Visualizing Cells Molecular Biology of the Cell - Chapter 9 Resolution, Detection Magnification Interaction of Light with matter: Absorbtion, Refraction, Reflection, Fluorescence Light Microscopy Absorbtion of Light (dyes, stains) Refraction methods Fluorescence Microscopy Green Fluorescent Protein Confocal Microscopy Total internal reflection fluorescence Electron microscopy Staining methods Scanning Electron Microscopy
Size > 2 meters (2x10 0 meters) Typical cell is 20 micrometers (um) = 20x10-6 meters 1.5 centimeter (1.5x10-2 meters) Large organelles, 2 micrometers (um) = 2x10-6 meters Average human hair (diameter).1 mm = 100 micrometers (um) = 100x10-6 meters Macromolecular complexes, 0.2 micrometers (um) = 2x10-7 meters Figure 9-1
Resolution vs Magnification
Resolution Resolution is determined by the wavelength of light and the numerical aperture of the objective lens. Resolution IMPROVES if D gets SMALLER Resolution is directly proportional to the lens numerical aperture Resolution is inversely proportional to the wavelength used for imaging Visible light wavelength are between about 360 and 780 nm The resolution limit of the light microscope (using near UV illumination) is about.2µm (200 nm). (A red blood cell is 7µm across)
Long Wavelength Light Long wavelength light is less damaging and less easily scattered than short wavelength light and can illuminate deep structures Cy5 dyes emit nearinfrared light
Resolution vs Detection Objects smaller than the resolution limit of a microscope can be detected MBoC supplemental video Microtubules24 nm diameter (DIC image) Actin (8nm) on myosin lawn (Fluorescent actin)
Objects smaller than the resolution limit can be detected but are not resolved
What is magnification good for? Imaging devices have discrete detector elements Rods and Cones in Eye Pixels in Digital Camera
Magnification The minimal adequate magnification is one that allows the smallest object you want to resolve to fall on 3 discrete elements of the imaging device.
Properties of Interaction Four properties of the interaction between light and matter influence the design of microscopes used to produce contrast.
Absorption methods: Stained tissue sections Stains are compounds that absorb light or electrons. Black stain absorbs all colors of light Colored stains absorb some colors of light, others to pass Kidney Collecting Duct Fig 9-11 Adipose Tissue Purkinje neuron
Different components of a cell can be selectively stained Chemical compounds of some stains bind preferentially to specific cellular components Hematoxylin (blue) stains nucleic acids Osmium stains lipids in neuron sheath Hematoxylin & eosin (proteins stain pink) Periodic acid Shiff s stains carbohydrates
Antibodies are used to detect specific cell components
Antibodies can be linked to enzymes that produce colored products Craniofacial neurons in E10.5 mouse (horseradish peroxidase labeling), Sahay et al., J. Neurosci 2003. 23:6671-80 Delta I gene expression in developing somites (beta-gal staining)
Refraction Techniques in Light Microscopy Very little incident light is absorbed, reflected or refracted by a living biological specimen Brightfield Imaging: refracted light from the specimen is poorly detected
Methods for imaging refracted light I. Darkfield Imaging
Living Cells Living cells are seen clearly using phase contrast microscopy to image the difference between refracted and non-refracted light. Phase Contrast Imaging
Features of living cells can be seen clearly using phase contrast microscopy. Shelden
Fluorescence microscopy: Fluorescent molecules absorb high energy light and then emit less energetic, longer wavelength light. The shift in wavelength between absorbed and emitted light is called the Stokes shift. Excitation spectrum maxima Emission spectrum Rhodamine anti-map in cultured neurons
Fluorescence Microscopes Fluorescence microscopes use excitation, emission and dichroic filters to take advantage of the Stokes shift Fig 9-11
Fluorescent Stains Chemical compounds of some fluorescent stains bind preferentially to specific cellular components Fluorescent phalloidin (red) stains actin DAPI (blue) stains nucleic acids Shelden
Fluorophores Antibody linked fluorophores detect specific cellular antigens Anti-tubulin (green) Anti-neurofilaments (green)
FISH Fluorescence in situ hybridization (FISH) uses synthetic fluorescent RNA probes to detect compatible mrna in cells and tissue mrna Fluorescent RNA probe Figure 9-12
Light emitting dyes reveal changes in ion concentrations Figure 9.32 Calcium changes during fertilization visualized with aequorin Calcium signaling in renal epithelial cells (fluo-5 indicator) Shelden
Fluorescent Protiens Green fluorescent proteins can be expressed in living organisms Aequorea victoria GFP
Gene Promoters Cell-type specific gene promoters can be used to express GFP in specific cells or tissues Neuron specific promoter GFP Coding Sequence Figure 9-25 (transgenic fruit fly larva)
Fluorescent Fusion Protiens Green fluorescent fusion proteins can be used to label proteins in living cells Recombinant DNA Relocation of GFP-tagged proteins in muscle cells
Fluorescent Probes Dynamic studies of fluorescent probes can be conducted using Fluorescence Recovery After Photobleaching (FRAP) Fig 9-31
Fluorescent Probes Dynamic studies of fluorescent probes can be conducted using photo-activatable probes Fig 9-30
Electron Microscope The electron microscope uses electrons to resolve fine structure of the cell The wavelength of an electron can be.004 nanometers, so the theoretical limit of resolution of an electron microscope is 1/20 angstoms, or 1/20 the diameter of a hydrogen atom. Electrons pass through the specimen in TEM Fig 9-42
EM Stains Stains used for electron microscopy (EM) are very dense (metals) so they absorb electrons EM stains are generally soluble, reactive forms of metals such as lead, uranium, gold, silver and tungsten Water Osmium tetroxide Uranyl acetate Shelden
Gaseous metals can be directionally applied (shadowing) Sputter coatter Fig 9-52
Metal Shadowing Metal shadowing can be applies to surface structures or interior structures using freeze fracture and freeze etching methods Cryoelectron microscopy of freezefractured intestinal microvilli Cryoelectron microscopy of freezeetched skeletal muscle fibers Specimens are frozen, then split using a microtome and exposed surfaces prepared and imaged.
EM Negative Staining EM Negative staining allows isolated macromolecules to be seen Actin filaments Macromolecules on substrate Stain in excess Fig 9-54 Bacteriophage virus Gene Meyer University of S. Carolina Excess stain removed and dried Shelden
Computational Methods Computational methods can produce three dimensional structural inormation from multiple views of an object Figure 9-55 reconstruction of the Hepatitis B virus
Antibodies Antibodies can be attached to metal particles to detect specific cellular components Immuno-gold labeling of microtubules in an interphase cells Immuno-gold labeling of centromere proteins. Fig 9-46
Pulse Chase Radio-Labeling Dynamic studies of molecules can be conduced at the EM level by pulse chase radio-labeling. Fig 9-38
Reflection techniques in microscopy: Scanning EM: Fig 9-49 Fig 9-50