Adobe Systems Adobe Systems Adobe Systems S5015 Light microscopy methods in biology Lecture 1: History and principles of light microscopy Milan Ešner Cellular Imaging Core Facility, Ceitec MU milan.esner@ceitec.muni.cz Adobe Systems S5015 : Light microscopy methods in biology Light microscopy methods in biology 2 Lecture 1: September 30, 2021 at 9:00 History of microscopy, fundamentals of light microscopy. Contrasting techniques in transmitted light microscopy – brightfield, darkfield, phase contrast, DIC. Widefield fluorescence microscopy - deconvolution, point spread function, point spread function measurements, description. Detectors in widefield microscopy, filters, light sources. Lecture 2: October 14, 2021 at 9:00 Confocal microscopy - principles, types of confocal microscopes – scanning, spinning disc, channel vs spectral imaging, linear unmixing. Lasers, detectors, beam splitters, optical sections. Lightsheet microscopy. Lecture 3: October 25, 2021 at 9:00 Imaging below diffraction limits – SIM, SMLM, STED Lecture 4: November 4, 2021 at 9:00 Basics of Image analysis – image types, pixels, voxels, basic filters, image processing, metadata, deconvolution, 3D visualization, objects detection, quantification. Practical 1: December 14, 2021 at 9:00 Transmitted light microscopy, widefield fluorescence, confocal, Apotome, SIM Practical 2: December 15, 2021 at 9:00 Sample preparation, single molecule localization microscopy acquisition and analysis, deconvolution. Adobe Systems Antonie van Leeuwenhoek (1632 – 1723) Dutch businessman and scientist. Best known for his pioneering work in microscopy. Used single lens microscope of his own design. [USEMAP] See caption Light microscopy methods in biology 3 Adobe Systems Robert Hooke (1635 – 1703) English scientist, philosopher, architect and polymath. Improved design of compound microscope and use it for scientific exploration – book Micrographia. Use term in biological context. Současný portrét Roberta Hookea Light microscopy methods in biology 4 Adobe Systems Carl Zeiss (1816 – 1888) German scientific instrument maker, optician and businessman. Founder of Carl Zeiss workshop in 1864 together with Ernst Abbe and Otto Schott in Jena, Germany. Die Anfänge des wissenschaftlichen Mikroskopbaus Light microscopy methods in biology 5 Adobe Systems Ernst Abbe (1840 – 1905) https://upload.wikimedia.org/wikipedia/commons/thumb/3/37/Ernst_Abbe_memorial.JPG/1280px-Ernst_Abbe _memorial.JPG German physicist, optical scientist, entrepreneur and social reformer. With Zeiss and Schott he developed numerous optical instruments. Among his inventions are apochromatic lens, Abbe condenser, refractometer. He defined numerical aperture -> mathematically describing optical resolution of light microscopes. He was also strongly implicated in unions and workers rights. In Zeiss he introduced 8-hour workday. Light microscopy methods in biology 6 Adobe Systems George Biddell Airy (1801 – 1892) English mathematician and astronomer. He established Greenwich as the location of the prime meridian. His main contribution to microscopy was explaining of formation of concentric diffraction pattern around the bright spot of light, named after him – Airy disc. [USEMAP] Light microscopy methods in biology 7 Adobe Systems August Koehler (1866 – 1948) German optical scientist. He developed and improved light illumination of sample, Koehler illumination. Light microscopy methods in biology 8 Adobe Systems Siedentopf and Zsigmondy First concept of light sheet microscope – illumination objective orthogonally to detection objective. Zsigmondy received Nobel prize for chemistry in 1925 for his research on colloid solutions. Light microscopy methods in biology 9 Adobe Systems Frits Zernike (1888 – 1966) Dutch physicist. Invented phase contrast microscopy. Producing high contrast images of transparent samples, hardly visible in standard bright field transmitted light imaging. Widely used technique of contrast enhancing. Received Nobel prize in 1953. Light microscopy methods in biology 10 Georges Nomarski (1919-1997) Polish physicist. Developed differential interference contrast (DIC) – contrast enhancing technique based on polarized light. Molecular Expressions: Science, Optics and You - Timeline ... Adobe Systems Marvin Minsky (1927-2016) American scientist, doing mainly research of artificial intelligence. He is Co-founder of Massachusetts Institute of Technology (MIT). He developed