2022-23 WEEK 29 BIOL25012 LASERS and its use in fluorescence.pdf

BIOL25012 Advanced Biochemistry WEEK 29 14/February/2023 Fluorescence instrumentation: Lasers and photomultipliers ALDO WEEKS 24 to 33: FLUORESCENCE Laser and photomultipliers LEARNING OUTCOMES After this lecture you should be able to : • Explain laser ’s 3 main features (mon...

BIOL25012 Advanced Biochemistry WEEK 29 14/February/2023 Fluorescence instrumentation: Lasers and photomultipliers ALDO WEEKS 24 to 33: FLUORESCENCE Laser and photomultipliers LEARNING OUTCOMES After this lecture you should be able to : • Explain laser ’s 3 main features (monochromatic, coherent and collimated) • Discuss the role of ‘ population inversion ’ in the generation of laser light. • Explain the importance of filters in fluorescence measurements. • Explain the role of photomultipliers and its mechanism. The history of LASER light and its effect in technology and society Duration: 5 minutes Lasers and the Cold War The technological revolution brought about by lasers in the 60’s and 70’s is frequently used to illustrate how the fate of a scientific idea may be affected by the prevailing social/political ideology in society. Excessive state control in the former Soviet Union is blamed for hindering the transfer from knowledge to innovative market applications….despite the fact that their basic science was truly outstanding. Instrumentation and set -up: Main differences between absorbance and fluorescence 1. Excitation sources: lamps and LASERS 2. Detection: Cuvette and cell configuration 3. Detection: Filters and Emission monochromators 4. Detection: Photomultipliers Signal detection (i.e. emitted photons at lower energy) is considerable more difficult in fluorescence than in absorbance There are not absolute fluorescence units: the recorded intensity of a fluorescence emission depends on the specific settings of a given instrumentation. 1. Excitation sources: lamps and LASERS Conventional fluorescence spectrophotometers uses a mercury lamp Advantage: multiple high -intensity peaks a different wavelengths high -intensity ➔ high excitation ➔ high emission ➔ EASY DETECTION In the last 20 years, cell biology research and medical imaging have adopted the use of LASER sources for better excitation – emission high -intensity ➔ high excitation ➔ high emission ➔ EASY DETECTION The aim is the same https://www.youtube.com/watch?v=oUEbMjtWc -A How a laser works Duration: 5 minutes 1. It is monochromatic. Only light of a single wavelength is produced in the whole process. This differs from ordinary light such as sunshine or lamplight, which are composed of different wavelengths of light, being close to white light. http://www.phy.cuhk.edu.hk/phyworld/articles/laser/laser_e.html LASER’s three major properties: 2. It is coherent. All photons have the same phase, hence they produce a very high intensity beam when they superimpose. The lights we see in daily life have random phases and polarization, so they are relatively much weaker. http://www.phy.cuhk.edu.hk/phyworld/articles/laser/laser_e.html LASER three major characteristics : 3. It has a very narrow and collimated ray , and hence it is very powerful. In contrast, lamplight diverges towards different directions and has a low intensity. http://www.phy.cuhk.edu.hk/phyworld/articles/laser/laser_e.html LASER three major characteristics : An electron absorbs energy and moves to an excited state. It stays in the excited state only momentarily: after 10 -7 seconds, it falls to an intermediate metastable state. The electron stays at the metastable state for a (relatively) long time: 10 -3 seconds or more. This lengthy time in the metastable state causes the number of electrons at the excited state to become larger than those at the ground state. This is called population inversion . Population inversion is key to producing laser, because it ensures that the number of electrons returning from the metastable state to the ground state is higher than those that are transiting from the ground state to the metastable state . Under this conditions, the number of photons in the medium will increase, and hence, a LASER output is produced. http://www.phy.cuhk.edu.hk/phyworld/articles/laser/laser_e.html A key concept: population inversion A key concept: population inversion Population inversion is key to producing laser, because it ensures that the number of electrons returning from the metastable state to the ground state is higher than those that are transiting from the ground state to the metastable state. Energy ( E = hv ) (Maxwell –Boltzmann distribution, Equipartition theorem BIOL14405 Molecular Principles for Biochemistry) A key concept: population inversion (Maxwell –Boltzmann distribution, Equipartition theorem BIOL14405 Molecular Principles for Biochemistry) Based on its power, laser can be divided into three types, 1. low power laser which uses gas as its laser medium. For example, the barcode scanner often used in supermarkets utilizes helium gas and neon gas as its laser medium. 