Basic Optics Initial Training for Optical Microscope Users Data Storage Store data on the data drive on the local machine: D\Data may be deleted at anytime. Do not leave your data here!    The core staff will clean the computer drives occasionally. This means that your profile, desktop and data from the data drive will all be removed. We will also remove other files that look like they are not needed. Core-Server: The core-server was set up by the IGB Computer Network and Research Group (CNRG) as a place for core users to store their data long term and move it back to your office. You do not need to bring thumb drives or other devices that could bring a computer virus into the core when you come to collect data. Move your data off of the local machine onto the core server at the end of each imaging session. Your PI will have a folder on the core-server and you can make a sub folder for your work. you will have access to all of the sub folders in your PI's folder but not other PI's folders. All of the data in your PI's folder was paid for by your PI and belongs to him/her. Your PI will be charged $8.75/terabyte every month  Email help@igb.illinois.edu   for information on charges and tape backup for long term storage.  Long Term Storage CNRG provides tape backup for long term storage for $200/ terabyte   Fluorescence Imaging Why Fluorescence: We can label what we want to see   Excitation and Emission Dapi           Widefield vs Confocal       https://www.journals.uchicago.edu/doi/full/10.1086/689588       IGB Core Instrument LSM 880     IGB Core Instrument LSM 900   405nm, 488nm, 561nm, 640nm excitation. Zen Blue file:///C:/Users/gfried/Downloads/EN_poster_Beampath-LSM-900_A1.pdf IGB Core Instrument V16 IGB Core Instruments Axiovert 200M Axiovert 200M Cameras cMOS           IGB Core Instruments LSM 700 LSM 700   http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/lsm700/indexflash.html IGB Core Instruments Zeiss LSM 710 Light Path https://www.gu.se/en/core-facilities/lsm-710-nlo Seven visible excitation lines: 405nm, 458nm, 488nm, 514nm, 561nm, 594nm, 633nm. Tisaphire laser 700nm to 980nm Spectral Unmixing http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html Multiphoton Microscopy http://zeiss-campus.magnet.fsu.edu/referencelibrary/multiphoton.html  Fluorescence Lifetime Imaging Microscopy (FLIM) http://www.iss.com/microscopy/components/FastFLIM.html   Objectives Optics Properties of light Wave particle duality: Light is a wave Absorption and Emission Beer's law Refraction Ray tracing   Resolution Resolution: The ability to separate two objects   A definition of Numerical Aperture   Fourier transform: Transform from real space to frequency space   Now we look at a real square wave and frequency space     Look at a simple optical system: Mathematical prediction of the Point Spread Function (PSF) on the left we have the mathematical point source know as a delta function. were the intensity at the Fourier plain can be found by taking the Fourier transform of this function. or    this has the same intensity at all points inside the aperture and zero outside. The second lens is now taking a Fourier transform on a box function the width of the aperture. or Substituting in the definition for NA Now we go back to Resolution. How close together we can position two points and still distinguish them           More intuitive approach     Notes from: http://web.mit.edu/2.710/Fall06/2.710-wk12-b-sl.pdf https://links.uwaterloo.ca/amath353docs/set11.pdf https://www.thefouriertransform.com/pairs/box.php http://www.phys.unm.edu/msbahae/Optics%20Lab/Fourier%20Optics.pdf How to Chose the Optimal Objective Dr. Sebastian Gliem Super Resolution Techniques   Sampling How does digital sampling affect resolution Look at imaging these object with a digital camera How close together do pixels need to be? Sampling over time: How often do you need to image a moving sample   Nyquist theory states that you should sample more than 2 X the frequency that you expect. Over sampling Nyquist sampling Under sampling causes aliasing Optical Transfer Function MTF         When objects get close together the contrast decreases. MTF = Image Modulation/Object Modulation MTF = 2(φ - cosφsinφ)/π   and φ = cos -1 (λν/2NA)   The Optical Transfer function is the Modulation transfer function times a phase component. OTF = MTF × eiφ(f)       Camera bit depth 0 or 1 00 or 01 or 10 or 11 000 or 001 or 010 or 011 or 100 or 101 or 110 or 111 and so on Jpg is 8 bit Tiff can be 16 bit   references https://imb.uq.edu.au/research/facilities/microscopy/training-manuals/microscopy-online-resources/image-capture/nyquist-conditions https://microscopy.berkeley.edu/courses/dib/sections/02images/sampling.html https://ocw.mit.edu/courses/mechanical-engineering/2-71-optics-spring-2009/video-lectures/lecture-22-coherent-and-incoherent-imaging/MIT2_71S09_lec22.pdf   Working in the IGB Core Expect to walk into a room with a fully functional instrument Let a core staff person know if you see a problem Clean up when you leave Acknowledge the IGB Core as: “Core Facilities at the Carl R. Woese Institute for Genomic Biology" Let us know when you publish Collaborations with the core facilities staff can be beneficial in the development of unique methods or capabilities.