Visualizing Cells - L2 Spring 2022 Lecture Notes PDF

Visualizing Cells Alberts MBotC Chapter 9  The Big Picture Cell biologists need techniques for visualizing individual cells. The small size and transparency of cells make this challenging. Light microscopy can image large-scale cellular structures, but resolution...

Visualizing Cells Alberts MBotC Chapter 9  The Big Picture Cell biologists need techniques for visualizing individual cells. The small size and transparency of cells make this challenging. Light microscopy can image large-scale cellular structures, but resolution is limited. Chemical stains and fluorescent molecules provide contrast and sensitivity. Electron microscopy allows much higher resolution than light microscopy, but requires special preservation and staining techniques. Flow cytometry uses fluorescent labels to measure levels of specific biomolecules and ions, and to sort cells based on expression levels.  Have you used response devices/clickers before? ? A. First time ever ? B. Second class ever C. I'm a pro (3 or more classes) ? ?  Have you registered with Top Hat? ? A. Yes ? B. No C. I don't know ? ? Figure 9-1 Molecular Biology of the Cell (© Garland Science 2008) Figure 9-2 Molecular Biology of the Cell (© Garland Science 2008)  Visualizing cells Many techniques are used to study cells, but visualizing cells and subcellular compartments is central to cell biology. Cellular structures (and most animal cells) are too small to see with the naked eye, and must be magnified for study. High quality light microscopes developed in 19th century allowed visualization of cells, and allowed Schleiden and Schwann to propose the Cell Theory. Developments over the past 200 years have increased its power and sensitivity of light microscopy. Figure 9-3a Molecular Biology of the Cell (© Garland Science 2008)  Light Microscopy Drawbacks Light microscopy is limited in resolution (the ability to distinguish 2 objects that are close to each other). The practical limit of resolution in light microscopy ~0.2 µm. Typical animal cell is 10-20 µm in diameter. Mitochondria, (~0.5 µm) are generally the smallest organelles that can be clearly seen by light microscopy. Important to distinguish resolution from magnification (we can magnify an image as much as we want, but it may be blurry) or detection (an item smaller than 0.2 µm can be detected if it emits light).  What sets the limit of resolution for microscopy? ? A. The wavelength of the light ? B. The size of the lens C. Both D. Neither ? E. I have no idea – that is physics, not biology ?  How Do You See a Tiny Bag of (Mostly) Water? Animal cells are essentially colorless and translucent, and are nearly invisible under light microscopy. How to get past this problem? Phase-contrast and differential-interference-contrast (DIC, aka Nomarski optics) microscopy use the change in phase of light waves as they pass through a cell: Visualization of live, unmanipulated cells, but with limited detail. Figure 9-7b Molecular Biology of the Cell (© Garland Science 2008) Figure 9-8 Molecular Biology of the Cell (© Garland Science 2008)  How Do You See a Tiny Bag of (Mostly) Water? Staining with chemical dyes can reveal details of cells, but generally requires fixation, which kills and preserves cells. Tissue samples are too large to be visualized by most microscopy techniques, and must be cut into thin sections.  Kidney tubule cells stained with hematoxylin and eosin, a commonly used dye combination Figure 9-11a Molecular Biology of the Cell (© Garland Science 2008)  Fluorscence Microscopy Chemical dyes produce color by absorbing specific wavelengths of light. Fluorescent molecules absorb light at one wavelength and emit light of a longer wavelength. Using specific filters, a cell can be illuminated with light of one color, and visualized at the fluorescence wavelength. Allows detection of very small numbers of fluorescent molecules, since they are viewed against a dark background. Fluorescent stains can be introduced by various methods: chemical dyes; fluorescent molecules attached to antibodies; or intrinsically fluorescent proteins (green fluorescent protein [GFP] and its variants) expressed by the cell itself. Figure 9-13 Molecular Biology of the Cell (© Garland Science 2008) Measuring changes in protein location over time B resting 2 min 5 min 30 min SYTO 13 (nucleus) NFAT2 merge Srinivasan et al. (2007), Journal of Immunology  GFP expressed in specific neurons in fruit fly embryo highlights neural connections in live animal Figure 9-25 Molecular Biology of the Cell (© Garland Science 2008)  Confocal Microscopy Fluorescence microscopy is limited by fluorescence coming from out-of-focus parts of cells. Confocal microscopy uses a scanning laser and pinhole apertures to limit detection to the focal plane – creation of an optical section with much better resolution. By taking images from various focal planes (“z-sections”), a 3-D image of the sample can be reconstructed. Figure 9-20 Molecular Biology of the Cell (© Garland Science 2008)  Conventional Confocal Figure 9-21 Molecular Biology of the Cell (© Garland Science 2008)  3D Optical “sections” reconstruction Pollen grain (endogenous fluorescence) Figure 9-22 Molecular Biology of the Cell (© Garland Science 2008)  Some other Cool Fluorescence Microscopy Techniques FRET (fluorscence resonance energy transfer) uses two fluorescent proteins where the excitation energy of the second matches the emission of the first. If (and only if) the 2 are very close together, exciting protein 1 will allow protein 2 to fluoresce. 2-photon microscopy uses 2 separate long-wave photons of light, instead of one short-wave photon, to excite the fluorophore. This allows much deeper penetration into a sample without sectioning (aka “vital microscopy”)  Protein A (Sla1p) Protein B (Abp1p) FRET signal Figure 9-28 Molecular Biology of the Cell (© Garland Science 2008)  Electron Microscopy A focused beam of electrons replaces light. Resolution more than 200x that of light microscopy can be achieved. Vacuum is required, so special techniques required. Cells must be fixed (glutaraldehyde, osmium tetroxide), and often desiccated, and then sliced into ultra-thin sections. Cells are mostly transparent to electrons, so electron-dense materials are used to stain cells. Can also use gold-tagged antibodies to mark specific proteins/structures. Scanning electron microscopy (SEM) images the outside surface (staining, but no sectioning); transmission electron microscopy (TEM) images internal structures.  Why does an electron beam give more resolution than light? ? ? A. The wavelength of an electron is much smaller than the wavelength of a photon B. Electrons are matter, not energy, so they are intrinsically different from photons C. Isn’t this physics again? I thought this was a biology course! ? ? Figure 9-42 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)  TEM reveals internal cellular structures with high resolution Figure 9-45 Molecular Biology of the Cell (© Garland Science 2008)  SEM generates 3D image of external surfaces Figure 9-50 Molecular Biology of the Cell (© Garland Science 2008)  3D Image of Mitochondrion Reconstructed From a Series of Sections Figure 9-47 Molecular Biology of the Cell (© Garland Science 2008) Antibodies conjugated with gold particles allow localization of specific molecules within cell by TEM  Flow Cytometry Live cells are analyzed in real-time in an aqueous stream as they pass through a laser Computer can collect fluorescence data (color, intensity), as well as laser scatter (reflecting size, shape, and internal architecture) Can be coupled with a cell sorter to allow cells to be separated based on fluorescence and scatter properties Basic principles behind flow cytometry and cell sorting  Cell number Fluorescence intensity Typical data from flow cytometry  Cell cycle analysis using DNA-staining dye(propidium iodide) in flow cytometry  The Big Picture Cell biologists need techniques for visualizing individual cells. The small size and transparency of cells make this challenging. Light microscopy can image large-scale cellular structures, but resolution is limited. Chemical stains and fluorescent molecules provide contrast and sensitivity. Electron microscopy allows much higher resolution than light microscopy, but requires special preservation and staining techniques. Flow cytometry uses fluorescent labels to measure levels of specific biomolecules and ions, and to sort cells based on expression levels.