "for the development of super-resolved in 2006 subsequently presented the ground-breaking work. fluorescence microscopy The real breakthrough came in 2005, when he stumbled across fluorescent proteins that could be activated at will, similar to those that W. E. Moerner had detected in 1997 at the level of a single molecule. Betzig realized that such a protein was the tool required to implement the idea that had come to him ten years earlier. The fluorescent molecules did not have to be of different colours, they could just as well fluoresce at different times. Surpassing Abbe s limit bylocalization superimposing images Photoactivated Microscopy Just one year later, Eric Betzig demonstrated, in collaboration with scientists working on excitable fluorescent proteins, that his idea held up in practice. Among other things, the scientists coupled the glowing protein to the membrane enveloping the lysosome, the cell s recycling station. Using a light pulse the proteins were activated for fluorescence, but since the pulse was so weak only a fraction of them started to glow. Due to their small number, almost all of them were positioned at a distance Figure 4 blurred images are processed using 2 The probability theory in order to render them The principle of single-molecule microscopy much sharper. Figure 5. The centre image shows lysosome membranes and is one of the first ones taken by Betzig using single-molecule microscopy. To the left, the same image taken using conventional microscopy. To the right, the image of the membranes has been enlarged. Note the scale division of 0.2 micrometres, equivalent to Abbe s diffraction limit. The resolution is many times improved. Image from Science 313: The distance between each protein > 0.2 µm Microscope The laureates are still mapping the innermost secrets of life weak light pulse 1 Aactivates a fraction of all The methods developed by Eric Betzig, Stefan Hell and W. E. Moerner have led to several nanoscopy techniques and are currently used all over the world. The three Laureates are still active When all images are 3researchers superimposed a high in the large and growing community of scientists spearheading innovation in the field resolution totality wherein ofappears, nanoscopy. When they direct their powerful nanoscopes toward the tiniest components of life individual proteins can be discerned. they also produce cutting-edge knowledge. Stefan Hell has peered inside living nerve cells in order to better understand brain synapses. W. E. Moerner has studied proteins in relation to Huntington s disease. Eric Betzig has tracked cell division inside embryos. These are just a few of many examples. One High-resolution thing is certain, the Nobel Laureates in Chemistry 2014 have laid the foundation for the image development of knowledge of the greatest importance to mankind. the fluorescent proteins. The distance between them is greater than Abbe s diffraction limit of 0.2 micrometres. They glow until bleached, at which point the procedure is repeated on a new subgroup of proteins. Single fluorescent protein Figure 8: (a) Low frequency spatial sampling, (b) Biased sampling, (c) Uniform sampling, (d) THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES : HTTP //KVA.SE Oversampling. 5(7) In collaboration with Lippincott-Schwarz and H. F. Hess, Betzig (Betzig et al., 2006) expressed fusions of a photoactivatable GFP (PA-GF Kaede), similar to that described above (Patterson and Lippincott-Schwartz, 2002) and a lysosomal transmembrane protein (CD63) to obtain the super-resolved structure of a thin section of a fixed mammalian lysosome (Fig. 9).

5 2014 Nobel Prize in Physics for the invention of efficient blue light-emitting diodes which has enabled bright and energy-saving white light sources Isamu Akasaki Meijo University, Nagoya, Japan, Nagoya University, Nagoya, Japan Hiroshi Amano Nagoya University, Nagoya, Japan Shuji Nakamura University of California, Santa Barbara, CA, USA

