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Fluorescence and Confocal Microscopy Dr. Fraser Coxon Bone Research Programme

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Presentation on theme: "Fluorescence and Confocal Microscopy Dr. Fraser Coxon Bone Research Programme"— Presentation transcript:

1 Fluorescence and Confocal Microscopy Dr. Fraser Coxon Bone Research Programme f.p.coxon@abdn.ac.uk

2 Microscopy- limits of resolution Fluorescence microscopy is a light microscopic technique

3 Fluorescence 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), Fluorescent minerals

4 Simplified Jablonski Diagram S0S0 S1S1 Energy S1S1 Hv ex – excitation from absorbed photon hv em The lower the energy, the longer the wavelength hv ex S – S 1 – rapid vibrational energy loss as a result of inter-molecular collisions Radiative emission of a lower energy photon as the species returns to the ground state

5 A 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. Fluorescent tubes

6 Typical emission spectrum from fluorescent light

7 Fluorophores Compounds that fluoresce are known as FluorophoresCompounds that fluoresce are known as Fluorophores Stokes Shift- Stokes Shift- energy difference between the peak energy absorbance and the highest energy emission 495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescence Intensity Wavelength This property can be exploited in microscopy by using filters that transmit selective wavelengths of lightThis property can be exploited in microscopy by using filters that transmit selective wavelengths of light Aromatic ring structures are generally responsible for fluorescence properties of compoundsAromatic ring structures are generally responsible for fluorescence properties of compounds

8 Stokes shift of some widely-used fluorophores Increasingwavelength Ultra-violet visible Infra-red

9 Some uses of fluorescence microscopy Localisation of specific proteins and other subcellular structures within cells –Live cells (dynamic effects) –Chemically fixed cells Identify which cell compartment a protein localises to, and whether it colocalises with other proteins Analysis of signalling pathways in individual cells (e.g. calcium imaging) Measuring intracellular pH/detecting acidic compartments Localize/measure enzyme activity, using substrates that are cleaved to a fluorescent product

10 Fluorescence microscopy Useful for very exact, even subcellular, localisation Requirements: Reflective light illumination High intensity light source: mercury lamp Lenses with high N.A.

11 Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 ) Xe Lamp Hg Lamp

12 Fluorescence microscopy Filter Block in fluorescent light path A = Excitation filter B = Dichroic beam splitter C = Emission (barrier) filter Em Ex

13 Filters 520 nm Long Pass Filter >520 nm 575 nm Short Pass Filter <575 nm Short Pass Filter Long Pass Filter Transmitted Light White Light Source 620 -640 nm Band Pass Filter 630 nm Band Pass Filter

14 Beam path of fluorescent light Typical green emission fluorophore

15 Filter Set 09 Ex - BP 450-490 Beam Splitter - FT 510 Em - LP 515 Alexa Fluor 488 (green emission) for typical green fluorophores excitation spectrum emission spectrum emission filter excitation filter

16 Fluorophores Fluorescein Alexa Fluor 488 488522 Fluorescein 488525 ProbeExcitation Emission

17 Probes for Ions (Ca 2+ ): INDO-1 E x 350E m 405/480 QUIN-2E x 350E m 490 Fluo-3 E x 488E m 525 Fura -2E x 330/360E m 510 pH Sensitive Indicators: SNARF-1488575 BCECF488525/620 440/488525 ProbeExcitation Emission C 27 H 20 O 11 C 27 H 19 NO 6

18 Specific Organelle Probes BODIPY Golgi505511 NBD Golgi488525 DPH Lipid350420 TMA-DPH Lipid350420 Rhodamine 123 Mitochondria 488525 DiOLipid488500 diI-Cn-(5)Lipid550565 diO-Cn-(3)Lipid488500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH – diphenylhexatriene TMA - trimethylammonium

19 Nuclear probes (stain DNA) Hoechst 33342 (uv)346460 DAPI (uv)359461 Sytox green498592 TOTO-1514533 Sytox orange 547570 PI (uv/vis)536620 TO-PRO-3642657 excitationemission Work in live cells

