Summary 34 1.Cells follow rules of chemistry; 2.Water is the most abundant substance proteins constitutes most of a cell’s dry mass; 3.Four major classes of small organic molecules make macromolecules; 4.Living cells undergo metabolism; 5.A reaction will happen if it can result in lower free energy in the system; 6.Proteins and protein complexes execute almost all cell functions.

Lecture 3 Microscopy

Birth of Cell Biology 1838 Schleiden and Schwann “cell doctrine”

A typical cell is um Light microscope sees 0.2 um 1 um=10 -6 m 1 mm=10 -3 m 1 nm=10 -9 m 1 A=Angstrom = m

Nondestructive Live imaging Challenge: sample preparation Computerized 3-D reconstruction Resolution is limited by the wavelength of radiation

Bright-field Normaski DIC Phase-contrastDark-field Four types of light microscopy

Images can be digitally enhanced Improves interpretation, resolution (0.025 um), but appears 0.2 um (can’t tell whether it is single or double MT) Eyes have limitation in seeing dim signals and resolve bright images 1.Camera (the same kind as in night surveillance) 2. Digitally extract info (contrast)

Sample preparation: sectioning

Sample preparation: staining to visualize cellular contents Hematoxylin Eosin: HE

Fluorescence microscope

Fluorescent dyes

Immunofluorescence imaging does not show actual sizes

Image deconvolution: a computational approach to remove blur from a stack of images taken in different focal planes (digital technique) Needs a fast computer, less bleaching, very sensitive 3-D imaging (esp fluorescence)

Confocal microscope produces optical sections by excluding out-of-focus light (analog technique) Expensive scope, more bleaching, limitations in depth

Two photon

Fast electrons has short waves: 100,000V and nm In theory nm resolution: 10,000 of light microscope In practice, 0.1 nM (1A) Difficulties: Specimen preparation Contrast Radiation damage Therefore, effective 2 nM (20 A) 100 better than light microscope Gold atoms (bright spots) 0.2 nm apart

Transmission EM (TEM)

Immunogold EM

3D reconstruction Distorions of immuno EM: 1.Large depth of field deep structures in the same layer 2.Ab and Gold-Ab don’t penetrate too deep Label before imbedding

Scanning EM for surface imaging Smaller cheaper (SEM) 10 nM resolution

Imaging surface DIC TEM SEM

Metal shadowing (platinum) and observe under TEM Individual marcomolecules can been seen

Freeze-fracture electron microscopy Intramembrane particles (large TM proteins) Chloroplast

Freeze-etch electron microscopy Interior of cells Crack the frozen block, lower ice level by subliming ice In a vacuum (freeze drying), shadow the exposed parts of cells and observe replica Protein filaments in muscle cells

Negative staining of actin filaments Helical chain of acitin monomers 8 nm Diameter

EM tomography for 3-D reconstruction Resolution rivals X-ray crytallography 0.3 nm for crystalline arrays 0.8 nm for single particle reconstruction (subunits, domains, 2nd structures) Hepatitis B virus TEM See details of macro- molecular Complexes!!!

Calcium imaging Aequorin, a luminescent protein Ion-sensitive indicators Fluorescent Ca indicator:fura um Not bright Brighter better resolution

Introducing membrane-impermeable substance into a cell

Caged molecules photolysis Ca++ can be caged too Tubulin labeled With caged fluoresent dyes

Jellyfish GFP Live imaging Cajal bodies

Pulse-chase experiments using radioisotopes

Summary 1.Cell doctrine; 2.Two major types of microscopes: light and electron; 3.Limitation of resolution: wavelength of radiation; 4.Advantage and disadvantage of light and electron MS 5.Different types of light microscopes: bright field, phase contrast, DIC, dark field,fluorescent, confocal 6.Image processing: digital enhancement 7.Two major types of EM: TEM and SEM 8.Additional tricks: shadowing, freeze-fracture, freeze etching, negative staining, tomography; 9.Live imaging, calcium indicators, caged compounds, GFP, pulse chasing