Presentation on theme: "Fluorescence and Confocal Microscopy"— Presentation transcript:
1 Fluorescence and Confocal Microscopy Dr. Fraser CoxonBone Research Programme
2 Microscopy- limits of resolution Fluorescence microscopy is alight microscopic technique
3 Fluorescence Fluorescent minerals An optical phenomenon in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.Usually the absorbed photon is in the ultraviolet range, and the emitted light is in the visible range.Fluorescence is named after the mineral fluorite (composed of calcium fluoride),The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence". Stokes observed that the fluorescing light has longer wavelengths than the excitation light, a phenomenon that has become to be known as the Stokes shift. Fluorescence microscopy is an excellent method of studying material that can be made to fluoresceFluorescent minerals
4 Simplified Jablonski Diagram Hvex – excitation from absorbed photonS1S’ – S1 – rapid vibrational energy loss as a result of inter-molecular collisionsEnergyhvexhvemRadiative emission of a lower energy photon as the species returns to the ground stateS0Lower enrgy photon Is emitted , therefore has longer wavelength.Difference betweenIn fluorescence, the species is first excited, by absorbing a photon of light, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy (squiggly line; internal conversion or vibrational relaxation) until it reaches the lowest vibrational state of the excited electronic state.The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the processThe lower the energy, the longer the wavelength
5 Fluorescent tubesA fluorescent lamp or fluorescent tube uses electricity to excite mercury vapour in argon or neon gas, producing short-wave ultraviolet light. This light then causes a phosphor coating to fluoresce, producing visible white light.
6 Typical emission spectrum from fluorescent light
7 Fluorophores Compounds that fluoresce are known as Fluorophores Aromatic ring structures are generally responsible for fluorescence properties of compoundsStokes Shift is 25 nmFluoresceinmoleculeStokes Shift- energy difference between the peak energy absorbance and the highest energy emission495 nm520 nmFluorescence IntensityWavelengthThis property can be exploited in microscopy by using filters that transmit selective wavelengths of light
8 Stokes shift of some widely-used fluorophores Ultra-violetIncreasingwavelengthvisibleInfra-red
9 Some uses of fluorescence microscopy Localisation of specific proteins and other subcellular structures within cellsLive cells (dynamic effects)Chemically fixed cellsIdentify which cell compartment a protein localises to, and whether it colocalises with other proteinsAnalysis of signalling pathways in individual cells (e.g. calcium imaging)Measuring intracellular pH/detecting acidic compartmentsLocalize/measure enzyme activity, using substrates that are cleaved to a fluorescent productThe use of fluorochromes has made it possible to identify cells and sub-microscopic cellular components and other entities with a high degree of specificity amidst non-fluorescing material. What is more, the fluorescence microscope can reveal the presence of fluorescing material with exquisite sensitivity. An extremely small number of fluorescent molecules (as few as 50 molecules per cubic micrometer) can be detected. In a given sample, through the use of multiple staining, different probes will reveal the presence of individual target molecules. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of the respective specimens, the presence of fluorescing molecules below such limits is made remarkably visible.
10 Fluorescence microscopy Useful for very exact, evensubcellular, localisationRequirements:Reflective light illuminationHigh intensity light source: mercury lampLenses with high N.A.
11 Arc Lamp Excitation Spectra Xe LampIrradiance at 0.5 m (mW m-2 nm-1)Hg Lamp
13 Filters Long Pass Filter Short Pass Filter Band Pass Filter White Light SourceTransmitted LightLong Pass Filter>520 nm520 nm Long Pass FilterShort Pass Filter<575 nm575 nm Short Pass FilterBand Pass Filternm630 nm Band Pass Filter
14 Beam path of fluorescent light Typical green emission fluorophore
15 for typical ‘green’ fluorophores Alexa Fluor 488 (green emission) spectrumAlexa Fluor 488(green emission)excitationspectrumFilter Set 09Ex - BPBeam Splitter - FT 510Em - LP 515excitationfilteremissionfilterfor typical‘green’ fluorophores
19 Nuclear probes (stain DNA) excitationemissionHoechst (uv)DAPI (uv)Sytox greenTOTOSytox orangePI (uv/vis)TO-PROWork in live cells
20 Fluorescent probes for cellular structures TRITC Phalloidin (F-actin)Fluorescent Phalloidin conjugates used to visualize the actin cytoskeletonPhalloidin is a fungal toxin (from Amanita phalloides) that binds to polymerised F-actinFluorescent conjugates of wheat germ agglutinin (WGA)WGA binds to glycosylated proteins, and therefore stains the plasma membrane and the Golgi apparatusWGA-AlexaFluor594Amanita phalloides also known as the death cap!
