Cell and Molecular Biology
               structure, function, & the molecules of cells

  Methodologies, Techniques, & Procedures
   for visualizing Cell Structure in CMB


History of Microscopy,    Optical Microscopy,    Microscopy on Web,
Nobel links to microscopy,   Wikipedia,   Virtual Library-microscopy,
Zeiss, Inc History of EM (CZ) the Transmission-EM,   EM-stock pics,
EM-Wikipedia TEM-Wikipedia,
Multimedia Production Harvard U.

    Cell Biology Dictionaries                 A Table of Glossaries        Glossary of Techniques
    National Human Genome Glossary           General Procedures & Protocols - Cell Bio
    General Procedures & Protocols - Molecular Biology                Mallery's CMB Resources

       mcb6e-pages 371-379 & 380-404*



Early Methodology in CMB  - 1910 to 2010
 Equipment advances especially  of last 50 years are the epitome of modern scientific age

Light Microscopy - produces magnified images of small objects with compound lens
                                         objective lens - next to object (
100x)  and ocular lens (10x) = 1,000x magnification
        types of light microscopy*       (technically complicated  &
  mcb fig 9.10*)
          1876  Abbe optimizes microscope designs (lens & condensers)
                  1886  Zeiss - lens RESOLUTION near limits of light   (0.2 um*)
        specimen preparation
                  1900's -
killing, fixing*, embedding & sectioning : microtome*    (1 to 10 um thin tissue sections)
                                                      selective staining :  stains attach to specific molecules   (picture*)
        tracing molecular precursors:
1924 Lacassagne    methods & preparation*   images*    tracking*
fluorescence microscopy* - 1941  Coons         mcb 9.opener*
                     immunofluorescence microscopy of rat intestine*   &   Green Fluorescent Protein
                     confocal fluorescence microscopy* - 1953  Minsky
-  provides a sharper image - ecb-confocal

                  1980  AlexrodTIRF (total internal reflection fluorescence)
eliminates background light (pics)
              1998  Live Cell Imaging   confocal microscopy by PerkinElmer, Inc.  (image of scope)

Electron Microscopy   mcb fig 9.20*  
      ecb panel 1.1
 TEM  1931  Ruska  - 1st  transmission  electron microscope           TEM-photo mcb9.5a*
                          TEM passes e's through a specimen  onto a viewing screen
                          (resolution  theoretical = 0.005nm, but effective resolution is =
0.1 to 0.2 nm*)       
             1952  Palade / Porter - 1st TEM pics & EM stains - image is due to differential scattering of e's by molecules
                           in specimen (stains as
heavy metals - osmium tetroxide for membranes) stain = dark.
                           specimens must be thin = 50 nm thick; cut via
             1957  Robertson - unit membrane hypothesis  (all membranes look alike in EM)
             2000  computer image averaging allows 3D modeling -  models of ribosome & Ca-ATPase pump
 fFEM (cryoelectron microscopy) - an aqueous specimen is frozen in liquid N2 (-1960C)
             1964  Steere & Muhlethaler - develops freeze fracture EM -  prep*  &   pics*  (scroll down)
             2004  cryoelectron tomography - specimen is rotated in electron beam & individual images are
                          computationally fit into 3D reconstruction (tomogram) - nuclear pores*

 SEM (scanning electron microscopy) - mcb fig 9.20*  =  picture of neuron* &   virtual SEM*
Charles Oatley - 1st scanning EM (Stereoscan)  -  uses metal shadowing to coat specimens coating*
& bombardment with e's releases 2ndary e's when focused onto detector reveals 3D surface details
 Tagging -    1981  antibody tagging with gold particle in electron microscopy - fig 9.21* 
            1974  Nobel Prize to  G. Palade, C. deDuve, A. Claude   -  for their "inner workings of cells"







Investigations of Cells - some major EUKARYOTIC ORGANELLES
                     electron microscopy has used fixed sectioned cells which are static
                      to describe organelles, mostly by presence or absence of membranes...


Single Membrane Bound (DIGESTIVE) Organelles:


1. endosomes... membrane bound vesicles of extra-cellular milieu internalized by ENDOCYTOSIS
        a.  endocytosis - cathrin protein "coated" membrane pits - pinch off endosome vesicles
        b. phagocytosis - whole cells engulfed & passed to lysosomes for digestion
        c. autophagy - worn-out organelles fuse with lysosome   mcb9.2a &  endosomes & lysosomes

2. lysosomes... hundreds single membrane bound vesicles* (exclusive to animals- plants use vacuoles)
        have acid pH environment to help denature proteins (H+ATPases* & Cl transporters --> HCl)
        contains hydrolytic enzymes (nucleases, proteases, phosphatases, glycosylases) for digestion.
                         Tay-Sachs (tt): defective lysosomal enzyme which degrades ganglosides,
                                                glysolipids buildup in neurons dementia, blindness, and death
        nuclear & some cytosolic proteins aren't digested within lysosomes, but rather proteasome*