20 Fluorescent probes for cellular structures Fluorescent Phalloidin conjugates used to visualize the actin cytoskeleton Phalloidin is a fungal toxin (from Amanita phalloides) that binds to polymerised F- actin TRITC Phalloidin (F-actin) Fluorescent conjugates of wheat germ agglutinin (WGA) WGA binds to glycosylated proteins, and therefore stains the plasma membrane and the Golgi apparatus WGA-AlexaFluor594

21 Probing acidic vesicles Lysotracker – weakly basic amine that selectively accumulates in compartments of low pH (e.g. endosomes/lysosomes) +50nM bafilomycin (inhibitor of V-ATPases) Ctrl Lysotracker-red 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 available Most fluorescence microscopes are equipped with 3 filter sets that are suitable for fluorophores that emit in the blue, green and red wavelengths E.g. DAPI; fluorescein; rhodamine Blue: nuclei (DAPI) Green: actin (FITC-phalloidin) Red: acidic vesicles (lysotracker red)

23 How can we detect specific proteins by fluorescence microscopy? Immunostaining in fixed cells Transfection 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 victoriaGreen fluorescent protein (GFP) isolated from jellyfish Aequoria victoria Excitation maxima at 470 nm; Peak emission at 509 nmExcitation maxima at 470 nm; Peak emission at 509 nm Coding sequence of GFP can be inserted adjacent to that of a protein of interest, or to an isolated signal sequenceCoding sequence of GFP can be inserted adjacent to that of a protein of interest, or to an isolated signal sequence Transfect such constructs into cells of interest; GFP-tagged protein will be produced and can be identified in living cells by fluorescence microscopyTransfect such constructs into cells of interest; GFP-tagged protein will be produced and can be identified in living cells by fluorescence microscopy Similar fluorescent proteins with different characteristics now available (e.g. YFP, RFP, mCherry)Similar fluorescent proteins with different characteristics now available (e.g. YFP, RFP, mCherry) GFP GFP-Rac nuclei GFP-Rab1a nuclei plasma membrane Golgi

25 Now even more fluorescent protein tags..... mCherry etcmCherry etc Prof. 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:

26 Immunostaining Detection of a protein within a cells/tissues using antibodies raised against that proteinDetection of a protein within a cells/tissues using antibodies raised against that protein The cells must be fixedThe cells must be fixed –E.g. aldehydes such as formaldehyde, which cross-links the proteins Cells must also be permeabilised (using low concentration of detergent, e.g. triton X100) to enable antibodies to gain access to the cellsCells must also be permeabilised (using low concentration of detergent, e.g. triton X100) to enable antibodies to gain access to the cells

27 Immunostaining Incubate with an antibody (Ab) specific for the protein of interest, followed by a secondary Ab specific to the primary Ab (i.e. species- specific)Incubate 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 lightThis secondary Ab is usually coupled to a fluorescent tag which fluoresces when exposed to a certain wavelength of light Fluorescent marker red- Rab6 (Golgi) Green- nuclei

28 Confocal Microscopy

29 What is confocal microscopy? Modification to reflected light (fluorescent) microscopy that enables optical sectioning of a sample, eliminating out of focus light Principle patented by Marvin Minsky in 1957, although laser scanning confocal microscopes not developed until 1980s Useful for analysing samples with significant depth e.g. tissue samples conventional confocal

30 Laser scanning confocal microscopy Laser excitation source provides high power point illumination of specific wavelength of light Sample is scanned line by line with the focused laser beam Emitted 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 Confocal Microscope

31 Wide-field microscopy Confocal microscopy Principles of confocal microscopy Solid lines- light in focus Dashed lines- out of focus light source dichroic objective focal plane camera PMT pinhole source

32 Wide-field fluorescent Microscope Confocal Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Camera Excitation Filter Objective Laser Emission Pinhole Excitation Pinhole Photomultiplier Tube (PMT) Emission Filter Black line = focal plane Red line = above focal plane Green 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 detector Compromise between resolution and signal

