Fluorescence Light Microscopy for Cell Biology Why use light microscopy? Traditional questions that light microscopy has addressed: Structure within a cell Locations of specific molecules within a cell Recent advances now allow these questions to be asked in live cells! 1
Why use light microscopy? Non-traditional biophysical questions that light microscopy can now address: Determine diffusion constants and binding affinities of a molecule at a specific site within a cell. Determine whether two molecules are interacting at a specific time and place within a cell. Much more information than just structure! Outline of topics Microscopes: Conventional fluorescence microscopy Confocal microscopy Deconvolution microscopy Two-photon microscopy Imaging techniques: 3D time-lapse FRAP FRET 2
Web sites for more information and tutorials Olympus microscopy resource center http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html Molecular Expressions Optical Microscopy Primer http://microscopy.fsu.edu/primer/index.html Molecular Probes www.molecularprobes.com Basic operating principles of a light microscope eyepiece objective lens specimen focus condenser 3
Compound microscope design yields magnification and resolution Magnification 5x, 10x, 20x, 25x, 40x, 60x, 100x typically available for microscope objectives 4
Resolution is determined in part by the imaging medium AIR OIL n = refractive index of medium: n=1.0 air; n=1.3 water; n=1.5 oil. (for glass n=1.5) The more light collected, the more complete is the image, and so resolution improves. Resolution quantified by numerical aperture (NA) sinnan 5
What s the resolution limit of light microscopy? ] d Rayleigh s criterion: N nn d = /(2)(NA) d/ n d500/.5 700. 7nmmμ Beware of empty magnification High NA Low NA 6
Inverted Research Microscope new detector original detector old detector Computer controlled Computer Microscopist Detector Microscope (Who needs a microscopist?) 7
Fluorescence Microscopy Predominant mode of light microscopy today Provides molecular specificity Yields high signal to background What is fluorescence? Absorption of a photon with emission of longer wavelength photon 8
Fluorescence is absorption of a higher energy photon with emission of a lower energy photon higher energy (shorter wavelengths) lower energy (longer wavelengths) Typical spectral curves for a fluorescent molecule used in microscopy How do we specifically excite the molecule, and then specifically detect its fluorescence? 9
Specificity provided by filters Filters plus a dichroic mirror Fluorescence Microscopy Emission filter Exciter filter Dichroic mirror Objective Specimen Specimen Filters plus a dichroic mirror 10
Components combined in a small filter cube Fluorescence Inverted Research Microscope excitation filters, dichroic 11
What can we see by fluorescence microscopy? Probes for specific biomolecules Probes for genes Probes for ions Probes for specific biomolecules Fluorescent antibodies (immunofluorescence) Fluorescent biomarkers conjugated fluorescent dye antibody or other binding molecule cellular molecule 12
Examples of immunofluorescence Chromosome axis with topo II Microtubules in an endothelial cell Cell walls in plant cells Histone proteins (green), EGF receptor (red) Examples of fluorescent biomarkers Metaphase chromosomes Nucleic acid (yellow) Actin filaments (tubeworm) Apoptotic cell: Lectin (green), nucleic acid (red) 13
Fluorescent probes for cellular organelles Endoplasmic reticulum Golgi Mitochondria Lysosomes (red), Nucleus (blue) Fluorescent probes for small signaling molecules calcium (pollen tube) phosphatidyl inositol (fibroblasts) camp (fibroblasts) 14
Conclusion: An assortment of fluorescent probes enables detection of a variety of cellular structures and organelles. A more limited assortment of fluorescent probes permits detection of small signaling molecules that regulate cell processes. But cells are 3D!!! How do we get a 3D image of a cell? focus Easy, change the focus. 15
This is called optical sectioning microscopy Acquire a series of focal plane images that span the depth of the cell or object of interest. Example: collecting a 3D image of a tiny fluorescent bead (~0.2 μm) z +4 μm +3 μm +2 μm +1 μm +0 μm -1 μm -2 μm -3 μm -4 μm focal planes 16
3D image stack from the small fluorescent bead focal planes (xy images) The 3D bead image viewed from the side xz section 17
But why didn t we get - xy sections xz section? Because the lens does not collect all of the light emitted by the specimen Therefore the image formed is imperfect. 18