6 2014 Nobel Prize in Physics for the invention of efficient blue light-emitting diodes which has enabled bright and energy-saving white light sources p-layer active layer n-layer hole electron The heart of the LED. A light-emitting diode consists of several layers of semiconducting materials. Electrical voltage drives electrons from the n-layer and holes from the p-layer to the active layer, where they recombine and light is emitted. The light s wavelength depends entirely on the semiconducting material used. The LED is no larger than a grain of sand. Blue LED lamp. The light-emitting diode in this lamp consists of several different layers of gallium nitride (GaN). By mixing in indium (In) and aluminium (Al), the Laureates succeeded in increasing the lamp s efficiency. anode (p-electrode) p-gan p-aigan Zinc-doped InGaN n-aigan n-gan GaN Buffer Layer Sapphire Substrate cathode (n-electrode) wire bond post anvil anode cathode Fig. 3. Structure of a blue LED with a double heterojunction InGaN/AlGaN. From [39] Double heterostructures and quantum wells The development of infrared LEDs and laser diodes had shown that heterojunctions and The principle for a light-emitting diode LED (upper left) and an example of a blue LED lamp. LEDs are also more long-lasting than other lamps. Incandescent bulbs tend to last 1,000 hours, as heat destroys the filament, while fluorescent lamps usually last around 10,000 hours. LEDs can last for 100,000 hours, thus greatly reducing materials consumption.

7 highly energy-efficient LED lamps contribute to saving the Earth s resources. A bright revolution The Laureates inventions revolutionized the field of illumination technology. New, more efficient, cheaper and smarter lamps are developed all the time. White LED lamps can be created in two different ways. One way is to use blue light to excite a phosphor so that it shines in red and green. When all colours come together, white light is produced. The other way is to construct the lamp out of three LEDs, red, green and blue, and let the eye do the work of combining the three colours into white Nobel Prize in Physics for the invention of efficient blue light-emitting diodes which has enabled bright and white LED lamps are thus energy-saving flexible light sources, already withlight severalsources applications in the field of illumination millions of different colours can be produced; the colours and intensity can be varied as needed. Colourful light panels, several hundred square metres in size, blink, change colours and patterns. And everything can be controlled by computers. The possibility to control the colour of light also implies that LED lamps can reproduce the alternations of natural light and follow our biological clock. Greenhouse-cultivation using artificial light is already a reality. 300 lm/w 16 lm/w The invention of the blue LED is just twenty years old, but it has already contributed to creating white light in an entirely new manner to the benefit of us all. 0,1 lm/w OIL LAMP (approx B.C.) The LED lamp also holds great promise when it comes to the possibility of increasing the quality of life for the more than 1.5 billion people who currently lack access to electricity grids, as the low power requirements imply that the lamp can be pow70 lm/w ered by cheap local solar power. Moreover, polluted water can be sterilised using ultraviolet LEDs, a subsequent elaboration of the blue LED. LIGHT BULB (19th century) FLUORESCENT LAMP (20th century) LED (21st century) LED lamps require less power to emit light than the older light sources. Efficiency is denoted in luminous flux (measured in lumen) per unit added power (measured in watt). As about one fourth of world electricity consumption is used for lighting purposes, the highly energy-efficient LED lamps contribute to saving the Earth s resources. A bright revolution Bright white signs for cyclists in Stockholm. The Laureates inventions revolutionized the field of illumination technology. New, more efficient, cheaper and smarter lamps are developed all the time. White LED lamps can be created in two different ways. One way is to use blue light to excite a phosphor so that THE it shines in red and green. When all colnobel PRIZE IN PHYSICS 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES ours come together, white light is produced. The other way is to construct the lamp out of three LEDs, red, green and blue, and let the eye do the work of combining the three colours into white. Fig. 4. Historical evolution of commercial LEDs. From [42]. PC-White stands for phosphor lamps are thus flexible light sources, already with several applications in the field of illumination ImagesLED from nobelprize.org converted white light, DH stands for double heterostructure. The wallplug efficiency is the ratio colours between emitted power and supplied electrical power. millions of different can belight produced; the colours and intensity can be varied as needed. Colourful light panels, several hundred square metres in size, blink, change colours and patterns. And everything can be controlled by computers. The possibility to control the colour of light also implies that LED lamps can reproduceapplications the alternations of natural light and follow our biological clock. Greenhouse-cultivation : HTTP //KVA.SE 4(5)