**7. References**

Batchelder, H.P., Swift, E. (1989). Estimated near-surface mesoplanktonic bioluminescence in the western North Atlantic during July 1986. Limnol. Oceanogr. 34: 113-128

Batchelder, H.P., Swift, E., Van Keuren, J.R. (1990). Pattern of planktonic bioluminescence in the northern Sargasso Sea: seasonal and vertical distribution. Mar. Biol. 104: 153- 164

observed the photosynthesis-irradiance relation to bioluminescence capacity. That is, cells grown at higher irradiance levels produce more photosynthetic products which may be diverted to the bioluminescence system (Sweeney et al., 1959; Sweeney 1981; Swift et al., 1981; Sullivan and Swift 1995). It is very possible that increased levels of nutrients from upwelling and storm runoff events may override diminished irradiance levels found during the fall and winter months to explain maximum bioluminescence observed in these species.

A significant portion of bioluminescence in all oceans is produced by dinoflagellates. The number of bioluminescent species and their relative abundance changes temporally and spatially. There is evidence that dinoflagellates exhibit changes in per cell bioluminescence magnitude which may be attributable to environmental conditions such as light, temperature, and nutrient history. In the present study, photosynthetic and heterotrophic dinoflagellates were collected and tested for bioluminescence on a quarterly basis from 1994-1996 at San Clemente Island, located 100 km off the Southern California coast. Per cell bioluminescence was measured for the phototrophs *Ceratium fusus, Pyrocystis noctiluca, Gonyaulax polyedra* as well as 3 other species of *Gonyaulax*, and in 6 species of the heterotroph *Protoperidinium*. Our data strongly suggests that dinoflagellates have a marked seasonality in the open ocean which may be attributable to regional environmental events such as upwelling and associated winter storm land runoff. We observed correlations between surface (0-50m) nitrates and Chl *a* with bioluminescence in *Pyrocystis noctiluca* and *Protoperidinium pellucidum*. Increased levels of Chl *a* measured in the winter and spring '95 correlated with increased *P. pellucidum* bioluminescence. Both nitrates and mean bioluminescence cell-1 in *P. noctiluca* showed similar trends temporally and in magnitude. Peak levels of nitrates were found in the Southern California Bight in the summer months followed by increases in bioluminescence during the fall months. Peak bioluminescence was observed to occur in the fall for both years in *P. noctiluca* whereas peak bioluminescence in

We gratefully acknowledge financial support from the Office of Naval Research, Code 322BC, Arlington, VA and the Naval Space and Warfare Systems Center, San Diego, CA for conducting these studies. Special thanks are due to Dr. Jim Case (University of California, Santa Barbara) for his continuing guidance in this study and Ms. Connie H. Liu (Naval Space and Warfare Systems Center, San Diego, CA) for providing mean Chl *a* and nitrate

Batchelder, H.P., Swift, E. (1989). Estimated near-surface mesoplanktonic bioluminescence in the western North Atlantic during July 1986. Limnol. Oceanogr. 34: 113-128 Batchelder, H.P., Swift, E., Van Keuren, J.R. (1990). Pattern of planktonic bioluminescence

in the northern Sargasso Sea: seasonal and vertical distribution. Mar. Biol. 104: 153-

*P. pellucidum* was measured in winter '95 and later in fall '95.

**5. Conclusion** 

**6. Acknowledgments** 

values for this study.

164

**7. References** 


**Part 2** 

**Bioluminescence Imaging Methods** 


**Part 2** 

**Bioluminescence Imaging Methods** 

46 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

Lapota, D., Duckworth, D., Groves, J., Rosen, G., Rosenberger, D., Case, J.F. (1997). Long

Latz, M.I., Jeong, H.J. (1993). Effect of dinoflagellate diet and starvation on the

Matheson, I.B.C., Lee, J., Zalewski, E.F. (1984). A calibration technique for photometers.