first scanning confocal microscope in 1957. Light microscopy methods in biology 11 Adobe Systems Mojmír Petráň (*1923) Czechoslovak scientist. Studied medicine at Charles university, Prague. With Milan Hadravsky he developed spinning disc confocal microscope in 1964, using Nipkow disc from TV. Light microscopy methods in biology 12 Adobe Systems Jan Huisken, Eric Stelzer Developed fluorescence light sheet microscope (selective plane illumination microscope) in 2004. Almost 100 years after Siedentopf and Zsigmony microscope they built first fluorescence microscope, using different optical paths for illumination and detection. Today exists many variants of lightsheet microscopes and several commercial systems are available. Ernst H.K. Stelzer | Physikalischebiologie.de Jan Huisken | Morgridge Institute for Research Light microscopy methods in biology 13 Adobe Systems Mats Gustafsson (1960-2011) Light microscopy methods in biology 14 Developped method of structured illumination microscopy SIM. SIM surpass diffraction limit of optical microscopes and is able to achieve resolution below 100 nm. Adobe Systems Eric Betzig (*1960) Inventor of single molecule localization technique STORM/PALM together with William Moerner and lattice light sheet technology. Received Nobel prize in 2014 together with Eric Betzig and Stephen Hell Light microscopy methods in biology 15 Adobe Systems William Moerner Light microscopy methods in biology 16 Contributed to development of sinlge molecule localization microscopy. Received Nobel prize in 2014 together with Eric Betzig and Stephen Hell Adobe Systems Steffen Hell Light microscopy methods in biology 17 Invented stimulated emission depletion (STED) microscopy. Received Nobel prize in 2014 together with Eric Betzig and Stephen Hell Adobe Systems A close up of a logo Description automatically generated Principles of light microscopy Light microscopy methods in biology 18 Adobe Systems -In microscopy you don’t observe an OBJECT, you observe an IMAGE of the object -1) Light diffraction by the specimen -2) collection of diffracted/non-diffracted rays by objective -3) interference of diffracted and non-diffracted rays in the image plane. Three steps in image formation •Magnification •Resolution •Contrast A close up of a logo Description automatically generated Image formation Light microscopy methods in biology 19 Adobe Systems http://www.eyelighting.com/_CE/pagecontent/Images/Lamp%20Technology%20Education/quality%20of%20a%20 light%20source.jpg Monochromatic - single wavelength (color). Polychromatic Coherent – waves of the same wavelength that maintain the same phase relation. Noncoherent Polarized – waves with E-vectors in parallel. Nonpolarized Collimated – waves with the same coaxial paths of propagation (same direction). Divergent Visible electromagnetic radiation with electric and magnetic fields whose amplitudes oscillate as a sine function over dimensions of space and time. Dual character: waves – amplitude and phase particles – photons. shorter wavelength = higher energy A close up of a logo Description automatically generated Light Light microscopy methods in biology 20 Adobe Systems Light diffraction When light passes at the edge of object (opening, aperture, specimen), light is bended on edges and passes to the area of geometrical shadow. -Due to the diffraction, Image of a point is not a point A close up of a logo Description automatically generated Light microscopy methods in biology 21 Adobe Systems Constructive interference Light diffraction and interference A close up of a logo Description automatically generated Light microscopy methods in biology 22 Adobe Systems Destructive interference A close up of a logo Description automatically generated Light diffraction and interference Light microscopy methods in biology 23 Adobe Systems 0 1st 1st 2nd 2nd 3nd 3nd A close up of a logo Description automatically generated Light diffraction and interference Light microscopy methods in biology 24 Adobe Systems Air objective Oil immersion objective Immersion objectives have higher NA and therefore higher resolution. Adjust refractive index of immersion to your medium!!! Mowiol, prolong, vectashield –> oil immersion Glycerol -> glycerol immersion PBS, medium –> water immersion Mismatch = high spherical aberrations From P. Evennett Výsledek obrázku pro numerical aperture www.zeiss-campus.magnet.fsu.edu A close up of a logo Description automatically generated