Abstract

Despite a growing need, oceanographers are limited by existing technological constrains and are unable to observe aquatic microbes in their natural setting. In order to provide a simple and easy to implement solution for such studies, a new Thin Light Sheet Microscope (TLSM) has been developed. The TLSM utilizes a well-defined sheet of laser light, which has a narrow (23 micron) axial dimension over a 1 mm × 1 mm field of view. This light sheet is positioned precisely within the depth of field of the microscope’s objective lens. The technique thus utilizes conventional microscope optics but replaces the illumination system. The advantages of the TLSM are two-fold: First, it concentrates light only where excitation is needed, thus maximizing the efficiency of the illumination source. Secondly, the TLSM maximizes image sharpness while at the same time minimizing the level of background noise. Particles that are not located within the objective’s depth of field are not illuminated and therefore do not contribute to an out-of-focus image. Images from a prototype system that used SYBR Green I fluorescence stain in order to localize single bacteria are reported. The bacteria were in a relatively large and undisturbed volume of 4ml, which contained natural seawater. The TLSM can be used for fresh water studies of bacteria with no modification. The microscope permits the observation of interactions at the microscale and has potential to yield insights into how microbes structure pelagic ecosystems.

© Optical Society of America

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References

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. Cell Biology (1)

J. G. White, W. B. Amos and M. Fordham, ?An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy,? J. Cell Biology 105, 41-48. (1987).
[CrossRef] [PubMed]

. Microscopy (1)

G. J. Brakenhoff, H. T. M. van der Voort, E. A. van Spronsen and A. Nanninga ?Three-dimensional imaging in fluorescence by confocal scanning microscopy,? J. Microscopy, 153, 151-159, (1989).
[CrossRef]

Adv. Microb. Ecol. (1)

T. Nagata and D. Kirchman, ?Roles of submicron particles and colloids in microbial food webs and biegeochemical cycles within marine environments,? Adv. Microb. Ecol. 15, 81-103 (1997).

Ann. Rev. Biophys. Bioeng. (1)

D. A. Agard, ?Optical Sectioning microscopy: Cellular architecture in three dimensions,? Ann. Rev. Biophys. Bioeng. 13, 191-219 (1984).
[CrossRef]

Appl. Enivron. Microbiol. (1)

G. Mitchell, L. Pearson, S. Dillon and K. Kantalis, ?Natural assemblages of marine bacteria exhibiting high-speed motility and large accelerations,? Appl. Enivron. Microbiol. 61, 4436-4440 (1995).

Appl. Environ. Microbiol. (1)

M. Karner and J. A. Fuhrman, ?Determination of active marine bacterioplankton: a comparison of universal 16srRNA probes, autoradiography and nucleoid staining,? Appl. Environ. Microbiol. 63, 1208-1213 (1997).
[PubMed]

Appl. Opt. (2)

Aquat. Microb. (1)

L. Legendre and J. LeFevre, ?Microbial food webs and the export of biogenic carbon in oceans,? Aquat. Microb. Ecol. 9, 69-77 (1995).
[CrossRef]

Aquat. Microb. Ecol. (2)

F.Schut, R. A. Prins and J. C. Gottschal, ?Oligotrophy and pelagic marine bacteria: facts and fiction,? Aquat. Microb. Ecol. 12, 177-202 (1997).
[CrossRef]

R. T. Noble and J. A. Fuhrman, ?Use of SYBR Green 1 for rapid epifluorescence counts of marine bacteria and viruses,? Aquat. Microb. Ecol. 14, 113-118 (1998).
[CrossRef]

Bioimaging (1)

F. Lanni, B. Bailey, D. L. Farkas and D. L. Taylor ?Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes,? Bioimaging 1, 187-196 (1993).
[CrossRef]

Deep-Sea Res. I (1)

A. L. Alldredge, U. Passow and B. E. Logan, ?The abundance and significance of a class of large, transparent organic particles in the ocean,? Deep-Sea Res. I 40, 1131-1140 (1993).
[CrossRef]

IEEE (1)

F. Macias-Garz, A. C. Bovik, K. R. Diller, S. J. Aggarwal and J. K. Aggarwal, ?Transactions on Acoustics, Speech and Signal Processing,? IEEE 36, 1067-1074 (1988).

