BIO 315 Lab Exam I. Section #: Name:

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1 Section #: Name: Also provide this information on the computer grid sheet given to you. (Section # in special code box) BIO 315 Lab Exam I 1. In labeling the parts of a standard compound light microscope diagrammed on the right, which of the following should be placed in Blank 2: A) ocular lens B) objective lens x C) condenser D) focus knob Which of the same components has the greatest impact in determining the degree of resolution achieved by the microscope: Figure 1. Sectional diagram through a standard compound light microscope. A) ocular lens B) objective lens x C) condenser D) focus knob 3. Which of the same components can be used to alter the amount of contrast visible in the specimen: A) ocular lens B) objective lens C) condenser x D) focus knob 4. Which pair of the options in question #1 is used to calculate the total magnification of a microscope: A) B and C B) B and D C) A and B x D) A and C

2 5. Which of the following units might be used to describe resolution: A) nm B) µ C) a unit of distance D) any of the above x 6.. Which of the following contribute to the resolution that can be discerned in a specimen? A) λ of light used B) N.A. of objective lens C) refractive index of medium between specimen and lens D) All of the above x 7. Which of the following conditions would result in the best resolution? A) blue light and 0.7 N.A. B) blue light and 0.9 N. A. x C) red light and 0.7 N.A. D) red light and 0.9 N.A. 8.. Which of the following contribute to the N.A. of an objective lens? A) half the cone angle of light collected by the objective lens B) refractive index of medium between specimen and lens (oil vs air) C) λ of light used D) both A and B x 9. Which of the following components of the fluorescence microscope selectively allows only the wavelength of light that excites the fluorescent label on the molecule of interest of a specimen to reach the specimen: A) Excitation filter x B) Emission filter C) Barrier filter D) Both B and C

3 10. Which of the following reagents provided the specificity needed to visualize histones only in the nuclei of the immunostained embryos of Lab 1B: A) Anti-mouse IgG 2 o antibody B) Anti-neuronal protein 1 o antibody C) Anti-histone 1 o antibody x D) Anti-histone 2 o antibody 11. Which of the following reagents allowed the fluorescent tag to be indirectly targeted to the histones in the immunostained embryos of Lab 1B: A) Anti-histone 1 o antibody B) Anti-neuronal protein 1 o antibody C) Anti-histone 2 o antibody D) Anti-mouse IgG 2 o antibody x Blue Green Red 12. Use the electromagnetic spectrum chart above to determine which color a molecule would appear when labeled with a fluorescent probe that absorbs at 500 nm and emits at 700 nm: A) Blue B) Green C) Red x D) Purple 13. Which of the following proteins is recognized by the 2 o antibodies used in the immunostaining protocol of Lab 1B: A) histones B) mouse IgG C) mouse 1 o antibody D) both B and C (They are the same thing.) x

4 14. Which of the following methods for labeling a protein with a fluorescent tag requires the use of recombinant DNA technology: A) Immunofluorescent staining of the protein in fixed cells B) Expression of a GFP-tagged version of the protein in a living cell x C) Direct attachment through covalent linkage and injection of labeled protein into cell D) DAPI staining 15. Which of the following methods could be used to monitor the dynamic behavior of a specific protein inside a living cell: A) Standard light microscopy of the cell B) Immunofluorescent staining of the cell C) Fluorescence microscopy of a GFP-tagged version of the protein expressed in the cell x D) Any of the above 16. Which of the following methods for labeling a protein with a fluorescent tag requires the use of a vertebrate species with an immune system: A) Immunofluorescent staining of the protein in fixed cells x B) Expression of a GFP-tagged version of the protein in a cell C) Direct attachment through covalent linkage and injection of labeled protein into cell D) DAPI staining 17. Which of the following methods is routinely used to label the DNA of the nucleus with a fluorescent tag: A) Immunofluorescent staining of the protein in fixed cells B) Expression of a GFP-tagged version of the protein in a cell C) Direct attachment through covalent linkage and injection of labeled protein into cell D) DAPI staining x 18. Which of the following non-fluorescent stains can be used to stain everything in a specimen to provide higher contrast: A) Janus Green B) Orcein stain C) DCIP D) Iodine stain x