21 Probing acidic vesicles Lysotracker – weakly basic amine that selectively accumulates in compartments of low pH (e.g. endosomes/lysosomes)CtrlLysotracker-red+50nM bafilomycin (inhibitor of V-ATPases)Other probes, such as lysosensor, emit wavelengths that is dependent on the pH
22 Imaging multiple fluorophores in a single sample Straightforward provided that the fluorophores have distinct excitation and emission spectra, and the appropriate filters are availableMost fluorescence microscopes are equipped with 3 filter sets that are suitable for fluorophores that emit in the blue, green and red wavelengthsE.g. DAPI; fluorescein; rhodamineBlue: nuclei (DAPI)Green: actin (FITC-phalloidin)Red: acidic vesicles (lysotracker red)
23 How can we detect specific proteins by fluorescence microscopy? Immunostaining in fixed cellsTransfection of cells with DNA constructs expressing protein of interest couple to an inherently fluorescent protein (can analyse live cells, OR cells after fixation)
24 Fluorescent protein tags Green fluorescent protein (GFP) isolated from jellyfish Aequoria victoriaExcitation maxima at 470 nm; Peak emission at 509 nmCoding sequence of GFP can be inserted adjacent to that of a protein of interest, or to an isolated signal sequenceTransfect such constructs into cells of interest; GFP-tagged protein will be produced and can be identified in living cells by fluorescence microscopySimilar fluorescent proteins with different characteristics now available (e.g. YFP, RFP, mCherry)GFPGFP-RacnucleiGFP-Rab1aplasmamembraneGolgiGFP rarely affects the normal function of the protein that is tagged
25 Now even more fluorescent protein tags..... mCherry etcProf. Roger Tsien, UC San Diego(Nobel Prize winner, 2009)Collage of histone H2B fusion proteins- amino acid sequence for human histone H2B fused to monomeric fluorescent protein sequences. Shows mitosis (anaphase) of cervical carcinoma cells:GFP rarely affects the normal function of the protein that is tagged
26 ImmunostainingDetection of a protein within a cells/tissues using antibodies raised against that proteinThe cells must be ‘fixed’E.g. aldehydes such as formaldehyde, which cross-links the proteinsCells must also be permeabilised (using low concentration of detergent, e.g. triton X100) to enable antibodies to gain access to the cellsAdvantage- enables localisation to be determinedDisadvantage- many antibodies don’t work. Non- quantitative
27 ImmunostainingIncubate with an antibody (Ab) specific for the protein of interest, followed by a secondary Ab specific to the primary Ab (i.e. species-specific)This secondary Ab is usually coupled to a fluorescent tag which fluoresces when exposed to a certain wavelength of lightred- Rab6 (Golgi)Green- nucleiFluorescentmarkerAdvantage- enables localisation to be determinedDisadvantage- many antibodies don’t work. Non- quantitative
29 What is confocal microscopy? conventionalModification to reflected light (fluorescent) microscopy that enables optical sectioning of a sample, eliminating out of focus lightPrinciple patented by Marvin Minsky in 1957, although laser scanning confocal microscopes not developed until 1980sUseful for analysing samples with significant depth e.g. tissue samplesconfocal
30 Laser scanning confocal microscopy MicroscopeLaser excitation source provides high power point illumination of specific wavelength of lightSample is scanned line by line with the focused laser beamEmitted fluorescence is detected pixel by pixel by means of a photomultiplier tube (PMT)Pinhole in front of the detector eliminates light originating from outside the plane of focus
31 Wide-field microscopy Principles of confocal microscopyobjectivefocal planedichroicsourceWide-field microscopycameraSolid lines- light in focusDashed lines- out of focus lightsourceConfocal microscopypinholePMT
32 Confocal Microscope Wide-field fluorescent Microscope Arc LampLaserExcitation PinholeExcitation DiaphragmExcitation FilterPhotomultiplierTube (PMT)CameraObjectiveObjectiveEmissionFilterEmission FilterEmission PinholeBlack line = focal planeRed line = above focal planeGreen line = below focal plane
33 Considerations with the pinhole size Diameter of the pinhole determines the optical thickness of the acquired image (smaller pinhole = thinner section i.e greater resolution)However, smaller pinhole reduces the amount of light reaching the detectorCompromise between resolution and signal
34 Scanning Galvanometer The Scan Path of the Laser BeamScanning GalvanometerStartxyLaser inPoint ScanningLaser out- toMicroscopeSpecimenFrames/Sec # Lines
