C8116 Immunochemical techniques Advanced microscopy III Spring term 2024 Hans Gorris Department of Biochemistry May 14th, 2024 1 Epifluorescence microscopy 2 3 Total internal reflection fluorescence mic. (TIRF) Total internal reflection leads to emergence of an evanescent field (with exponential decay of intensity): => reduces the excitation volume to a depth of ca. 100 nm TIRF is suitable for investigating phenomena close to the glass slide => e.g. cell membranes 4 Confocal microscopy Additional features of confocal microscopy 5 β€’ improved contrast / multiphoton microscopy β€’ optical sectioning (z stacks) β€’ multiple fluorescence measurements can be performed in individual points (e.g. spectra, lifetime, FRET, FCS) 5 Spectral imaging: lambda stack anatomy Time domain Frequency domain Advantages: - Extremely low optical background - independent of fluorophore concentration 6 Fluorescence lifetime measurements (FLIM) Analyzing protein-protein interactions by FRET 7 𝐸! = 1 1 + 𝑅"# 𝑅$ % R0: ET = 50% FRET microscopy: Experimental setup 8 pulsed laser beam splitter microscopepinhole donor filter detector 1 acceptor filter detector 2 time-resolved single photon counting color beam splitter Intramolecular FRET Intermolecular FRET Single-molecule FRET 9 - Protein-protein interactions are investigated in their natural environment - Fusion with fluorescent proteins (e.g. GFP) are used - The location of the interaction can be determined (=> super-resolution microscopy) - Real-time imaging - Heterogeneous and dynamic biological processes can be observed Requires dedicated equipment: Þ Strong background reduction (autofluorescence): confocal microscopy or TIRF Þ Sensitive cameras or avalanche photodiodes Þ Reduction of photobleaching (GFP is not very photostable) Single-molecule FRET in vivo 10 Fluorescence correlation spectroscopy 11 12 Fluorescence correlation spectroscopy PC/Correlator low fluorophore concentration (~ 0.1 nM = 10-10 M) + very small focal volumen (fL = 10-15 L) => single molecule in focal volume Confocal optics => Compare to single molecule tracking Fluorescence correlation spectroscopy Time Fluorescence Raw data Each time when a fluorescent molecule passes through the confocal volume, there is a burst of light 13 Data analysis: autocorrelation 90 s90 s90 s SignalF(t) 30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s30 s60 s60 s60 s60 s 60 s60 s 60 s60 s60 s60 s60 s60 s60 s60 s60 s60 s90 s90 s90 s90 s90 s90 s90 s90 s 90 s90 s90 s90 s Time lag (Δτ) correlation no correlation 30 s 88 % 12 % 60 s 75 % 25 % 90 s 60 % 40 % Describes how strongly two data points with a given time lag correlate t (s) 𝐹 𝑑 14 Fluorescence correlation spectroscopy - brackets: averaging over time - F(t): fluorescence signal at time t - Ξ΄F(t): deviation of the fluorescence signal at time t from the average fluorescence signal Calculation of G(Ο„) for the time series of the increments: 𝐺 𝜏 = 𝛿𝐹 𝑑 βˆ— 𝛿 𝑑 + 𝜏 𝐹 𝑑 & Calculation of increments: 𝛿𝐹 𝑑 = 𝐹 𝑑 βˆ’ 𝐹 𝑑 15 Fluorescence correlation spectroscopy Time Fluorescence Raw data Autocorrelation function 𝜏": diffusion time through confocal volume 16 Investigating the mobilitiy of biomolecules Proinsulin C-peptide (with fluorescent label) focal volume: in solution on membrane 17 Investigating the mobilitiy of biomolecules tetramethylindocarbocyanine (DIL) in cell membrane fluorescent protein (EGFP) in cytoplasm 18 Investigating the mobilitiy & emission fluctuation 𝜏&: diffusion time through confocal volume 𝜏': fluctuation time in confocal volume 19 Interaction analysis The size of a molecular complex changes the diffusion time 20 Cross correlation spectroscopy Interaction analysis with two fluorophores 21 Interaction analysis with two fluorophores Both binding partners carry a fluorescent label 22 Interaction analysis with two fluorophores with Ca2+ without Ca2+ Binding of calmodulin to CaM-dependent protein 23 Cross-correlation spectroscopy: immunoassay The size of a molecular complex changes the diffusion time 24 1. High background fluorescence: Conventional wide field microscopy => A single fluorophore molecule cannot be detected (ultimate detection limit) 2. Diffraction limit of light: The image resolution was defined by Ernst Abbe (1873): ca. 200 nm a l sin2n d = Numerical apperture (NA) 25 Limitations of fluorescence microscopy Near-field optical microscopy (NSOM) 26 27 Coating Core Tip => Near field illumination (evanescent field) Usually metal coating to avoid stray light Near-field scanning optical microscopy (NSOM) Diffraction only occurs in far-field imaging, where spherical wave-fronts leaving the aperture can be regarded locally as plane waves => β€œSimpleβ€œ solution: avoid diffraction in the first place not diffraction limited coming close to the sample 28 Near-field scanning optical microscopy (NSOM) various operation modes – purely near-field or combining near-/far-field excitation/emission or vice versa 29 Fluorescent sample Near-field scanning optical microscopy (NSOM) Scanning approach 30 Combination: NSOM and shear force feedback (tuning fork): Topography und optical information Not diffraction limited: d β‰ˆ 30 nm Near-field scanning optical microscopy (NSOM) piezo control tuning fork objective piezo control Surface of sample SNOM tip glass fiber collimater argon laser oscillator 31 Near-field fluorescence image (4.5 mm by 4.5 mm) of single oxazine 720 molecules dispersed on the surface of a poly(methylmethacrylate) film. Each subdiffraction peak (full width at half maximum, 100 nm) comes from a single molecule (X. S. Xie, Acc. Chem. Res. 29, 598 (1996)). Near-field scanning optical microscopy (NSOM) Detection of single fluorescent molecules 32 Near-field scanning optical microscopy (NSOM) Advantages: Ø resolution ~ 20 nm in lateral (depending on tip size) and ~ 2-5 nm in axial direction Ø optical and topological information Limitations: Ø only applicable to surfaces Ø tip may break in contact with specimen (scanning) Ø far-field microscopy has many advantages (except the diffraction limit) Far-field optical microscopy => Using freely propagating light waves 33 34 Optical resolution of light microscopy a l sin2n d = Ernst Abbe: Diffraction of waves at a cleft central bright strip light intensity Light from a point source (e.g. a fluorophore) is diffracted by the inner rim of the objective and forms an Airy disc. The size of the Airy disc depends on Ξ» and NA of the objective:Airy disc Airy pattern a l sin 61.0 n d = => 2 points are resolvable if the maximum of one Airy disc coincides with the first minimum of the next Airy pattern. Corresponding intensity profile FirstminimumMaximum 35 Optical resolution of light microscopy Rayleigh criterion: when are two objects visible as separate points Source Nikon: http://www.microscopyu.com e.g. a fluorescent molecule 36 Optical resolution of light microscopy red waves: positive interference green waves: negative interference Point spread function: Max. axial und lateral resolution Diffraction limited spot: Point spread function 37 Optical resolution of light microscopy Stefan Hell STED William Moerner - Detection of single fluorescent molecules - switchable fluorophores Eric Betzig - Near field microscopy - STORM 38 Nobel prizce for Chemistry in 2014 (Far-field) microscopy beyond the diffraction limit A) Single molecule localisation (2006) e.g. STORM (STochastic Optical Reconconstruction Microscopy) B) Structured illumination (1994/1999) z.B. STED (STimulated Emission Depletion)39 Microscopy beyond the diffraction limit Using non-linear optical processes STochastic Optical Reconconstruction Microscopy STORM => based on wide-field microscopy (frequently in combination with TIRF) 40 Single Molecule Tracking Γ³ Imaging (STORM) => Rather than using a highly diluted solution of fluorophores, individual fluorophores are switched on/off in a sequential manner 41 STORM microscopy => Maximum of the point spread function of a single fluor. molecule can be determined precisely But: 1000-10.000 images required to put together a high-resolution image => Need for high