Adobe Systems Adobe Systems Adobe Systems S5015 Light microscopy methods in biology Lecture 2: Confocal and lightsheet 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 Ligh microscopy methods in biology 3 Widefield fluorescence imaging +fast and photo efficient +sensitive detectors – sCMOS with 95%QE, +variable LED sources +easy and accessible technique of imaging +relatively cheap instruments −blurred images due to out of focus light OSRAM HXP 120W/45 CVIS Emission filter Detector Excitation filter Light source Dichromatic mirror http://zeiss-campus.magnet.fsu.edu/articles/basics/images/fluorescencefigure6.jpg A close up of a logo Description automatically generated Adobe Systems A close up of a logo Description automatically generated Confocal microscopy Ligh microscopy methods in biology 4 Adobe Systems Scanning mirrors OSRAM HXP 120W/45 CVIS confocal pinhole A close up of a logo Description automatically generated Confocal microscopy Ligh microscopy methods in biology 5 Coherent light source lasers -Pinhole physically blocking out of focuse light -Define optical thickness of scan -Diameter in Airy units, depends on wavelengths and NA -Impact resolution -Multicolor imaging - same opening produce different optical thickness -Adjustable for each dye in point scanning microscopes -Spinning disc confocals have fixed pinhole diameter Pinhole Adobe Systems Scanning mirrors OSRAM HXP 120W/45 CVIS confocal pinhole A close up of a logo Description automatically generated Point scanning confocal microscopy Ligh microscopy methods in biology 6 Coherent light source lasers Nyquist-Shannon criterion Pixel size should be at least 2.3 times smaller than actual optical resolution of the system. Adobe Systems http://zeiss-campus.magnet.fsu.edu/tutorials + optical sectioning + fast -pinholes optimized for one objective -(interchangeable discs) -sensitive to dust Spinning disc confocal microscopy Ligh microscopy methods in biology 7 A close up of a logo Description automatically generated Adobe Systems A close up of a logo Description automatically generated Resolution Ligh microscopy methods in biology 8 Confocal microscope 1 AU pinhole Confocal microscope 0.25 AU pinhole Conventional microscope Lateral resolution Axial resolution Confocal microscope 1 AU pinhole Confocal microscope 0.25 AU pinhole Conventional microscope Only slightly increased resolution compare to widefield system Main advantage – increase contrast and optical sections Adobe Systems Ligh microscopy methods in biology 9 A close up of a logo Description automatically generated Light source Ølasers polarized, coherent, monochromatic, collimated ØCw – constant wave lasers, mW range Ø405, 488, 561, 640 – standard wavelengths Ø350, 458, 514 and others ØArgon laser source (458, 488, 514) – outdated ØDiode lasers most often ØWhite laser tunable ØPulsed lasers, cw lasers ØLasers are combined together by laser combiner ØDichromatic beam splitter, or AOBS Ø Adobe Systems Ligh microscopy methods in biology 10 A close up of a logo Description automatically generated Detectors photons electrons photodiode Capture light emitted from sample -> Point scanning confocal microscopes: Photon Multiplying Tube, Avalanche PhotoDiode detectors Widefield, spinning disc microscopes: CCD, EM-CCD, sCMOS sensors Numbers, digits, gray levels Výsledek obrázku pro gray levels 0 255 electric current ADC converter Adobe Systems Ligh microscopy methods in biology 11 A close up of a logo Description automatically generated Detectors Photomultiplier tube detector (PMT) photons electrons photocathode anode dynode (electrodes) Photocathode determine QE and spectral sensitivity. GaAsP (Galium, Arsenid, Phosphid) are more sensitive then standard multialkali PMTs, but are sensitive to overexposure. QE up to 50% in 550 nm. Adobe Systems Ligh microscopy methods in biology 12 A close up of a logo Description automatically generated Detectors photons electrons GaAsP photocathode avalanche photodiode GaAsP (Galium, Arsenid, Phosphid), but are sensitive to overexposure. QE up to 50% in 550 nm. Avalanche diode Avalanche photodiode detector (APD) – hybrid detectors (Leica) Adobe Systems Ligh microscopy methods in biology 13 A close up of a logo Description automatically generated Detectors CCD and CMOS devices CCD – Charge-Coupled Device Pixels read out by line (interline CCD). Single output node and ADC, lower frame rate. Consume more electricity. CMOS – Complementary Metal-Oxide-Semiconductor Faster read out then CCD, each pixels read out individually. Each row has its own output node with ADC. More noise compare to CCD, but faster fps. Major improvements in last years. Photoactive part releasing electrons and generating charge, which is converted into a voltage. Analog signal (voltage) is converted to discrete signal (numbers). Array