Numerical aperture Light microscopy methods in biology 25 Adobe Systems Refractive index Medium R. Index (n) Air 1 Water 1.333 Glycerol 1.46 Immersion Oil 1.515 Cover glass 1.515 - Determines how much light is bent (refracted), when entering the material n1 * sin(a1) = n2 * sin(a2) Snell’s law: n1 n2 a1 a2 RI is different for every wavelength – light dispersion on prism. A white beam of light dispersed into different colors when passing through a triangular prism. air water Light entering to different medium changes the speed A close up of a logo Description automatically generated Refraction 26 Image Adobe Systems Ernst Karl Abbe https://upload.wikimedia.org/wikipedia/commons/thumb/3/37/Ernst_Abbe_memorial.JPG/1280px-Ernst_Abbe _memorial.JPG Ernst Abbe.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/4/46/Ernst-Abbe-Denkmal_Jena_F%C3%BCrstengrabe n_-_20140802_125709.jpg/170px-Ernst-Abbe-Denkmal_Jena_F%C3%BCrstengraben_-_20140802_125709.jpg (1840 – 1905) Microscope Numerical aperture A close up of a logo Description automatically generated Resolution 27 Adobe Systems By Spencer Bliven - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=31456019 https://upload.wikimedia.org/wikipedia/commons/a/ae/Airy_disk_spacing_near_Rayleigh_criterion.png Two point sources are regarded as just resolved when the principal diffraction maximum of one image coincides with the first minimum of the other. Rayleigh criterion R: resolution/distance λ: wavelength of light NA: numerical aperture A close up of a logo Description automatically generated Resolution Light microscopy methods in biology 28 Adobe Systems Magnification does not play a role in resolution! Only NA and l are taken into account! For green light (550 nm and 1.4 NA objective) d @ 240 nm Limiting resolution of light microscopes (diffraction limited). Objective Resolved distance d (nm) 5x/0.15 2200 10x/0.3 1100 20x/0.5 700 40x/0.75 450 40x/1.3 260 63x/1.4 240 100x/1.3 260 Confocal microscope 1 AU pinhole Confocal microscope 0.25 AU pinhole Conventional microscope A close up of a logo Description automatically generated Lateral resolution 29 Adobe Systems ⍺ A close up of a logo Description automatically generated Objective coding Light microscopy methods in biology 30 Adobe Systems Chromatic aberrations Spherical aberrations F F F F A close up of a logo Description automatically generated Most frequent lens abberations Apochromatic objectives Use correct coverslip Refraction media match Light microscopy methods in biology 31 Adobe Systems OSRAM HXP 120W/45 CVIS A close up of a logo Description automatically generated Transmitted light microscopy Light microscopy methods in biology 32 Adobe Systems A picture containing sitting, indoor, table, clock Description automatically generated Conjugated planes A close up of a logo Description automatically generated Light microscopy methods in biology 33 Field set Aperture set Adobe Systems A close up of a person Description automatically generated Light passing directly through specimen to eys ot other detector BF – bright field, some microscopes H on condensor + Easy to setup, no special requirements - Low contrast Bright field A close up of a logo Description automatically generated Closed illuminating aperture Light microscopy methods in biology 34 Adobe Systems Only diffracted light from specimen passing to objective DF – bright field + Easy to setup, no special requirements, for high NA Special condenser required Dark field Dark field A picture containing clock Description automatically generated Light microscopy methods in biology 35 Adobe Systems A screenshot of a cell phone Description automatically generated Transparent objects not visible in the transmitted light mode still diffract light path and cause phase shift Phase contrast imaging transform phase differences into amplitude differences. Ph1, Ph2, Ph3 – phase contrast + Easy to setup, nice contrast, working on plastic dish - Special objectives required, halo effect around objects Phase contrast Light microscopy methods in biology 36 Adobe Systems Differential interference contrast (DIC) Based on polarised light. Transform local gradient in optical path length (refractive index and thickness) in an object into regions of contrast in the object image. Amplitude differences in the image due to refractive index and/or thickness of the specimen. + nice contrast, 3D perspectives, no halos around objects - Works on glass only, not