J. Opt. Soc. Am. A (1)

Mar. Ecol. Prog. Ser. (2)

A. Heissenberger, G. G. Leppard G. J. and Herndl, ?Ultrastructure of marine snow. II. Microbiological considerations,? Mar. Ecol. Prog. Ser. 135, 299-308 (1996).
[CrossRef]

L. M. Proctor and J. A. Fuhrman, ?Roles of viral infection in organic particle flux,? Mar. Ecol. Prog. Ser. 69, 133-142 (1991).
[CrossRef]

Mar. Ecolo. Prog. Ser. (1)

A. Shibata, K. Kogure, I. Koike and K. Ohwada, ?Formation of submicron colloidal particles from maring bacteria by viral infection,? Mar. Ecolo. Prog. Ser. 303-307 (1997).
[CrossRef]

Marine Ecology Progress Series (1)

P. J. S. Franks J. S. Jaffe, ?Microscale distributions of phytoplankton: initial results from a two dimensional imaging Flourometer,: OSST,? Marine Ecology Progress Series, 220, (2001).
[CrossRef]

Meas. Sci. Technol. (1)

C. D. Meinhart, S. T. Wereley and M. H. B. Gray ?Volume illumination for two-dimensional particle image velocimetry,? Meas. Sci. Technol. 11, 809-814 (2000)
[CrossRef]

Microb. Ecol. (1)

F. Azam, D. C. Smith, G. F. Steward and ?. Hagstr?m, ?Bacteria-organic matter coupling and its significance for oceanic carbon cycling,? Microb. Ecol. 28, 167-179 (1993).
[CrossRef]

Nature (3)

I. Koike, H. Shigemitsu, T. Kazuki and K. Kogure, ?Role of sub-micrometre particles in the ocean,? Nature 345, 242-244 (1990).
[CrossRef]

D. C. Smith, M. Simon, A. L. Alldredge and F. Azam, ?Intense hydrolytic enzyme activity on marine aggregates and implications for rapid particle dissolution,? Nature 359, 139-142 (1992).
[CrossRef]

B. Bailey, D. L. Farkas, D. L. Taylor and F. Lanni ?Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,? Nature 366, 44-48 (1993)
[CrossRef] [PubMed]

Oceanography (1)

J. S. Jaffe, P. J. S. Franks and A. W. Leising. ?Simultaneous imaging of phytoplankton and zooplankton distributions,? Oceanography, 11, 24 ? 29 (1998).
[CrossRef]

Opt. Commun. (1)

H. K. Stelzer and S. Lindek ?Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,? Opt. Commun. 111, 536-547 (1994)
[CrossRef]

Particle analysis in oceanography (1)

F. Azam and D. C. Smith, ?Bacterial influence on the variability in the ocean's biogeochemical state: A mechanistic view,? In: S. Demers (ed.) Particle analysis in oceanography. (Springer-Verlag, 213-236 1991).
[CrossRef]

Proceedings of SPIE (1)

J. A. Conchello, C. M. Cogswell, A. G. Tescher and T. Wilson, ?Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII,? Proceedings of SPIE Volume 3919 (2000).

Prog. Oceanogr. (1)

A. L. Alldredge and M. Silver, ?Characteristics, dynamics and significance of marine snow,? Prog. Oceanogr. 20, 41-82 (1988).
[CrossRef]

Science (3)

N. Blackburn, T. Fenchel and F. Mitchell, ?Microscale nutrient patches in planktonic habitats shown by chemotactic bacteria,? Science 282, 2254-2256 (1998).
[CrossRef] [PubMed]

A. L. Alldredge and Y. Cohen, ?Can microscale chemical patches persist in the sea? Microelectrode study of marine snow, fecal pellets,? Science 235, 687-691 (1987).
[CrossRef]

F. Azam, ?Microbial control of oceanic carbon flux: The plot thickens,? Science 280, 694-696 (1998).
[CrossRef]

Other (5)

T. Fenchel, G. M. King and T. H. Blackburn, Bacterial Biogeochemsitry: The Ecophysiology of Mineral Cycling, 2nd Edition. (Academic Press, New York 1998).

E. Gratton, ?Laser sources for confocal and two-photon microscopy,? Chapter in Confocal and Two- Photon Microscopy: Foundations, Applications and Advances. Ed., Alberto Diaspro, (Wiley & Sons, Inc. 2000).

S. Inou? and K. Spring, ?Video Microscopy: The Fundamentals,? (New York: Plenum, 1997).

D. Hanselman and B. Littlefield, Mastering MATLAB: A Comprehensive Tutorial and Reference, (Prentice-Hall, Inc., 1996).

T. Wilson and C. Sheppard. ?Theory and practice of scanning optical microscopy,? (Academic Press, London, 1984).

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Figures (4)

Figure 1:
Figure 1:

Thin light sheet microscopy (TLSM), schematic diagram.

Figure 2:
Figure 2:

Thin light sheet - beam characteristics.

Figure 3
Figure 3

(a): Sample illuminated with the thin light sheet illumination. (b) Same sample with a broad beam illumination. Marine bacterial isolates in 0.22 μm filtered seawater, stained. Particle size cannot be scaled due to sensor blooming. Particles appear about 1.7 times larger than their actual size.

Figure 4:
Figure 4:

Different size organisms imaged with the TLSM of total seawater. Particles appear about 1.7 times larger than their actual size.

Equations (2)

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δ = n λ 0 N A 2 + n e NA M
d = K · λ · f #

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