So in 3D the image of a point source always looks like this: out-of-focus light Out-of-focus light creates blur in a 3D image Image Formation Selected focal plane image 19
Image of many real point sources in focus 1 μm out-of-focus A real specimen is composed of many more such point sources 3D microscopy methods to reduce out-of-focus light Confocal microscopy Two-photon microscopy Deconvolution microscopy 20
Conventional fluorescence microscopy excites the whole specimen and collects emitted light from the whole specimen excitation light 3D specimen Confocal microscopy excites the whole specimen but collects emitted light primarily from the focal point 3D specimen 21
A typical research confocal microscope The result is an image with reduced haze, improved contrast and better resolution Confocal Conventional 22
3D images can also be generated Dim specimens are harder to image by confocal because the pinhole rejects a lot of light In practice, the pinhole is often made larger to generate partially confocal images 23
Partially confocal images are a compromise conventional fully confocal (small pinhole) partially confocal (medium pinhole) partially confocal (large pinhole) Conclusion Confocal microscopy can generate high contrast 3D images of specimens. It has been the predominant instrument for high resolution light microscopy in the past 10 years. Dimmer specimens are more challenging. 24
Cells are not only 3D, they are also alive. How do we image changes over time? Computer Detector Shutter Microscope Microscopist Easy, computer-controlled time-lapse imaging But the cells need to be happy too. Sophistication of the chamber depends on the sensitivity of the cells and the duration of the imaging experiment. ph and temperature are key variables. 25
Sometimes the cells are very happy (But our budget is not) Even with perfect incubation conditions, repeated light exposure causes problems heating (?) photobleaching dimmer signal free radicals 26
Photobleaching is always a problem 1 2 3 4 5 6 Bleaching rate depends on the dye Fluor, Cy3 Alexa Dyes have been optimized for bleaching and for the laser lines available on typical confocal microscopes 27
How are living cells labeled with dyes? cell permeable dyes microinjection of dyes endogenous dyes Cell permeant dyes Fura2 Cleaved by non-specific esterases in the cell to become impermeant, and locked in the cell. 28
Calcium concentration can then be measured in space and time Fertilization-induced calcium wave in a starfish oocyte. Confocal images every 5 sec Microinjection microinjection needle patch-clamped neural cell 29
Microinjection is difficult and time-consuming Only a limited number of cells can be injected per experiment (~100) Endogenous probes are now the most convenient and most widely used -barrel chromophore 27 kd Crystal structure of GFP green fluorescent protein 30
Proteins of interest can be tagged with GFP and observed in live cells Gene for Your Protein GFP Transform, transfect cells Translocation of the GFP-tagged glucocorticoid receptor from cytoplasm to nucleus Surprising features often discovered by time-lapse 31
Double or triple labeling of live cells is feasible with GFP variants A spectrum of markers: blue, cyan, green, yellow, red Time-lapse imaging: a case study How is chromatin packaged at the Mbp level in the interphase nucleus? from Molecular Biology of the Cell (1998) Bray et al. And how does this packing change when a region becomes activated? 32
Live-cell imaging done in a specially developed cell line Cells contain ~200 tandemly repeated copies of {MMTV - ras - BPV} and are stably transfected with GFP-GR under tetracycline regulation. The tandem array is visualized as a bright structure above the background of GFP-GR in the nucleus GFP-GR RNA FISH OVERLAY These bright structures are the tandem array because they produce the predicted transcript. 33
How does this structure change over time as transcription is induced? Problems Method: Time-Lapse Imaging Very rapid bleaching Cells become sick after only 10-20 images Cells drift out of focus Changes occur on slow time scale (~4 h) but need ~100 movies Solutions 2D time lapse, 1 image every 15 min Non-confocal images Microscopist manually corrects focus during experiment ~5 data sets collected in parallel The microscope set up Tools: Chambered cover slip, stage heater, conventional fluorescence microscope with xy controlled stage 34
Results Unfolding and refolding suggests fiber packing at different densities Conclusion GFP and its variants have made it possible to easily tag proteins with fluorescent markers, and then observe the behavior of specific cellular structures over time. 35