computational power / appropriate β€žswitchableβ€œ fluorophores 42 STORM microscopy Pointillism in modern art 43 STORM microscopy Microtubule Clathrincoated pit 44 STORM microscopy Wide-field image STORM image Fibrobalast in the kidney Enlarged section Mikrotubule 45 STORM microscopy: images Variation: PALM (Photoactivated Localization Microscopy) => based on FP Switching on by UV-Licht Switching off by photobleaching Yield: ~500 photons Switching on to fluorescent state by supporting dye (e.g. Cy3) Switching off to dark state: spontaneously Yield: 6000 photons per activated fluorescent molecule 46 STORM microscopy photoswitchable fluorophores are required: Nn d a l sin2 = Resolution: 2 points that can just be distinguished by using STORM: N: Number of photons emitted by a single fluorophore molecule that can be detected In praxis, resolution of 10 - 20 nm: > factor 10! Other factors are limiting: e.g. antibodies have a diameter of 15 nm Different sizes of molecules 47 STORM microscopy STimulated Emission Depletion Microscopy STED => is based on Confocal Microscopy 48 2. Spontaneous emission Abs. Fluorescence tfl = 1–10 ns 3. Stimulated emission Abs. Stimulated Emission Dt Β» 1 ps Light can interact with matter: 1. Absorption 49 STED microscopy EXC und STED are pulsed lasers with defined timing of pulses 50 STED microscopy: instrumental setup 51 Excitation spot Depletion spot Remaining spot STED microscopy: improved lateral resolution 52 STED microscopy: improved lateral resolution Conventional confocal microscopy STED microscopy 53 STED microscopy: improved lateral resolution 54 STED microscopy: STED pulse intensity 55 STED microscopy: STED pulse intensity 56 STED microscopy: STED pulse intensity 57 STED microscopy: STED pulse intensity I: Intensity of the STED laser Is: Required intensity to completely deplete the excited state 58 STED microscopy: STED pulse intensity Intensity Intensity I: Intensity of the STED laser Is: Required intensity to completely deplete the excited state In praxis, resolution of < 10 nm 59 STED microscopy: STED pulse intensity Conventional CLSM 60 STED microscopy: scanning Conventional CLSM 61 STED microscopy: scanning STED-CLSM (low power STED) 62 STED microscopy: scanning STED-CLSM (high power STED) 63 STED microscopy: scanning STED-CLSM => A higher resolution requires more scanning steps 64 STED microscopy: scanning 65 STED microscopy: images 66 STED microscopy: images 67 STED microscopy: images 68 STED microscopy: images 69 STED microscopy: images Light Sheet Microscopy => based on wide-field microscopy 70 71 => Separate light paths for excitation and emission light excitation light em ission light Light sheet microscopy => Separate light paths for excitation and emission light 72 Light sheet microscopy: planar illumination 73 Intrinsic optical sectioning => only the focal plane is illuminated => avoids photobleaching outside the sheet Fast image acquisition => Whole image taken in a single exposure (no scanning required, but scanning techniques also exist) => more than 100 full images can be taken per second (depending on camera) Applicable to larger biological samples => 3-D imaging => small living organisms => embryo development Light sheet microscopy: advantages 74 A sea horse: detection of autofluorescence for imaging Light sheet microscopy: images 75 pink: progenitor cells green: aorta blue: vein Light sheet microscopy: images Formation of lymph vessels in a mouse embryo 76 red: (1) red blood cells (2) myocard (heart muscle) cyan: endocard (inner lining of heart) Light sheet microscopy: video Heartbeat of zebrafish Expansion Microscopy => Blows up sample before imaging 77 78 Expansion microscopy 79 2 Β΅m Expansion microscopy: images Different types of microtubles 80 4x expanded => 60 nm resolution Gao et al. (2019) Science 363, 245 Brain of fruit fly Drosophila: Mapping of more than 40 million synapses in 62 hours Expansion + light sheet microscopy: images 81 Expansion + light sheet microscopy: video Labeling of neuronal cells in the brain