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Seminar Series About Optics and Microscopy

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Seminar Series About Optics and Microscopy Fundamentals in optics and microscopy NIF Optical Seminar Jan 16th 2019 - Aurelien Breakdown Detections Illuminations Magic NIF Optical Seminar Jan 16th 2019 - Aurelien Illumination: What is light ? Wave Particle NIF Optical Seminar Jan 16th 2019 - Aurelien Light as a wave: properties Intensity I α A2 Wavelength (λ) Frequency ν=λ/c Amplitude (A) z direction of propagation Phase (φ) π/2 z0 t1203 π 0 z 3π/2 NIF Optical Seminar Jan 16th 2019 - Aurelien Light as a wave: properties LINEAR POLARIZATION CIRCULAR POLARIZATION ELLIPTICAL POLARIZATION Polarization becomes crucial for techniques such as DIC or if you use lasers as an illumination source. NIF Optical Seminar Jan 16th 2019 - Aurelien Light as a particle: why ? "It seems as though we must use sometimes the one theory and sometimes the other, while at times we may use either. (…) We have two contradictory pictures of reality; separately neither of them fully explains the phenomena of light, but together they do" PHOTOELECTRIC EFFECT Frequency threshold : below this threshold, no electrons are emitted, even if intensity is increased e e e BUT METAL Light propagates as discrete packets of energy called PHOTONS: Wave theory of light: increasing either the frequency or the intensity of light would increase electron Energy = hν h: Plank’s constant emission rate NIF Optical Seminar Jan 16th 2019 - Aurelien Light interactions Law of reflection: θ1 θ1' θ = θ ’ Reflection 1 1 n1 n2 θ2 n1 < n2 Law of refraction (Snell’s law): n1 sinθ1 = n2 sinθ2 Multicolor Refraction (n is dependent on n1 = c/v1, n2=c/v2 λ) is called dispersion Refraction NIF Optical Seminar Jan 16th 2019 - Aurelien Diffraction π/2 π 0 3π/2 constructive interference 0-2π destructive interference π intermediate interference π/2 Superposition of light waves generates interference patterns. NIF Optical Seminar Jan 16th 2019 - Aurelien Absorption (visible and fluorescence) NIF Optical Seminar Jan 16th 2019 - Aurelien NIF Optical Seminar Jan 16th 2019 - Aurelien UPRIGHT MICROSCOPE camera epifluorescence eyepiece/occulars pathway Tube lens fluorescence lamphouse objective condenser turret trans-illumination (condenser lens, lamphouse aperture diaphragm) field diaphragm NIF Optical Seminar Jan 16th 2019 - Aurelien Illuminations • Uniform illumination of the sample (widefield): • Trans illumination: white light (LED or incandescent) or can add filters if needed (i.e. optogenetics experiments). • Fluorescence lightpath source can be an arc lamp (older systems) or LED-based to tune specific wavelengths. • Other systems use lasers as illumination. Generally those are used in point scanning illumination (confocal microscopes in the core) or more complex schemes (beam shaping methods). NIF Optical Seminar Jan 16th 2019 - Aurelien Detectors • Chip sensors such as CCD, cMOS. Takes a snapshot of a plane, holds spatial location, usually monochromatic (except for histology), has a certain pixel size (spatial resolution constraint) and refresh rate (kHz or less), needs brighter signal . Used for widefield imaging. • Photodiodes and PMTs: no intrinsic spatial location, very sensitive (can detect single photons) and fast (MHz/GHz). Used in confocal and multiphoton microscopes. NIF Optical Seminar Jan 16th 2019 - Aurelien Lenses • Many different types of lenses: convex, concave, plano-, bi- …. • In microscopes, lenses are used to collect and optimize light transmission through distances by relaying important spatial planes (meaning the plane where your sample lies) • For simplicity, we will only consider convex lenses. 1 1 1 = + 푓 표 푖 NIF Optical Seminar Jan 16th 2019 - Aurelien Ray Diagram a-levelphysicstutor.com NIF Optical Seminar Jan 16th 2019 - Aurelien “4f” system and magnification M= f2/f1 NIF Optical Seminar Jan 16th 2019 - Aurelien Trans illumination/ Brightfield CRITICAl ILLUMINATION KÖHLER ILLUMINATION sample plane f CO condenser lens resolution f contrast aperture CO aperture diaphragm diaphragm depth of field fF field lens determine the filling of the condenser aperture fF PROBLEM OF CRITICAL field contrast (preventing ILLUMINATION: diaphragm glare) an image of the light source is fCL formed at the sample plane collector lens collimate light fCL NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: the critical and expensive part of the microscope 4 1 2 3 NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: Numerical aperture D pupil plane (back focal plane) objective α f n focal plane Angular aperture: sin α Numerical aperture: NA = n sin α Pupil diameter: D = 2 f NA = 2 n f sin α NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: point