Chapter 1 Tools of the Cell Biologist 1. Cell Structure 2. Tools of the cell biologist

Modern Cell Biology genes proteins functions & localization pinktentacle.com

Cell Structure A cell is the smallest living functional unit separated from its environment by a plasma membrane. A cell is the smallest living functional unit separated from its environment by a plasma membrane. Prokaryotic CellEukaryotic Cell Prokaryotic cellEukaryotic cell

Nucleus and Nucleolus

Endoplasmic Reticulum and Ribosomes

Golgi Apparatus

Clathrin-coated Vesicles and Lysosomes

Peroxisomes

Mitochondria

Cytoskeletons Intermediate filament

Centrosome Centriole Halo (Centriolar material)

Plasma Membranes

Development of Muticellular Organisms A fertilized egg Embryonic Stem Cells Various Types of Cell Cell-specific Gene Expression Differentiation Specialization

Tools of the cell biologist Microscopes Microscopes Immunocytochemistry Immunocytochemistry Cell culture Cell culture Flow cytometry Flow cytometry Cell fractionation Cell fractionation Column chromatography Column chromatography Electrophoresis Electrophoresis Recombinant DNA technology Recombinant DNA technology

Microscope Light microscope Light microscope Electron microscope Electron microscope

Light Microscope

Resolution, Numerical Aperture & Wavelength d=0.61 /nSin  0.61  NA d: resolution : wavelength n: refractive index NA: numerical aperture

Power of magnification - The relative enlargement of the specimen when seen through the microscope. The power of magnification can be calculated by multiplying the power of the eye piece lens by the power of the objective lens. Inversion - The reversal of the specimen image by the microscope lenses. A specimen that appears upside down when being viewed is actually right-side up on the slide. Working distance - The distance between the front of the objective and the top of the cover glass on the slide. The higher the magnification the smaller the working distance. Resolution (resolving power) - The least distance between two points or lines at which they are seen as two, rather than a single blur. The greater the numerical aperture the greater the resolution. Depth of focus - The thickness of a specimen which may be seen in focus at one time. The greater the power of magnification the lesser the depth of focus. Field of vision - The surface area which can seen when looking through the light microscope. The area decreases with increasing power of magnification. Numerical aperture (N.A.) - A designation, usually engraved on objectives and condensers, expressing the amount of light reaching the specimen. The greater the N.A. the greater the resolving power. Objectives - The device which holds the lenses of the microscope. Engraved on the object is its power of magnification, and usually the numerical aperture. The larger number is the power of magnification. The longer the objective the more power of magnification. Diaphragm - a device under the stage of a microscope that can regulate the amount of light reaching a specimen. The more power of magnification the more the diaphragm is opened. Terminology for Microscope

Fluorescence Microscopy

Fluorescence Excitation and Emission GFP (green fluorescence Protein)

Immunostaining Mitotic cell in prometaphase (left) and metaphase (right). Labeled with anti- tubulin antibody and fluorescent secondary antibody. Courtesy of Dr. Gary Gorbsky Ph.D., University of Oklahoma Health Sciences Center. primary antibody secondary antibody antigen

FRET (Fluorescence Resonance Energy Transfer)

Light Microscopy Variations Phase Contrast Microscopy Phase Contrast Microscopy Differential Interference Contrast (DIC) Microscopy Differential Interference Contrast (DIC) Microscopy Polarizing Microscopy Polarizing Microscopy Dark Field Microscopy Dark Field Microscopy Confocal Scanning Microscopy Confocal Scanning Microscopy

Transmission Electron Microscopy (TEM)

Colloidal gold-labeling for EM

EM Images of Negative Staining and Metal Shadowing Negative staining Metal shadowing

Freeze-fracture EM

Cryoelectron Micrographs

Scanning Electron Microscopy (SEM)

Scanning Electron Micrographs

Atomic-force Microscopy

Cell Culture Cell Culture Media Cell Culture Media

Growth Pattern of Primary Cells and Established Cell Lines

Fluorescence-activated Cell Sorter (FACS) CD4 CD8

Cell Fractionation by Differential Centrifugation

Density Gradient Centrifugation Low buoyant-density Component High buoyant-density Component

Column Chromatography

Gel Filtration Chromatography

Ion-exchange Chromatography UV absorbance Increasing salt concentration

Hydrophobic Interaction Chromatography decreasing salt concentration UV absorbance Hydrophobic ligand (Butyl, Ethyl, Isopropyl, Octyl, Phenyl, Propyl, etc)

Affinity Chromatography UV absorbance Free ligand concentration

Electrophoresis Principle

An Example of Electrophoresis

Western Blotting

Two Dimensional Gel Electrophoresis 2. SDS-PAGE *Isoelectric point 1. Isoelectric Focusing

Microelectrodes Microelectrodes

Patch clamping Patch clamping

Microinjection Microinjection

Pulse-chase experiment Pulse-chase experiment 1. Seed cells (5×10 6 ) on petridish. 2. Incubate for at least 2 hr with 10 ml of complete medium. 3. Wash cells with warmed DPBS. 4. Incubate for 30 min with 10 ml of warmed Met-free medium. 5. Add 100 µCi [ 35 S]Met, and then incubate for min (PULSE). 6. Wash cells with cold DPBS. 7. Add 10 ml of warmed medium containing L-methionine (CHASE). 8. Incubate for appropriate time (CHASE time). 9. Wash cells with cold DPBS, and then resuspend with 1ml of DPBS. 10. Spin down, and resupend with µl of lysis buffer. 11. Incubate for 30 min on ice. 12. Perform the Immunoprecipitation protocol as described.

Keep it up!!!

Comparison of Microscopes