Seliger, H.H., Biggley, W.H., Swift, E. (1969). Absolute values of photon emission from the

Seliger, H.H., Biggley, W.H. (1982). Optimization of bioluminescence in marine

Sullivan, J.M., Swift, E. (1995). Photoenhancement of bioluminescence capacity in natural

Sweeney, B.M., Haxo, F.T., Hastings, J.W. (1959). Action spectra for two effects of light on

Sweeney, B.M. (1971). Laboratory studies of a green *Noctiluca* from New Guinea. J. Phycol. 7:

Sweeney, B.M. (1981). Variations in the bioluminescence per cell in dinoflagellates. In:

Swift, E., Meunier, V.A., Biggley, W.H., Hoarau, J., Barras, H. (1981). Factors affecting

Tett, P.B., Kelly, M.G. (1973). Marine bioluminescence. Oceanogr. Mar. Biol. Annu. Rev. 11:

Yentsch, C.S., Laird, J.C. (1968). Seasonal sequence of bioluminescence and the occurrence

Limnology and Oceanography, AGU, San Francisco, CA, December. Seliger, H.H., Fastie, W.G., Taylor, W.R., McElroy, W.D. (1961). Bioluminescence in

(Ehrenb.) Dujardin. J. Geophy. Res. 100 (C4): 6565-6574

luminescence in *Gonyaulax polyedra.* J. Gen. Physiol. 43: 285-299

Bioluminescence Symposium, November 1993, Maui, abstract p. 61

correlates in southern California coastal waters (submitted)

Ocean Optics 7 (489): 380-381

*lunula*. Photochem. Photobiol. 10: 232-277

Chesapeake Bay. Science 133: 699-700

53-58

89-173

Res. 26: 127-133

Minneapolis, p 90-94

J. mar. biol. Ass. U.K. 51: 183-206

term dinoflagellate bioluminescence, chlorophyll, and their environmental

bioluminescence of the heterotrophic dinoflagellate, *Protoperidinium divergens*. ONR

marine dinoflagellates *Pyrodinium bahamense, Gonyaulax polyedra, and Pyrocystis* 

dinoflagellates. Paper presented at Annual Meeting, American Society of

and laboratory populations of the autotrophic dinoflagellate *Ceratium fusus*

Nealson, K.H.(ed.)., Bioluminescence Current Perspectives. Burgess Publishing,

bioluminescent capacity in oceanic dinoflagellates. In: Nealson, K.H. (ed.)., Bioluminescence Current Perspectives. Burgess Publishing, Minneapolis, p 95-106 Swift, E., Sullivan, J.M., Batchelder, H.P., Van Keuren, J., Vaillancourt, R.D. (1995).

Bioluminescent organisms and bioluminescent measurements in the North Atlantic Ocean near latitude 59.5N, longitude 21W. J. Geophy. Res. 100 (C4): 6527-6547 Tett, P.B. (1971). The relation between dinoflagellates and the bioluminescence of sea water.

of endogenous rhythms in oceanic waters off Woods Hole, Massachusetts. J. mar.

**3** 

*USA* 

**Living Animals** 

Ramasamy Paulmurugan

*Stanford University School of Medicine,* 

**Bioluminescent Proteins: High Sensitive** 

**Interactions and Protein Foldings in** 

**Optical Reporters for Imaging Protein-Protein** 

Bioluminescence is the production and emission of light by a living organism. Bioluminescence imaging was developed over the last decade as a tool for studying biological processes in living small laboratory animals by molecular imaging. The bioluminescence-based optical imaging is highly sensitive, low-cost, and non-invasive, enabling the real-time analysis of disease processes within the cell at a molecular level in living animals. Recent advances in protein complementation strategies have further expanded its applications by quantitatively monitoring several sub-cellular processes such as protein-protein interactions, protein dimerizations, and protein foldings. In this chapter, we provide a brief introduction to bioluminescence imaging technology and discuss its applications in studying protein-protein interactions, protein dimerizations, and protein foldings, which are some of the most important cellular processes that occur in the heart signal transduction network within the cells, by non-invasively imaging living animals.

Molecular imaging offers many unique opportunities to study biological processes in intact organisms. Bioluminescence imaging (BLI) is one of several molecular imaging strategies currently in use for studying different biological processes. It is based on the sensitive detection of visible light produced during luciferase enzyme mediated oxidation of substrate luciferin in the presence of several co-factors. The luciferase enzyme can be expressed in cells as an indicator of cellular process, and can be used to image living animals by developing tumor xenografts, or developing transgenic animals either to selectively express in a particular type of tissue using a tissue specific promoter, or in the entire animal by a constitutive promoter, to study different cellular diseases. The expressed luciferase enzyme can be imaged with an optical cooled charge coupled device (CCD) camera by injecting the substrate luciferin. Several bioluminescence reporters with a wide range of emission wavelengths are currently identified from insects and crustacean copepods **(Table 1)**. Some of the proteins were even modified by changing from a few to several amino acids by *in vitro* manipulations, and achieved considerably altered proteins with

**1. Introduction** 

**1.1 Bioluminescence** 