  Other Single Membrane digestive-like Organelles:
  3. plant vacuole...  membrane limited interior space (up to 80% cell volume) containing
            membrane transport proteins accumulate ions, nutrients, & wastes.                 mcb9.7*
            lumen holds digestive enzymes (has acid pH optima - acts like lysosomes).
            tonoplast membrane permeable to water influx, helps establish turgor pressure (5-20 ATM)

  4. peroxisomes... spherical (0.2-1.0 Ám) organelle containing oxidases (catalase) that use O2
                           to oxidize (remove e-'s from) molecules as H2O2 (& other toxins).  mcb9.4*
           degrades FA's to acetyl groups - used to make cholesterols (esp. impt in liver/kidney cells).
                                  X-linked adrenoleukodystrophy (ADL): no FA digestion occurs,
                                  leads to several neuro-linked defects and death.                 

           plants contain glyoxysomes which oxidize stored lipids (very similar to peroxisomes).






  Membrane Complexes...   as Organelles

  5. endoplasmic reticulum... network of closed-flattened membrane sacks called cisternae
         found in al nucleated cells; involved in protein/lipid biosynthesis
         2 types:  SER (smooth) - lacks ribosomes                                                       mcb9.5
                                            - makes FA & phospholipds (esp. in hepatocytes)
                                            - detoxifies hydrophobic chemicals including carcinogens & pesticides
                        RER (rough)  - membranes bound with ribosomes                              mcb9.4*
                                            - makes plasma membrane proteins & exportable proteins of ECM
                                            - abundant in cells making  -  antibody proteins (plasma cells)
                                                                                   - pancreas (digestive enzymes & hormones)

 6. Golgi Complex... series flattened membrane sacks (cisternae) that take up ER transport vesicles
         and process contents via glycosylation (addition of carbohydrate residues).
         3 divisions:  cis - where ER vesicles enter                                                       mcb9.5b*
                           medial - where modifications (glycosylations) occur
                           trans - vesicle packages & budded off here for secretion               mcb9.6* 






 Double Membrane Bound Organelles:

 7. nucleus...  largest; double membrane bound - outer membrane contiguous with ER
         synthesizes DNA, rRNA, tRNA, primary transcript (mRNA preccursor)    
         peri-nuclear space (2-5nm) is contiguous with lumen of ER                  nucleus em*
pores of protein nucleoporins (mcb 8.20a*) regulates nucleoplasm-cytoplasm exchange
via NLS* of 7 aa sequence @ C-terminus (N-pro-pro-lys-lys-lys-arg-lys-val-C)
         nucleolus - regions of rDNA that makes rRNA (but, ribsosomal proteins made in cytoplasm)
         nucleoplasm - 'cytoplasm' of the nucleus
            heterochromatin - condensed (darker EM color) = inactive DNA -                 mcb6.33a*
            euchromatin - non-condensed (lighter EM color) = active DNA
            lamins - fibrous proteins adjacent to inner nuclear membrane = frame for nuclear shape

 8. mitochondria... conducts ATP production of cell via oxidative metabolism of glucose & fatty acids
      outer membrane (50:50 lipid/protein) contains porin (mcb10.18*) transports most ligands < 10K
      inner membrane (25:75 lipid/protein) strictly regulates most transport into mitoplasm
      cristae - infoldings of inner membrane (mcb9.8* &  mcb12.6*    pic1* & pic2*)       





 9. chloroplast... largest green plant cell organelle (0.5-2.0 Ám by 10 Ám)
      has double membrane with extensive inner membrane-limited sacks called thylakoids (mcb9.9*)
      absorbs light energy via chlorophyllous pigments & makes ATP & NADPH (chemiosmosis)
      reduces CO2 into CH2O
          Similarities of Mitochondria & chloroplasts...
             1.  make ATP/NAD(P)H via similar mechanisms
                      - chemiosmosis: oxidative creation of H+ gradient coupled to ATP synthase
             2.  show mobility throughout cell
             3. divide by fission independent of whole cell's division
             4. autonomously replicate their own DNA [mito: 16,569 nucleotide pairs: about 37 genes]
[chlp: 10fg or 120 genes - highly supercoiled & repetitive-up to 6 copies]
             5. both contain 70s - bacterial size ribosomes  [eukaryotes have 80s ribosomes]
             6. synthesize their own proteins on their own protein synthesizing machinery 

                "The Inner Life of a Cell " - a movie animation by John Liebler & Harvard U.
                           REVIEW: major organelles of animal & plant cells
                      mcb5.19*(ans) & mcb5.19*(ans) - Quick Review of Major Eukaryotic Cell Organelles









Skip the material below:

   Where do we get homogenous cell populations for microscopy?  
Cell Culture- growing isolated cells in defined media
  single cells in controlled conditions... form colonies (clones)
req: 370C, pH, salts, essential amino acids, vitamins,
  glucose, serum (growth factors - insulin & transferrin [Fe])


* & mcb22.3*      American Type Culture Collection
  Society for in vitro Biology & Plant Tissue Culture

Single Cell Analysis...
  Cytometer -
instruments for analyzing cells and
Sorting Cells* - via fluorescent tags &  core facility 
Microspectroscopy... a technique for 
         measurements of spectral absorption of stained
         microscopic material in cells.  
microspec apparatus