trivial to setup https://www.olympus-lifescience.com Light microscopy methods in biology 37 Adobe Systems http://zeiss-campus.magnet.fsu.edu/articles/basics/images/fluorescencefigure6.jpg Reflected light A close up of a logo Description automatically generated Fluorescence microscopy Light microscopy methods in biology 38 Emisní filtr Excitation filter light Adobe Systems Fluorescein A close up of a logo Description automatically generated Fluorescence Light microscopy methods in biology 39 Adobe Systems -widefield (epifluorescence) - -confocal microscopes -point scanning microscopes -spinning disc microscopes - -lightsheet microscopes Diffraction limited resolution Resolution below diffraction limit - nanoscopes -Structured illumination (SIM) - -Stimulated emission depletion (STED) - -Stochastic optical reconstruction (STORM) Types of fluorescent microscopes Light microscopy methods in biology 40 Adobe Systems Fluorescein https://en.wikipedia.org/wiki Difference between positions of the band maxima of the absorption and emission spectra Stokes shift A close up of a logo Description automatically generated Fluorescence Light microscopy methods in biology 41 Adobe Systems Fluorescein Jablonski diagram S0 S1 S2 ground state excited state Radiation - fluorescence A close up of a logo Description automatically generated Fluorescence Light microscopy methods in biology 42 Adobe Systems -George Biddell Airy (1801-1892) -Best focused point source of light won’t appear as a point, but as a spot surrounded by series of concentric bright rings – Airy pattern. -Size of Airy disc depend on the light wavelength and the size of aperture. Imaging Device (circular aperture) Airy disc (d) https://upload.wikimedia.org/wikipedia/commons/thumb/2/21/George_Biddell_Airy2.jpg/220px-George_Bid dell_Airy2.jpg - Airy disc contain ~84% of the light from the point source A close up of a logo Description automatically generated Airy disc Light microscopy methods in biology 43 Adobe Systems -Three dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to image plane object Point Spread Function (PSF) Image convolution A close up of a logo Description automatically generated Point spread function Light microscopy methods in biology 44 Adobe Systems -Three dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to image plane object Point Spread Function (PSF) Image deconvolution A close up of a logo Description automatically generated Deconvolution Light microscopy methods in biology 45 A picture containing text, light, lit, star Description automatically generated A map of the world Description automatically generated with low confidence convolution Adobe Systems https://upload.wikimedia.org/wikipedia/commons/thumb/f/f6/Diversity_of_fluorescent_patterns_and_col ors_in_marine_fishes_-_journal.pone.0083259.g001.png/800px-Diversity_of_fluorescent_patterns_and_co lors_in_marine_fishes_-_journal.pone.0083259.g001.png https://en.wikipedia.org/wiki/Fluorescence https://stephenslab.files.wordpress.com/2012/12/aequorea-spectra.jpg Fluorescent proteins in living organisms Roger Y. Tsien Roger Tsien Nobel prize in chemistry 2008 Discovery and development of the green fluorescent protein GFP Martin Chalfie Osamu Shimomura Martin Chalfie Osamu Shinomura Aequorea victoria http://www.photobiology.info/Zimmer_files/Fig6.png A close up of a logo Description automatically generated Fluorescence Light microscopy methods in biology 46 Adobe Systems OSRAM HXP 120W/45 CVIS Excitation light Emission spectrum Sample Excitation filter Emission filter Dichromatic mirror Detector A close up of a logo Description automatically generated Components of fluorescent microscope Light microscopy methods in biology 47 Adobe Systems Související obrázek Výsledek obrázku pro chroma filters Single bandpass (limitations min and max) Longpass filter Shortpass filter Multi bandpass filter A close up of a logo Description automatically generated Filters and beam splitters Light microscopy methods in biology 48 Adobe Systems Crosstalk/bleed through A close up of a logo Description automatically generated Filters and beam splitters Light microscopy methods in biology 49 Adobe Systems Mercury short arc lamp: Obsolete, short life time – 200 hours, toxic wapors Manual alignmnent http://zeiss-campus.magnet.fsu.edu/articles/lightsources/images/mercurylampsfigure1.jpg A close up of a logo Description automatically generated Light sources Light microscopy methods in