of multiple units (pixels). Adobe Systems Ligh microscopy methods in biology 14 A close up of a logo Description automatically generated Detectors photons electrons photodiode Photoactive part (photodiode) Isolated interline device (shift register) charge transfer charge transfer charge transfer charge transfer charge transfer charge transfer Output node Pixel charge fill to neighborhood pixels horizontally and vertically (interline CCD) 54, 651,21,10,54 A/D converter CCD sensor Adobe Systems Ligh microscopy methods in biology 15 A close up of a logo Description automatically generated Detectors Electron multiplying charge-coupled device – on-chip amplification Advantage - Extremely sensitive, near 100% QE Disadvantage – low pixel number, big pixel size Output node 320, 25, 56, 890 A/D converter photons electrons photodiode High Voltage Serial Multiplication Register EM-CCD sensor Adobe Systems Ligh microscopy methods in biology 16 A close up of a logo Description automatically generated Detectors CMOS sensor photons electrons photodiode 5,40,5680,651 2,50,6800,640 5047,45,6,610 Adobe Systems Ligh microscopy methods in biology 17 A close up of a logo Description automatically generated NOISE Dark current noise – randomly generated electrons without photons (thermally generated electrons). Depends on temperature. Read noise – several sources, errors during quantification of electrons to current (voltage) and subsequent analog-digital (A/D) conversion, higher at faster pixel read-out rates. Hamamtsu ORCA Fusion Adobe Systems A close up of a logo Description automatically generated Confocal image acquisition Ligh microscopy methods in biology 18 Confocal microscope have usually multiple detectors for fluorescence and one detector for transmitted light. Based on fluorochromes we can acquire multicolor image in different ways ØSequential scanning with interchangeable hardware setting between tracks ØSequential scanning without interchangeable setting hardware between tracks Ø ØSimultaneouse scanning Ø ØLambda scanning Diagram Description automatically generated Adobe Systems A close up of a logo Description automatically generated Channel imaging Ligh microscopy methods in biology 19 Sequential acquisition Detector 1 405 Detector 2 488 Detector 3 561 Adobe Systems A close up of a logo Description automatically generated Channel imaging Ligh microscopy methods in biology 20 Detector 2 Detector 1 Detector 3 Simultaneous acquisition 405 488 561 + speed - crossexcitation and bleedthrough Adobe Systems Detector lambda A close up of a logo Description automatically generated Lambda imaging Ligh microscopy methods in biology 21 405 488 561 + speed + separation of similar spectras -Mathematical separation -of fluorochromes 1 2 3 4 5 6 7 8 9 10 11 13 12 Adobe Systems A close up of a logo Description automatically generated Linear unmixing Ligh microscopy methods in biology 22 Acquire lambda scan and define spectra of each fluorophore separately Spectra saved in databes Using the same setings acquire mixture of fluorophores Different algorhitms to separate lambda scan to individual fluorophores – multichannel image Defined spectra or unknown spectra Adobe Systems Pinhole diameter: ↓ diameter = ↓ light = ↓ optical thickness and ↑ lateral resolution Detector gain: ↑ gain = ↑ noise = ↓ laser power Pixel dwell time (𝜇sec): time of measurement of each pixel Uni-direction or bi-directional scan Number of pixels: size an numbers freely adjustable in point scanning systems Bit depth: adjustable Averaging: measurement of one pixel several time A close up of a logo Description automatically generated Parameters of confocal imaging Ligh microscopy methods in biology 23 Adobe Systems Limitations of confocal microscope Ligh microscopy methods in biology 24 A close up of a logo Description automatically generated -speed, phototoxicity, photobleaching, penetration depth - Adobe Systems Ligh microscopy methods in biology 25 Two photon confocal microscopy Excitation with longer wavelength laser, than emmision Using near infrared femtoseconds pulsed laser Longer wavelength = lower energy, two photons required for exciation Near IR laser penetrates deeper in tissue, less scattering Confocality assured by excitation point spread function – no pinhole Superior to standard one photon confocal microscopes for deep tissue imaging A close up of a logo Description automatically generated Adobe Systems A close up of a logo Description automatically generated Fluorescence recovery after photobleaching - FRAP Ligh microscopy methods in biology 26 Methods to study the mobility of fluorescently labeled molecules inside cells Photobleach selected region and measure restoration of signal intensity over time Active transport or diffusion Adobe Systems A close up of a logo Description