34 Scanning Galvanometer x y Laser in Point Scanning Laser out- to Microscope The Scan Path of the Laser Beam Start Specimen Frames/Sec # Lines 1512 2256 4128 8 64 16 32

35 Advantages Reduced blurring of the image from light scattering Optical sectioning of thick specimens Detection uses highly sensitive photomultipliers, improving signal to noise ratio Z-axis scanning enabling generation of 3D datasets Magnification can be adjusted electronically Disadvantages Slow scan speeds Limited use in dynamic tracking studies Photobleaching from laser excitation Lasers may damage living cells, limiting use in live cell studies Lower resolution than camera detection Laser scanning confocal microscopy

36 LSM510 META system in the IMS Argon and HeNe lasers giving lines at wavelengths allowing excitation of visible-light fluorophores: Argon458 nm (cyan) Argon476 nm (green) Argon488(green) Argon514 (orange) HeNe543 (red) HeNe633nm(far red) 3 detection channels, therefore 3 fluorophores in a specimen can be captured simultaneously

37 Effect of pinhole size on z resolution WIDE PINHOLE 13 m optical section NARROW PINHOLE 1 m optical section Sample 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 samples Can 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 Single scan Mean of 8 scans Effect of averaging multiple scans Lysotracker red GFPRab18 Human osteoclast adenovirally transduced with WT GFPRab18

40 Studies of colocalisation to subcellular organelles NE10790 Ctrl Rab6 WGA (Golgi)merge

41 Nuclei merge Plekhm1-FLAGGFPRab7 Studies of colocalisation between proteins Transfected cells expressing GFP-Rab 7 and Plekhm-dsRed Yellow colour in merged image indicates colocalisation Transfected cells expressing GFP-LC3 and Plekhm-dsRed:

42 Imaging in 3 dimensions From Source To Detector x VOXEL 3D space PIXEL 2D space z y y x z z Sequential scans through sample:

43 Imaging z-series Samples up to 100 m thick can be analysed (although quenching of fluorescence signal can occur in thick tissue specimens) z (axial) resolution as little as 0.5 m Wavelength of fluorescent light and the numerical aperture of the objective lens determine the limits of this resolution Motorised stage crucial for capturing z-series

44 Z-series of an osteoclast resorbing dentine Scans covers 26 m in the z (axial) dimension Blue- cell membrane Red- F-actin Green- substrate surface

45 Orthogonal views generated from the 3D data set Blue- cell membrane Red- F-actin Green- substrate surface xy xz xy yz depth = 26 m

46 Importance of z-scanning for determining localisation Fluorescent conjugates of WGA- binds to glycosylated proteins, and therefore stains the Golgi and plasma membrane Wheat germ agglutinin tubulin F-actin Human osteoclast on glass zx

47 Animation of resorbing osteoclast

48 Isosurface rendering (red and green fluorescence only) Green- bisphosphonate Red- F-actin Blue- osteoclast membrane (left only) Max intensity projection 3D reconstruction of osteoclast resorbing dentine

49 3D imaging using confocal microscopy © 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories

50 Live cell imaging Useful for analysing fluorescent probes in living organisms in real time e.g. a GFP- tagged expression construct Z series can be collected then resolved post-acquisition using complex algorithms Lasers used in confocal microscopy may damage living organisms Confocal microscopy has some difficulties dealing with weak fluorescence Live cell imaging also limited by scan times DeltaVision Alternative- wide-field microscopy with deconvolution

51 Widefield microscopy with deconvolution Conventional and Confocal microscopy Two different ways of reducing blur in fluorescent images Also structured illumination (e.g. Zeiss Apotome system)

52 Summary Fluorescence 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 reconstructions Advanced applications possible with laser-scanning confocal systems, e.g. analysis of protein:protein interactions using FRET Resolution not as good as electron microscopy! Immuno-EM approaches required to look at protein localisation at the ultrastructural level


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