Confocal Microscopy & Imaging Technology. Yan Wu

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1 Confocal Microscopy & Imaging Technology Yan Wu Dec. 05, 2014

2 Cells under the microscope What we use to see the details of the cell? Light and Electron Microscopy - Bright light / fluorescence microscopy - Confocal Laser Scanning microscopy - Transmission Electron Microscopy (TEM) - Scanning Electron Microscopy (SEM)

3 Light microcopy Light microscopes - Epi-light microscope - Inverted microscopes - Dissection microscopes Fluorescent microscope - incorporated in light microscopes

4 Confocal Laser Scanning Microscopy A fluorescence microscope with a laser as its source of illumination actin dynamic A guard cell development in Arabidopsis

5 Filter cube D.M. The excitation light reflects off the surface of the dichroic mirror into the objective. the fluorescence emission passes through the dichroic to the eyepiece or detection system. Dichroic mirror separates excitation and emission light paths.

6 Optical system of a fluorescence microscope Human eyes emitter v exciter only BLUE light is allowed to pass through Dic Figure 9-13 Molecular Biology of the Cell ( Garland Science 2008)

7 The most used fluorescence dyes DAPI CFP GFP YFP Rhodamine B Cy3 Alexa 568 RFP Cy5

8 Confocal Laser Scanning Microscopy (CLSM) Confocal Laser Scanning Microscopy (CLSM) one of a series of methods to generate slices from microscopic samples by means of optics. The sample stays intact, and the slicing may be repeated many times. The benefit of confocal imaging is a dramatically increased contrast by removal of out-of-focus haze. Z-sequences of optical slices (3D image stacks) are sources for subsequent rendering as anaglyphes, depth-coded maps or 3D movies.

9 Confocal Microscope Inverted microscope Upright microscope Detailed info about FV1000: on/product.asp?product=1008&c=6 Imaging of complex 3D objects is possible with the Confocal Scanning Microscopy

10 光栅 Pinhole PMT 狭缝 Laser types: 1. Argon (gas) 2. LD

11 FV1000 ASW software

12 SIM - SIMultaneous scaner Evolved light stimulation The stimulation/imaging positions and laser wavelengths can be set separately with two independent beams.

13 Confocal Microscopy

14 光学切片 -3D 结构的准确定位 Projection - 3D

15 The confocal microscopes in College of Life Sciences FV 1000 linked with IX81 (inverted microscope) FV 1000 linked with BX61 (upright microscope) Leica TCS SP8 with inverted microscope Analyzing software, MetaMorph (Molecular Devices, USA)

16 Super-resolved fluorescence microscopy Surpassing the limitations of the light microscope The limitation of the light microscope, - ABBE S diffraction limit (0.2 µm)

Stimulated Emission Depletion, developed by Stefan Hell in 2000 Two laser beams utilized, one stimulates fluorescent molecules to glow, another cancels out all

17 Stimulated Emission Depletion, developed by Stefan Hell in 2000 Two laser beams utilized, one stimulates fluorescent molecules to glow, another cancels out all fluorescence except for that in a nanometresized volume. yields an image with a resolution better than Abbe s stipulated limit. Scanning over the sample, nanometre for nanometre

18 The diffraction limit PSF (point spread function): - The 3D intensity distribution of the image of a point object is called PSF The size of the PSF determines the resolution of the microscope: - Two points closer than the full width at half-maximum (FWHM) of the PSF will be difficult to resolve because their images overlap substantially. The diffraction limit of resolution in light microscopy does not affect most imaging at the organ or tissue level. However, when zooming into cells, where a large number of subcellular structures are smaller than the wavelength of the light, it becomes an obstacle for studying these structures in detail. Therefore, it is important to develop techniques that improve the spatial resolution of light microscopy without compromising its noninvasiveness and biomolecular specificity.

19 The Abbe diffraction limit for a microscope Ernst Abbe found in 1873 that light with wavelength λ, traveling in a medium with refractive index n and converging to a spot with angle θ will make a spot radius, d = λ/2n sin θ denominator n sin θ is called the numerical aperture (NA) can reach about in modern optics, hence the Abbe limit is d = λ/2x1.4 = λ/2.8 Considering green light around 500 nm and a NA of 1, the Abbe limit is roughly d = λ/2 = 250 nm (0.25 µm) To increase the resolution, shorter wavelengths can be used such as UV, X-ray microscopes Nowadays, Super-resolved microscopy is invented

20 Stimulated emission depletion microscopy The concept of STED microscopy was first proposed in 1994 and subsequently demonstrated experimentally it uses a second laser (STED laser) to suppress the fluorescence emission from the fluorophores located off the center of the excitation. This suppression is achieved through stimulated emission: When an excited-state fluorophore encounters a photon that matches the energy difference between the excited and the ground state, it can be brought back to the ground state through stimulated emission before spontaneous fluorescence emission occurs. This process effectively depletes excited-state fluorophores capable of fluorescence emission

single-molecule microscopy by Eric Betzig and William Moerner in 2006 Upon the possibility to turn the fluorescence of individual molecules on and off, scientists image the

21 single-molecule microscopy by Eric Betzig and William Moerner in 2006 Upon the possibility to turn the fluorescence of individual molecules on and off, scientists image the same area multiple times, letting just a few interspersed molecules glow each time. Superimposing these images yields a dense super-image resolved at the nanolevel. nanoscopy

22 Principles of super-resolution singlemolecule active control microscopy In conventional fluorescence microscopy, all molecules emit simultaneously, so their diffraction-limited images overlap on the detector (camera) and information about the underlying structure is irretrievably lost. Addition of on-off control, toggling any one singlemolecule emitter between a dark and a fluorescent state. If individual sparse subsets of single molecules that are spatially separated further than the diffraction limit are made to emit, their positions may be extracted in a time-sequential manner by finding the center position of a mathematical fit of the single-molecule images.

23 Exemplary imaging technology - to study gene function in the cell FRET Ratio imaging Bimolecular fluorescence complementation (BiFC)

24 Why FRET Dynamical detection of protein-protein interaction Visualization of protein-protein interaction in live It is important in study of special temporal activities of protein protein interactions Intramolecular association

25 Why Ratio Imaging Localization = activation Ratio imaging eliminates volume and concentration artifacts Overexpression of target not necessary direct excitation of dye (bright signal) flexible wavelengths Endogenous protein activation

Super-resolution Microscopy

Semr oc kwhi t epaperser i es : 1. Introduction Super-resolution Microscopy Fluorescence microscopy has revolutionized the study of biological samples. Ever since the invention of fluorescence microscopy