automatically generated Fluorescent lifetime imaging - FLIM Ligh microscopy methods in biology 27 Lifetime = time fluorophor remains in excited state (before starting emmiting photons) Each molecule has different lifetime – picoseconds to nanoseconds Time between excitation and arrival of photon to detector – transition to ground state Sensitive to local environment – pH, temperature, ion concentration Requirements Pulsed laser – femtoseconds lasers Single photon detectors – single photon avalanche diode, PMT, hybrid detectors Special timing electronics for data registration Multiple fluorophores separation Environmental sensing – pH, ion concentration, protein interactions Conformational changes S0 S1 S2 excited state level emitted fluorescence light absorbed excitation light ground state level Fluorescence lifetime Adobe Systems A close up of a logo Description automatically generated Fluorescent lifetime imaging - FLIM Ligh microscopy methods in biology 28 Picoquant Adobe Systems Ligh microscopy methods in biology 29 SIM Apotome for optical sectioning Illumination of sample with a known spatially structured pattern Grid structure is projected on focal plane of sample Grid is moving in one direction only, several images acquired with different grid position Different grid structures – magnification and wavelength Final image calculated from several phase shifted images A picture containing green, dark, ocean floor Description automatically generated Nela Jandová, Marcela Buchtová, ÚŽFG AVČR Brno A close up of a logo Description automatically generated Adobe Systems Ligh microscopy methods in biology 30 107-1-488-ApoTome deconvolution-01-Stitching-06a_1b.mp4 spider-3.mp4 Nela Jandová, Marcela Buchtová, ÚŽFG AVČR Brno Economic solution compare to confocal microscope and faster. Not suitable for all samples. Same sample preparation as for widefiled, or confocal microscopy. SIM Apotome for optical sectioning A close up of a logo Description automatically generated Adobe Systems + optical sectioning + minimal photobleaching + fast -Sample preparation -Image analysis and storage Other optical sectioning methods Ligh microscopy methods in biology 31 A close up of a logo Description automatically generated Adobe Systems Ligh microscopy methods in biology 32 Z sectioning and photobleaching all planes are excited, also out of focus. Lightsheet illuminates only planes in focus, much more gentle to sample A close up of a logo Description automatically generated Adobe Systems Gaussian beam lightsheet microscopes Ligh microscopy methods in biology 33 Gaussian beam – diffraction limited Multiple optical setups possible and available Optical thickness defined by thickness of lightsheet Multiple excitation objectives and detection objectives Imaging of large specimens – water, clearing objectives Major drawback – data processing A close up of a logo Description automatically generated Adobe Systems Ligh microscopy methods in biology 34 OSRAM HXP 120W/45 CVIS Illumination Objective 2 Illumination Objective 1 Detector 2 Detection Objective 2 Detection Objective 1 Detector 1 Sample rotation A close up of a logo Description automatically generated Adobe Systems Refractive index match importance Ligh microscopy methods in biology 35 A close up of a logo Description automatically generated Adobe Systems Gaussian beam lightsheet microscopes Ligh microscopy methods in biology 36 A picture containing light, cake, outdoor, green Description automatically generated 210819_CO_II_20x_detail_4_DSFe V. Pospíšilová, D. Bohačiaková, LF MU A close up of a logo Description automatically generated Adobe Systems Ligh microscopy methods in biology 37 Example of lighsheet for live organoid imaging Similar geometry to inverted microscope, special culture dishes Samples in liquid media, not need to special mounting into agar Easy multi positions imaging Alternative lightsheet geometry A close up of a logo Description automatically generated Adobe Systems Ligh microscopy methods in biology 38 Oblique plane microscope Resolution comparable to lattice light sheet Single objective lightsheet microscopes A close up of a logo Description automatically generated Adobe Systems Ligh microscopy methods in biology 39 Lattice light sheet Most suitable for fast and gentle super-resolution live cell imaging in 3D Developped initially by E. Betzig Using non diffracting Bessel beam instead of classical Gaussian beam Non-diffracting light sheet formed by interference patterns 1257998s13 Protozoa Tetrahymena thermophila A close up of a logo Description automatically generated Adobe Systems A close up of a logo Description automatically generated Thank you for your attention Light microscopy methods in biology 40 Lecture 3: October 25, 2021 at 9:00 Imaging below diffraction limits – SIM, SMLM, STED