35 Laser scanning confocal microscopy AdvantagesDisadvantagesReduced blurring of the image from light scatteringOptical sectioning of thick specimensDetection uses highly sensitive photomultipliers, improving signal to noise ratioZ-axis scanning enabling generation of 3D datasetsMagnification can be adjusted electronicallySlow scan speedsLimited use in dynamic tracking studiesPhotobleaching from laser excitationLasers may damage living cells, limiting use in live cell studiesLower resolution than camera detection
36 LSM510 META system in the IMS Argon and HeNe lasers giving lines at wavelengths allowing excitation of visible-light fluorophores:Argon 458 nm (cyan)Argon 476 nm (green)Argon 488 (green)Argon 514 (orange)HeNe 543 (red)HeNe 633nm (far red)3 detection channels, therefore 3 fluorophores in a specimen can be captured simultaneously
37 Effect of pinhole size on z resolution WIDE PINHOLE13mm optical sectionNARROW PINHOLE1mm optical sectionSample of whole mouse retina; cells expressing GFP
38 Improving signal-to-noise ratio in confocal images Problem of high noise (low signal-to-noise ratio) in weakly fluorescent samplesCan reduce by:Slowing scan speed (increasing pixel time)Signal averaging from repeated scans (noise will appear only randomly, whereas genuine signal should be consistent and appear in every scan)Photobleaching may be a limitation with these approaches
39 Effect of averaging multiple scans Single scanMean of 8 scansHuman osteoclast adenovirally transduced with WT GFPRab18Lysotracker redGFPRab18
40 Studies of colocalisation to subcellular organelles CtrlRab6WGA (Golgi)merge
41 Studies of colocalisation between proteins NucleiGFPRab7Plekhm1-FLAGmergeTransfected cells expressingGFP-LC3 and Plekhm-dsRed:Transfected cells expressing GFP-Rab 7 and Plekhm-dsRedYellow colour in merged image indicates colocalisation
42 Sequential scans through sample: Imaging in 3 dimensionsFromSourcePIXEL2D spaceSequential scans through sample:xyzxzVOXEL3D spaceyzTo Detector
43 Imaging z-seriesSamples up to 100mm thick can be analysed (although quenching of fluorescence signal can occur in thick tissue specimens)z (axial) resolution as little as 0.5mmWavelength of fluorescent light and the numerical aperture of the objective lens determine the limits of this resolutionMotorised stage crucial for capturing z-series
44 Z-series of an osteoclast resorbing dentine Blue- cell membraneRed- F-actinGreen- substrate surfaceScans covers 26mm in the z (axial) dimension
45 Orthogonal views generated from the 3D data set Blue- cell membraneRed- F-actinGreen- substrate surfacexzxzdepth =26mmxyyzxyyz
46 Importance of z-scanning for determining localisation Wheat germ agglutinintubulinF-actinHuman osteoclast on glassFluorescent conjugates of WGA- binds to glycosylated proteins, and therefore stains the Golgi and plasma membranezxAmanita phalloides also known as the death cap!
47 Animation of resorbing osteoclast Mutations are in ClCN7 geneParents each have different homozygous mutations. Mother’s mutation indicates that the protein will retain activityNot sure about father’s. This could mean that CLCN-7 is not involved at all?!Mutations must interact in some way?
48 3D reconstruction of osteoclast resorbing dentine Isosurface rendering(red and green fluorescence only)Max intensity projectionGreen- bisphosphonateRed- F-actinBlue- osteoclast membrane (left only)
50 Alternative- wide-field microscopy with deconvolution Live cell imagingLasers used in confocal microscopy may damage living organismsConfocal microscopy has some difficulties dealing with weak fluorescenceLive cell imaging also limited by scan timesAlternative- wide-field microscopy with deconvolutionUseful for analysing fluorescent probes in living organisms in real time e.g. a GFP-tagged expression constructZ series can be collected then resolved post-acquisition using complex algorithmsDeltaVision
51 Two different ways of reducing “blur” in fluorescent imagesConventional andConfocal microscopyWidefield microscopywith deconvolutionAlso structured illumination (e.g. Zeiss Apotome system)
52 SummaryFluorescence microscopy is a powerful technique for visualizing proteins, subcellular structures and cellular processes in intact cells (live or fixed)Confocal microscopy provides additional resolution in the z-dimension, enabling optical slicing of thicker specimens and 3D reconstructionsAdvanced applications possible with laser-scanning confocal systems, e.g. analysis of protein:protein interactions using FRETResolution not as good as electron microscopy! Immuno-EM approaches required to look at protein localisation at the ultrastructural level