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MCB5068 Intro Goals For MCB 5068 1. Obtain a solid foundation of knowledge in cell biology 2. Obtain a working knowledge of available techniques. 3. Be able to critically read and evaluate the scientific literature. 4. Be able to define and investigate a biological problem. Molecular Cell Biology 5068 To Do: Visit website: www.mcb5068.wustl.edu Sign up for course. Check out Self Assessment homework under Mercer- Introduction Visit Discussion Sections: Read “Official” Instructions TA’s: 1st Session: Nana Owusu-Boaitey [email protected] 2nd Session: Shankar Parajuli [email protected] 3rd Session: Jeff Kremer [email protected] What is Cell Biology? biochemistry genetics cytology physiology Molecular Cell Biology CELL BIOLOGY/MICROSCOPE Microscope first built in 1595 by Hans and Zacharias Jensen in Holland Zacharias Jensen CELL BIOLOGY/MICROSCOPE Robert Hooke accomplished in physics, astronomy, chemistry, biology, geology, and architecture. Invented universal joint, iris diaphragm, anchor escapement & balance spring, devised equation describing elasticity (“Hooke’s Law”). In 1665 publishes Micrographia CELL BIOLOGY/MICROSCOPE Robert Hooke . “I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey- comb, but that the pores of it were not regular. these pores, or cells, . were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this. .” CELL BIOLOGY/MICROSCOPE Antony van Leeuwenhoek (1632-1723) CELL BIOLOGY/MICROSCOPE Antony van Leeuwenhoek (1632-1723) a tradesman of Delft, Holland, in 1673, with no formal training, makes some of the most important discoveries in biology. He discovered bacteria, free- Red blood cells living and parasitic microscopic protists, sperm cells, blood cells and more. All of this from a very simple device that could magnify up to 300X. Spiral bacteria THE CELL THEORY Matthias Jakob Schleiden 1804-1881 Theodor Schwann 1810-1882 Schleiden Schwann THE CELL THEORY First coined by Theodore Schwann in 1839, and formed from the ideas of Matthias Schleiden, Schwann, and Rudolf Virchow. The theory proposes that: 1. Anything that is alive is made up of cells. 2. The chemical reactions that occur in organisms occur in cells. 3. All cells come from preexisting cells. SPONTANEOUS GENERATION From ancient time, through the Middle Ages, and until the late nineteenth century, it was generally accepted that some life forms arose spontaneously from non-living organic matter. Jan Baptista van Helmont (1577-1644) Flemish physican, chemist and physiologist. Invented the word “gas”. Recipe for mice: Place a dirty shirt or some rags in an open pot or barrel containing a few grains of wheat or some wheat bran, and in 21 days, mice will appear SPONTANEOUS GENERATION (1668-1859) Although the belief in the spontaneous generation of large organisms wanes after 1668, the invention of the microscope serves to enhance the belief in spontaneous generation. Microscopy revealed a whole new class of organisms (animalcules) that appeared to arise spontaneously. It was quickly learned that you needed only to place hay in water and wait a few days before examining your new creations under the microscope. This belief persisted for nearly two centuries. SPONTANEOUS GENERATION (1668-1859) In 1859, after years of debate The French Academy of Sciences sponsors a contest for the best experiment either proving or disproving spontaneous generation. The French chemist, Louis Pasteur (1822-1895) uses a variation of the methods of Needham and Spallanzani. He boils meat broth in a flask, heats the neck of the flask in a flame until it became pliable, and bent it into the shape of an S. Air could enter the flask, but airborne microorganisms could not - they would settle by gravity in the neck. As Pasteur had expected, no microorganisms grew. When Pasteur tilted the flask so that the broth reached the lowest point in the neck, where any airborne particles would have settled, the broth rapidly became cloudy with life. Pasteur had both refuted the theory of spontaneous generation and convincingly demonstrated that microorganisms are everywhere - even in the air. CELL BIOLOGY/MICROSCOPE Camillo Golgi (1843-1926) In 1898, Golgi develops a staining technique (silver nitrate) that allows the identification of an "internal reticular apparatus" that now bears his name: the "Golgi complex” or the “Golgi”. CELL BIOLOGY/MICROSCOPE By the late 1800’s to the early 1900’s the limits to the light microscope had been reached. Resolving ability roughly 1/2 λ of light used: ≈ 0.2 µm In 1930 A.A. Lebedeff designs and builds the first interference microscope. In 1932 Frits Zernike (1888-1966) invents the phase-contrast microscope. It is first brought to market in 1941 in Germany. Both microscopes aid in elucidating