5 19. The diagram on the right shows how differential centrifugation can be used to separate organelles in Lab 2. This method separates organelles on the basis of their: A) diameter B) size C) color D) density x 20. Which of the four fractions labeled in the diagram corresponds to the High Speed Supernatant fraction: Fraction 1> Fraction 2> Fraction 3> Fraction 4> A) Fraction 1 B) Fraction 2 C) Fraction 3 x D) Fraction In which of these four fractions would you expect to find a cytosolic protein, such as an enzyme of glycolysis: A) Fraction 1 B) Fraction 2 C) Fraction 3 x D) Fraction Which of these four fractions contained the nuclei: A) Fraction 1 B) Fraction 2 x C) Fraction 3 D) Fraction What was the source of your mitochondria in lab 2: A) Onion B) Drosophila embryo C) Chicken liver x D) Beef liver

6 24. Which of the following was used to identify the fractions containing mitochondria: A) Janus Green stain B) Orcein stain C) SDH activity D) Both A and C x 25. Which of the following provided a colorimetric indicator of the REDOX reaction occurring in your Succinate Dehydrogenase enzyme assays: A) Succinate B) DCIP x C) Janus Green stain D) Azide 26. Which of the following was used to stain mitochondria in Lab 2: A) Succinate B) DCIP C) Janus Green stain x D) Azide 27. Which of the following was used to stain nuclei in Lab 2: A) Succinate B) Orcein stain x C) Janus Green stain D) DAPI 28. Which of the following can be used to fluorescently stain nuclei: A) Janus Green stain B) Orcein stain C) DAPI x D) Either B or C

7 29. Succinate provided which of the following functions in your Succinate Dehydrogenase assays: A) Inhibitor B) Colorimetric Indicator C) Substrate x D) Buffer 30. Which of the following acted as an inhibitor of Succinate Dehydrogenase in your Succinate Dehydrogenase assays: A) Succinate B) DCIP C) Malonate x D) Azide 31. Which of the following poisoned the normal flow of electrons through the Electron Transfer Chain, forcing the electrons to go to the artificial electron acceptor in your Succinate Dehydrogenase assays: A) Buffer B) DCIP C) Malonate D) Azide x 32. Which of the following components of your Succinate Dehydrogenase assays was present to maintain a constant ph: A) Buffer x B) DCIP C) Malonate D) Azide 33. Which of the following would be most likely to be disrupted by fluctuations in ph: A) Ionic bonds x B) Covalent bonds C) Peptide bonds D) Van der Waals Interactions

8 In Lab 2, you used your data from the Succinate Dehydrogenase assays to construct Lineweaver Burke plots and used this to determine the enzyme s K M and V max in the presence and in absence of an inhibitor. Lineweaver Burke plots from similar assays of a HYPOTHETICAL enzyme in the presence (dotted line) and in the absence (solid line) of an inhibitor are shown below. (Each mark on each axis = 1.0) 1/v 1/[substrate] 34. Based on these data, the K M for the hypothetical enzyme in the absence of inhibitor is: A) 0.5 x B) 0.67 C) 1.0 D) Based on these data, the K M for the hypothetical enzyme in the presence of inhibitor is: A) 0.5 x B) 0.67 C) 1.0 D) 2.0

9 36. Based on these data, the V max for the hypothetical enzyme in the absence of inhibitor is: A) 0.5 B) 0.67 C) 1.0 D) 2.0 x 37. Based on these data, the V max for the hypothetical enzyme in the presence of inhibitor is: A) 0.5 B) 0.67 x C) 1.0 D) Based on the values for K M and V max in the presence and absence of inhibitor, the inhibitor can be classified as: A) a competitive inhibitor B) a non-competitive inhibitor x C) both a competitive and non-competitive inhibitor D) neither a competitive nor a non-competitive inhibitor 39. Based on this classification, which of the following would you predict to happen upon the addition of an excess of substrate to the inhibited reaction: A) The inhibition would NOT be overcome by excess substrate. x B) The inhibition would be overcome by excess substrate. C) The inhibition would be enhanced. D) The inhibitor would become an activator. 40. K M is given in units of: A) Substrate concentration x B) Substrate converted to product per unit of time C) Product per reaction volume D) None of the above

