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

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

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 fluoresce Fluorescent minerals

Simplified Jablonski Diagram Hvex – excitation from absorbed photon S1 S’ – S1 – rapid vibrational energy loss as a result of inter-molecular collisions Energy hvex hvem Radiative emission of a lower energy photon as the species returns to the ground state S0 Lower enrgy photon Is emitted , therefore has longer wavelength. Difference between In 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 process The lower the energy, the longer the wavelength

Fluorescent tubes 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.

Typical emission spectrum from fluorescent light

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

Stokes shift of some widely-used fluorophores Ultra-violet Increasing wavelength visible Infra-red

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 The 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.

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

Arc Lamp Excitation Spectra Xe Lamp   Irradiance at 0.5 m (mW m-2 nm-1)  Hg Lamp     

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

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

Beam path of fluorescent light Typical green emission fluorophore

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

Fluorophores Alexa Fluor 488 488 522 Fluorescein Fluorescein 488 525 488 522 Fluorescein Compare photostability and quantum yield? Fluorescein 488 525 Probe Excitation Emission

pH Sensitive Indicators: Probes for Ions (Ca2+): INDO-1 Ex350 Em405/480 QUIN-2 Ex350 Em490 Fluo-3 Ex488 Em525 Fura -2 Ex330/360 Em510 pH Sensitive Indicators: Probe Excitation Emission SNARF-1 488 575 BCECF 488 525/620 440/488 525 C27H19NO6 C27H20O11

Specific Organelle Probes Probe Site Excitation Emission BODIPY Golgi 505 511 NBD Golgi 488 525 DPH Lipid 350 420 TMA-DPH Lipid 350 420 Rhodamine 123 Mitochondria 488 525 DiO Lipid 488 500 diI-Cn-(5) Lipid 550 565 diO-Cn-(3) Lipid 488 500 BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH – diphenylhexatriene TMA - trimethylammonium

Nuclear probes (stain DNA) excitation emission Hoechst 33342 (uv) 346 460 DAPI (uv) 359 461 Sytox green 498 592 TOTO-1 514 533 Sytox orange 547 570 PI (uv/vis) 536 620 TO-PRO-3 642 657 Work in live cells

Fluorescent probes for cellular structures TRITC Phalloidin (F-actin) Fluorescent Phalloidin conjugates used to visualize the actin cytoskeleton Phalloidin is a fungal toxin (from Amanita phalloides) that binds to polymerised 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 Amanita phalloides also known as the death cap!

Probing acidic vesicles Lysotracker – weakly basic amine that selectively accumulates in compartments of low pH (e.g. endosomes/lysosomes) Ctrl Lysotracker-red +50nM bafilomycin (inhibitor of V-ATPases) Other probes, such as lysosensor, emit wavelengths that is dependent on the pH

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)

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)

Fluorescent protein tags Green fluorescent protein (GFP) isolated from jellyfish Aequoria victoria Excitation 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 sequence Transfect 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) GFP GFP-Rac nuclei GFP-Rab1a plasma membrane Golgi GFP rarely affects the normal function of the protein that is tagged

Now even more fluorescent protein tags..... mCherry 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: GFP rarely affects the normal function of the protein that is tagged

Immunostaining Detection of a protein within a cells/tissues using antibodies raised against that protein The 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 cells Advantage- enables localisation to be determined Disadvantage- many antibodies don’t work. Non- quantitative

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) This secondary Ab is usually coupled to a fluorescent tag which fluoresces when exposed to a certain wavelength of light red- Rab6 (Golgi) Green- nuclei Fluorescent marker Advantage- enables localisation to be determined Disadvantage- many antibodies don’t work. Non- quantitative

Confocal Microscopy

What is confocal microscopy? conventional 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 confocal http://www.bio.brandeis.edu/marderlab/microscopy5.html

Laser scanning confocal microscopy Microscope 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

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

Confocal Microscope Wide-field fluorescent Microscope Arc Lamp Laser Excitation Pinhole Excitation Diaphragm Excitation Filter Photomultiplier Tube (PMT) Camera Objective Objective Emission Filter Emission Filter Emission Pinhole Black line = focal plane Red line = above focal plane Green line = below focal plane

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

Scanning Galvanometer The Scan Path of the Laser Beam Scanning Galvanometer Start x y Laser in Point Scanning Laser out- to Microscope Specimen Frames/Sec # Lines 1 512 2 256 4 128 8 64 16 32

Laser scanning confocal microscopy Advantages Disadvantages 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 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

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

Effect of pinhole size on z resolution WIDE PINHOLE 13mm optical section NARROW PINHOLE 1mm optical section Sample of whole mouse retina; cells expressing GFP

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

Effect of averaging multiple scans Single scan Mean of 8 scans Human osteoclast adenovirally transduced with WT GFPRab18 Lysotracker red GFPRab18

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

Studies of colocalisation between proteins Nuclei GFPRab7 Plekhm1-FLAG merge Transfected cells expressing GFP-LC3 and Plekhm-dsRed: Transfected cells expressing GFP-Rab 7 and Plekhm-dsRed Yellow colour in merged image indicates colocalisation

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

Imaging z-series Samples up to 100mm thick can be analysed (although quenching of fluorescence signal can occur in thick tissue specimens) z (axial) resolution as little as 0.5mm 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

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

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

Importance of z-scanning for determining localisation Wheat germ agglutinin tubulin F-actin Human osteoclast on glass Fluorescent conjugates of WGA- binds to glycosylated proteins, and therefore stains the Golgi and plasma membrane zx Amanita phalloides also known as the death cap!

Animation of resorbing osteoclast Mutations are in ClCN7 gene Parents each have different homozygous mutations. Mother’s mutation indicates that the protein will retain activity Not sure about father’s. This could mean that CLCN-7 is not involved at all?! Mutations must interact in some way?

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

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

Alternative- wide-field microscopy with deconvolution Live cell imaging 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 Alternative- wide-field microscopy with deconvolution 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 DeltaVision

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

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