How do scientists study cells and the processes going on inside them? Along with growth in scientific knowledge come new technologies. *Development of new instruments. *Development of new methods in scientific research. •The invention of the microscope has greatly expanded our understanding of cell structure and function.

Microscopes The invention of the microscope in the 17th century led to the discovery of the cell. Robert Hooke described cells using this light microscope.

Modern Microscopes Visible light is focused on the specimen with the condenser lens. • Light passing through the specimen is refracted with an objective and an ocular lens (magnify and invert the image for the observer).

Two Important Properties of Microscopes: Magnification – how much larger an object is made to appear. Resolving power – minimum distance between two points that can still be distinguished as two separate points. *limited by the wavelength of light (inversely proportional). *maximum resolution of a LM is 0.2μm. *highest magnification is 1,000X.

Cell Theory The cell is the fundamental unit of structure and function in all organisms. All organisms are made of one or more cells. All cells come from pre-existing cells through cellular division. Theodor Schwann and Mattheis Schleiden

Electron Microscopes Instead of light, electron microscopes use electron beams which have much shorter wavelengths (resolving power of 0.2nm). Enhanced resolution and magnification led to the identification of subcellular organelles. There are two types of electron microscopes: transmission and scanning electron microscopes.

Transmission Electron Microscope Electromagnetic lenses. Electrons transmitted through the specimen are focused onto a viewing screen or film. • Used to study internal cellular structure.

Transmission Electron Microscope

TEM Images

Scanning Electron Microscope Electron beam scans the surface of the specimen. Excites secondary electrons on sample. Collects secondary electrons and focuses onto a viewing screen. Great depth of field – 3D image.

Gallery of Cells What can you infer about function from the structure of these cells?

Cell Types Given the diversity of life on earth, there is an endless variety of cell types. *Prokaryotes vs. eukaryotes. *Single-celled organisms vs. multi-celled organisms. *The human body alone contains over 200 different cells that vary in size, shape, and function. *Size: prokaryotes, 1-10μm; eukaryotes, 10-150+ μm. *Shape: relates closely to its function. Ex: skin cells are flat – protection; neurons are even more specialized. • Cells are different because they are making different proteins. *DNA directs protein synthesis - determines function and structure of cells.

Function is determined by cell protein production In order to determine function, we need to find out what kinds of proteins are produced by that cell (i.e., hormones, enzymes, fibers, etc.).

Problem: TEM, SEM, and most LM techniques “fix” or kill cells. Then how did scientists learn about the functions of the various organelles? Scientists first had to develop methods to isolate the different types of organelles without destroying their function. How do you think scientists were able to get the organelles out of live cells?

Scientists first had to learn how to grow cells outside the organism. These are live tissue culture cells grown in the laboratory (in vitro). • The next problem was how to isolate the organelles that produce proteins in the different cell types.

In order to get to the organelles, the cells need to be lysed or broken open. Methods of cell lysis: •Homogenation – pressure or grinding •Detergents – dissolve lipid membranes •Sonication – liquid shear •Enzymes – digest cell membranes and peptidoglycans •Freezing and grinding •Hypotonic medium

Once the cells are lysed, how do scientists separate the organelles? The various cell components can be isolated by centrifugation. Which cell components would you use to determine protein production?

Density gradient separation is another method used by scientists to isolate different cell fragments.

Electrophoresis separates solutes based on charge. Gel electrophoresis can be used to separate different proteins produced in the cell Electrophoresis separates solutes based on charge. *First linearize proteins with SDS. *SDS also attaches negative charges to the polypeptides. *The larger the peptide, the greater the charge on it. *An electric current is applied to separate the molecules based on charge.

Another method for separating biomolecules is liquid chromatography Relies on differential adsorption of solutes to a bed material. *Can be separated based on charge or size.

In gas chromatography the sample is partitioned between a carrier gas and a thin liquid layer. Very sensitive – detects very low concentrations of the molecules of interest.

Proteins can be identified using antibodies The specific protein is injected into animals initiating antibody production. The next step is to attach a marker to the antibody (can be radioactive, fluorescent, heavy metal, etc.) Apply antibody to gels, cell fragments or tissue sections. High affinity for the specific antigen – can detect very low concentrations. Can use several different markers in combination.

Examples:

Immunodiffusion Antigen and antibody diffuse into each other from separate wells in an agar dish. Forms a visible line of precipitate when an antibody-antigen reaction takes place.

X-ray diffraction is used to determine the internal structure of molecules This is the x-ray diffraction photo involved in the elucidation of the structure of DNA.

“Heavy” isotopes can be used to label molecules (ex: 15N, 13C, 2H, 18O). Meselson and Stahl used heavy isotopes to prove Watson and Crick’s model of semi-conservative DNA replication. • Extracted DNA from E. coli grown in medium containing ammonium ions (NH4+)

Summary: Along with growth in scientific knowledge come new technologies used to delve deeper into the workings of the cell. Cells are different because they are making different proteins. Many different cell biology techniques have been developed to characterize cells based on size, charge, and the specific molecules they produce. Newborn mice, two of which are transgenic mice with GFP fused to an epithelial protein.