biology 50 Adobe Systems Metal halide lamp: Longer life time – 2000 hours No alignment, contains also mercury http://zeiss-campus.magnet.fsu.edu/articles/lightsources/images/metalhalidelampsfigure1.jpg OSRAM HXP 120W/45 CVIS A close up of a logo Description automatically generated Light sources Light microscopy methods in biology 51 Adobe Systems http://zeiss-campus.magnet.fsu.edu/articles/lightsources/images/xenonlampsfigure1.jpg Xenon lamp A close up of a logo Description automatically generated Light sources Light microscopy methods in biology 52 Adobe Systems LED – light emmiting diodes Single wavelength, or combination of several LED‘s Instantly switching ON/OFF Výsledek obrázku pro zeiss colibri A close up of a logo Description automatically generated Light sources Light microscopy methods in biology 53 Adobe Systems Widefield microscopes – CCD or sCMOS sensors Pixel number, pixel size, bit depth, quantum efficiency, spectral response photons electrons photodiode Photoactive part (photodiode) Isolated interline device (charge transfer) - Smallest unit of sensor with defined size (x,y dimensions) - Sensor consists of many pixels A close up of a logo Description automatically generated Detectors Light microscopy methods in biology 54 Adobe Systems Binning and resolution -Several neighborhoods pixels are accumulated together and readed as one unit -Increasing sensitivity, decreasing resolution, decreasing size of image Binning 1 – full resolution Resolution = 12 x 12 pixels Pixel size = 6 x 6 μm Binning 2 Resolution = 6 x 6 pixels Pixel size = 12 x 12 μm Binning 4 Resolution = 3 x 3 pixels Pixel size = 24 x 24 μm Pixels A close up of a logo Description automatically generated Detectors Light microscopy methods in biology 55 Adobe Systems Spectral response and quantum efficiency A close up of a logo Description automatically generated Detectors Light microscopy methods in biology 56 Adobe Systems Analog to digital converter (ADC) – convert analog signal (current in Voltage) to discrete digital values - number of grey values. Number of bits 1 2 4 8 12 14 16 Number of grey values 2 4 16 256 4,096 16,384 65,536 Bit depth A close up of a logo Description automatically generated Detectors Light microscopy methods in biology 57 Adobe Systems Correct exposure – read histogram A close up of a logo Description automatically generated Exposure Light microscopy methods in biology 58 Adobe Systems 1 sec 2 sec 4 sec 8 sec Saturation - blooming Correct exposure – read histogram A close up of a logo Description automatically generated Exposure Light microscopy methods in biology 59 Adobe Systems -Important to know resolution of the system, avoid oversampling and undersampling -Pixel size should be 2-3 x smaller than the actual resolution of the system Objectiv: Zeiss Plan-Apochromat 100x/1.4 Optovar:1.6x C-mount: 1x Camera: Hamamatsu Flash 4.0 https://upload.wikimedia.org/wikipedia/commons/thumb/4/46/Ernst-Abbe-Denkmal_Jena_F%C3%BCrstengrabe n_-_20140802_125709.jpg/170px-Ernst-Abbe-Denkmal_Jena_F%C3%BCrstengraben_-_20140802_125709.jpg Resolution (GFP) = 0.51 * 550/1.4 = 200 nm Camera pixel size = 6.5 x 6.5 μm Magnification = 100 x 1.6 x 1 = 160 Oversampled: Max. resolution = 200 nm, image pixel = 40 nm Nyquist requirement = 2.3 times the resolution = 85 nm A close up of a logo Description automatically generated Digital sampling Light microscopy methods in biology 60 Adobe Systems -Important! Acts as external part of the objective - -Standard thickness is 0.17 mm (170 mm) == grade 1.5, best 1.5H (high precision 170 mm ± 0.05 mm) - -Variability should not exceed ±0.1 for objective with high NA - -Some objectives have correction collar to adjust for different thickness of cover slip Grade Number Thickness (mm) 0 83-130 1 130-160 1.5 160-190 2 190-250 Refraction index should be close to refraction index of glass == 1.5168 Home made – Mowiol solution Commercial solutions – VectaShield, Prolong glass Mounting media A close up of a logo Description automatically generated Coverslips Light microscopy methods in biology 61 Adobe Systems A close up of a logo Description automatically generated Thank you for your attention Light microscopy methods in biology 62 Lecture 2: October 14, 2021 at 9:00 Confocal microscopy - principles, types of confocal microscopes – scanning, spinning disc, channel vs spectral imaging, linear unmixing. Lasers, detectors, beam splitters, optical sections. Lightsheet microscopy.