2. medium power laser , such as the laser pointers used in classrooms. 3. high power laser which uses semiconductors as laser medium. Its power output can reach 500 mW. LASER power is given in Watts units A watt is a measure of the amount of energy converted or transferred Flow cytometry: Counting and sorting cells LASER APPLICATIONS Dr Sergio Colombo. IT sessions (WEEKS 32 -33) Virtual lab LABSTER: Flow -cytometry FACS, Fluorescence -automated cell sorting Easy, direct access to LABSTER from NOW module page: Just click the link over there. Virtual lab LABSTER: Flow -cytometry FACS, Fluorescence -automated cell sorting Cuvette and cell configuration Signal detection (i.e. emitted photons at lower energy) is considerable more difficult in fluorescence than in absorption spectroscopy. Cuvettes for fluorescence measurements should be made of high crystal (quartz) and be clear in all sides In conventional fluorescence spectrophotometer, emission detection is done at 90 ° degrees angle from the exciting bean. This is done to reduce interference (optical noise) from the exciting light beam Leading provider for research -grade cuvettes for fluorescence measurements Detection: Optical Filters and Emission monochromators Optical Filters Optical filters selectively transmit light in a particular range of wavelengths while blocking all others. Monochromating the emission signal: Optical filters 1. “long pass” filter: allows passing long wavelengths only. 2. “short pass” filter: allows passing short wavelengths only 3. “band pass” filter : blocks both longer and shorter wavelengths Monochromating the emission signal: Optical filters Why you need emission filters http://www.microscopyu.com/tutorials/flash/spectralprofiles/index.html Usually the absorption spectrum of a fluorophore overlaps with its emission spectrum, this creates two practical problems - Quenching: emission is re -absorbed, decreasing intensity - Emission spectrum has a broad wavelength, increasing noise (“stray light”). http://micro.magnet.fsu.edu/primer/java/filters/absorption/ Very good learning tool: Optical filters. Interactive tutorial light is produced by the lamps Visible light is decomposed by a grating prism A single -frequency wave (monochrome) is selected and divided After passing through the sample, light intensity is compared and absorbance calculated Monochromating emitting light: Automatic monochromators (last week) LAMP Grating (from Lecture WEEK 26) Monochromating emitting light: Automatic monochromators High -throughput microplate reader for fluorescence: State -of -the -art monochromators. Monochromating emitting light: Automatic monochromators Duration: 5 minutes (no narration) SAMPLE Monochromating emitting light: Automatic monochromators Monochromating emitting light: Automatic monochromators Detection: Photomultipliers There are not absolute fluorescence units: the recorded intensity of a fluorescence emission depends on the specific settings of a given instrumentation. Photomultiplier: amplifying emission signal VOLTAGE (0 -1000 V) Very good web page: Spectroscopy’s fundamental concepts There are not absolute fluorescence units: the recorded intensity of a fluorescence emission depends on the specific settings of a given instrumentation, especially the voltage used by a photomultiplier !!!!!!!!! Photomultiplier Photomultiplier: amplifying emission signal Actual setting for a fast -reaction kinetics optical unit “Detection, detection, detection!!” If lasers are beams of very high energy, So, why are they safe for using in a nightclub?? Laser and photomultipliers CONCLUSIONS • A laser is a light beam which possess three fundamental properties: monochromatic, coherent and collimated. • These properties make laser light an excellent excitation source for fluorescence measurements. • Electrons’ population inversion accounts for laser’s high intensity. • The use of photomultipliers is fundamental to enhance any emission signal from most fluorophores.