10 41. The epi-fluorescence microscope allows you to observe the cellular distribution of a specific molecule, such as a protein, that has been appropriately labeled. A) Describe how this works, including names of the specific components of the epi-fluorescence microscope not present in the traditional light microscopy that allow it to do this. (4 pt) pg. 5 The epifluorescence microscope uses light of a single wavelength instead of the full light spectrum. (+1) The light wavelength used excites the fluorescent label on the molecule of interest. (+1) An excitation filter between the light source and the sample allows only that wavelength of light to reach the sample. (+1) Then a barrier filter between the sample and the eye allows only light of the wavelength that is emitted from the excited fluorescent label to reach the eye. (+1) B) Describe two different methods for labeling a protein so that it can be visualized by epifluorescence microscopy. (4 pt) pg. 6 Any 2 of the following: 1) Covalently linking a fluorescent probe directly to the purified molecule in vitro. 2) Immunofluorescene-use an antibody that recognizes the molecule of interest that has a fluorescent probe covalently linked to it. GFP tagging of a protein-express a recombinant form of the gene encoding a protein of interest that has been fused to the gene encoding Green Fluorescent Protein (GFP) 40) The electron microscope offers higher resolution than the light microscope. A) Explain what is meant by the term higher resolution. (2 pt) pg. 2 Resolution is measured as the distance between 2 pts that can be discriminated from one another. The distance is smaller with higher resolution. (2 pts) (1pt credit given for answers saying that higher resolution means you can see more details in the image.) B) List two variables you could control to maximize the resolution of your light (or epi-fluorescence microscope. (2 pt) pg. 2 2 or more of the following: Use the lowest wavelength of light in the spectrum. Use a lens with a high numerical aperture.

11 Use a mounting medium that reduces scatter of light diffracted from specimen. Have ocular and objective lens closer together (to reduce light scatter). Etc. Not accepted: focus the microscope, use a higher magnification lens, etc. C) Explain why the electron microscope is able to offer higher resolution than the light microscope. (2 pt) pg. 2, 4 The accelerated electrons in an electron beam has a lower wavelength than the shortest wavelength of light in the spectrum. (2 pts) For Discussion Essays be Prepared to Answer the following (no key provided): -Explain how the confocal fluorescence microscope achieves higher resolution than the widefield fluorescence microscope. Be precise about what specific aspects of the micrcoscope achieve this. (3 pt) -Explain how lattice light sheet microscopy achieves higher resolution than widefield fluorescence microscopy. Be precise about what specific aspects of the micrcoscope achieve this. (4 pt) -Explain how lattice light sheet microscopy reduces the phototoxicity and photobleaching during imaging of live specimens. (3 pt)

12 -Explain how multiphoton micrsocopy (as used in Article #4) achieves higher resolution than widefield fluorescence microscopy. (2 pt) -Explain how multiphoton microscopy reduces the phototoxicity and photobleaching during imaging of live specimens. (2 pt) -Explain how multiphoton microscopy allows imaging at greater depth in a specimen. (2 pt) -In Article #4, which fluorescent label allowed mitochondria to be observed and how? (2 pt) -In Article #4, which fluorescent labels allowed the mitochondrial membrane potential of mitochondria to be examined and how? (2 pt)

13 -In Article #4, which fluorescent labels allowed the oxidative status of neurons to be examined and how? (2 pt) -Summarize what was learned about mitochondrial function in the mouse model of Alzheimer s Disease in the study reported in Article #4. (3 pt) (no need to use the whole page.be brief.)

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