spread function (PSF) NIF Optical Seminar Jan 16th 2019 - Aurelien Diffraction π/2 π 0 3π/2 constructive interference 0-2π destructive interference π Superposition of light waves generates interference patterns. NIF Optical Seminar Jan 16th 2019 - Aurelien PSF: why do you care ? NIF Optical Seminar Jan 16th 2019 - Aurelien Two point sources are resolved if the distance between the two maxima of the PSFs is equal or bigger than the distance between the maximum of a PSF with the first minimum NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: why you don’t put oil on certain objectives…. Numerical aperture Snell’s law NA = n sin α n1 sinθ1 = n2 sinθ2 n2 ~ 1 n2 ~ 1.5 n1 ~ 1.5 n1 ~ 1.5 NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: coverslips choice. Spoiler alert should be #1.5 (170 μm) correction ring The index of refraction of glass and oil is not equal. The thicker the glass the more aberrations. Most objectives are designed for 170 μm glass and you can play with the correction collar to improve the contrast of your image. NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: working distance for in vivo and ephys people. α working distance f n (w.d.) focal length (f) f = nominal tubelens focal length / nominal magnification Ex: Olympus LUMPlanFln, W f = 180 / 40 = 4.5 mm w.d. = 3.3 mm NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: aberrations Planapochromat: max degree of aberration correction – spherical and chromatic- correction (4 colors) - Planapo The tubelens might Planfluorite: less corrected (4 colors) - PlanFl contribute to aberration compensation Planoachromat: less corrected (3 colors) - Plan NIF Optical Seminar Jan 16th 2019 - Aurelien Objectives: chromatic and spherical aberrations. NIF Optical Seminar Jan 16th 2019 - Aurelien Objective and Tube lens f fT M T fO Changing fT, M changes too! http://www.microscopyu.com/articles/optics/cfintro.html fO http://www.olympusmicro.com/primer/anatomy/tubelength.html NIF Optical Seminar Jan 16th 2019 - Aurelien Trans illumination through eye piece S sample plane S S fCL fCL fF fF fCO fCO fO ∞ fT fE ? eyepiece field field lens objective tube tube lens light source light collector lens collector condenser lens condenser field field diaphragm aperture aperture diaphragm final in thefinal image retina intermediate imaging plane imaging intermediate NIF Optical Seminar Jan 16th 2019 - Aurelien Trans illumination with a camera S sample plane S fCL fCL fF fF fCO fCO fO ∞ fT field field lens objective tube tube lens light source light collector lens collector condenser lens condenser field field diaphragm aperture aperture diaphragm intermediate imaging plane imaging intermediate NIF Optical Seminar Jan 16th 2019 - Aurelien Fluorescence NIF Optical Seminar Jan 16th 2019 - Aurelien Fluorescence scales Glutamate Ribosome PSD-95 actin NIF Optical Seminar Jan 16th 2019 - Aurelien Fluorescence: parameter space Efficiency of fluorophores depend on 2 main parameters: • Extinction coefficient: how much light can be absorbed at a wavelength • Quantum yield: how many photons are emitted vs how many are absorbed Those two factors will determine the fluorophore’s brightness. Other parameters such as pH, temperature can also influence the brightness observed. Excitation from ground state to an excited state can happen in a single step or multiple steps (single or multi photon excitation) NIF Optical Seminar Jan 16th 2019 - Aurelien Pros and cons of fluorescence • Excellent SNR (theoretically) • Large number of fluorophores available • Usually up to 4 lines/colors at the same time • Can use the properties for more specialized microscopy such as FRET, FLIM, FRAP…. • No correlation between fluorophore intensities • Bleaching and dark state. • Phototoxicity • Autofluorescence (especially in tissue) NIF Optical Seminar Jan 16th 2019 - Aurelien Bleaching NIF Optical Seminar Jan 16th 2019 - Aurelien Fluorescence selection in a microscope A filter set is composed of: • Excitation filter: to limit the wavelengths reaching your sample. • Emission filter: to only collect the emitted wavelengths you’re interested in • Dichroic mirror that reflects certain wavelengths and let others pass through NIF Optical Seminar Jan 16th 2019 - Aurelien Fpbase and filter choices Created and maintained by Talley Lambert To help you choose filters for your here at HMS fluorophores www.fpbase.org www.semrock.com NIF Optical Seminar Jan 16th 2019 - Aurelien Beyond the tube lens: detection NIF Optical Seminar Jan 16th 2019 - Aurelien Ressources: • microscopyU • Olympus microscopy resource center • Leica Science lab Next week: • comparison between scopes • discussion of specific illuminators and detectors • Scan heads NIF Optical Seminar Jan 16th 2019 - Aurelien.
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