BIOL 4610 1st Edition Lecture 8 Outline of Current Lecture I. Fluorescence microscopyII. Dyes to detect intracellular ion concentrationsIII. Electron microscopyIV. Cell organelles Current LectureFluorescence microscopy:• A molecule absorbs light at one wavelength and gives off (emits) it at a different (longer) wavelength• Often used to detect ion concentrations, immunofluorescence, and make recombinant green fluorescent protein1) Dyes to detect intracellular ion concentrations: • Calcium levels higher (10,000X) outside of cell• When a nerve impulse comes, Cal floods into cell. We measure this by using a dye called Fura Dyes- (ester) can passively diffuse into cell• Inside cell, have esterase that trap Fura inside the cell• 1. stimulation to increase Cal levels inside cell• 2. Fura binds to Calcium and emits light at a yellow wavelength 2) Immunofluorescence: • Relying on the use of antibodies to detect the location of specific protein inside the cell• 10-12 AA sequence that recognizes a specific protein These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.• 1st Antibody: recognizes the specific protein• Use primary antibody from diff species (rabbits) that recognize a certain human protein• 2nd antibody: made to recognize all rabbit antibodies, and has fluorescent tag on it• (antibodies bound 2 each other)• Able to visualize structures that you wouldn’t before3) Make recombinant green fluorescent protein (GFP) gene: - In plasmid, insert gene X of interest AND green protein DNA (GFP) - Make a transgenic cell with fusion gene- Every time cell makes gene X, it will have GFP protein attached- So every time it is made in an organism, it will have green flourescence4) Forster resonance energy transfer (FRET)• Test whether 2 proteins interact with each other make 2 fusion proteins• If the 2 proteins interact use emitted wavelength of 1st 10 excite second proteins• YFP only works if excited by light at 4809.3. Electron Microscopy: High resolution imagingResolution- how low can you go to see two distinct objects before it’s just a big blurry one. Light microscope -About 0.2nmElectron microscope- 0.1nm (2000X ^^ in resolution)1) Transmission electron microscopy (TEM): • Use magnet (not glass lens) to focus the beam ofelectrons and magnify sample• Good for visualizing bacteria and specific structuresIn the cell, and viruses• But, don’t get big picture, only small part of it• Can’t look at anything living, must stain *Osmium2) Scanning electron microscopy (SEM):• Moved location of the sample• Still staining it with a metal• Get a 3D appearance• ** look at surface of cell **9.4 Cell Organelles1) Endosomes: Membrane bound compartment that comes from plasma membrane and it brings in molecules from outside via endocytosis2) Lysosomes: organelles within cells (only in animal cells) degrade macromolecules- have enzymes (lipases, nucleases) and reduced pH (4-5)3) Smooth endoplasmic reticulum-Large network of membranes that are folded into flattened sacs (cisternae)Use for synthesis of lipids, vitamins, detoxification enzymes4) Rough endoplasmic reticulum- Has ribosomes attachedProtein synthesis of proteins destined to go to: 1. Plasma membrane2. Outside of cell 3. Specific organelles 5) Golgi complex: where proteins go to after the ERFold proteins- disulfide bonds and carbohydrate molecules- Leave golgi by vesicles – dictate protein