the details in unstained living cells. CELL BIOLOGY/MICROSCOPE In 1932 Zernike traveling from Amsterdam, visits the Zeiss factory in Germany to present his method of phase contrast microscopy. After reviewing Zernike's method an older scientist said: "If this really had any practical value, then we would have invented it a long time ago." In 1953 Zernike was awarded the Nobel Prize for his phase contrast work. Light behaves as a Wave Wavelength sets limits on what one can see Lower limits on spatial resolution are defined by the Rayleigh Criterion Resolution = 0.61 x wavelength of light NA (numerical aperture) NA = nsinθ n = refracve index of the medium θ = semi-angle of an objec6ve lens The effect of NA on the image of a point. θ θ θ The need for separa6on to allow resoluon Contrast in the Image is Necessary: Types of Optical Microscopy Generate Contrast in Different Ways •Bright field - a conventional light microscope •DIC (Differential Interference Contrast - Nomarski) •Phase contrast •Fluorescence •Polarization •Dark field Bright-field Optics: Light Passing Straight Through the Sample •Most living cells are optically clear, so stains are essential to get bright field contrast •Preserving cell structure during staining and subsequent observation is essential, so cells must be treated with “fixatives” that make them stable •Fixing and staining is an art Generating Contrast Staining Coefficients of absorption among different materials differ by >10,000, so contrast can be big Without staining Everything is bright Most biological macromolecules do not absorb visible light Contrast depends on small differences between big numbers Need an optical trick Mammalian Cell: Bright-field and Phase-contrast Optics Principles of bright field and phase contrast optics Differential Interference Contrast (DIC) • Optical trick to visualize the interference between two parts of a light beam that pass through adjacent regions of the specimen • Small amounts of contrast can be expanded electronically • Lots of light: Video camera with low brightness & high gain Brightfield vs DIC Fluorescence Microscopy •Absorption of high-energy (low wavelength) photon •Loss of electronic energy (vibration) •Emission of lower-energy (higher wavelength) photon Design of a Fluorescence Microscope Green Fluorescent Protein - Considerations • Color - Not just green • Brightness • Size/Location 26.9 kDa • Time for folding • Time to bleaching GFP-Cadherin in cultured epithelial cells Immunofluorescence • Primary Abs recognize the antigen (Ag) • Secondary Abs recognize the primary Ab • Secondary Abs are labeled Immunofluorescence Example •Ab to tubulin •Ab to kinetochore proteins •DNA stain (DAPI) Biological microscopy problem: Cells are 3D objects, and pictures are 2D images. • Single cells are thicker than the wavelength of visible light, so they must be visualized with many “optical sections” • In an image of one section, one must remove light from other sections • Achieving a narrow “depth-of-field” • A “confocal light microscope” Laser-Scanning Confocal Light Microscopy •Laser thru pinhole •Illuminates sample with tiny spot of light •Scan the spot over the sample •Pinhole in front of detector: Receive only light emitted from the spot Light from points that are in focus versus out of focus Spinning-disk confocal microscopy: Higher speed and sensitivity Example: Confocal imaging lessens blur from out-of-focus light Optically Sectioning a Thick Sample: Pollen Grain Multiple optical sections assembled to form a 3D image Fluorescence can Measure Concentration of Ca2+ Ions in Cells: Sea Urchin Egg Fertilization Phase Contrast Fluorescence Total Internal Reflection Fluorescence (TIRF) Microscopy The penetration depth of the field typically ranges from 60 to 100 nm www.leica-microsystems.com Total Internal Reflection Fluorescence (TIRF) Microscopy www.leica-microsystems.com Summary • Light microscopy provides sufficient resolution to observe events that occur inside cells • Since light passes though water, it can be used to look at live as well as fixed material • Phase contrast and DIC optics: Good contrast • Fluorescence optics: Defined molecules can be localized within cells • “Vital” fluorescent stains: Watch particular molecular species in live cells CELL BIOLOGY/MICROSCOPE Louis de Broglie (1892-1987) In 1924 at the Faculty of Sciences at Paris University he delivers a thesis Recherches sur la Théorie des Quanta (Researches on the quantum theory), which earned him his doctorate. This thesis contained a series of important findings that he had obtained in the course of about two years. This research culminated in the de Broglie hypothesis stating that any moving particle or object had an associated
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