Abstract

Fluorescence spectroscopy has been used to measure fluorescence quantum efficiency (QE) of dried Bacillus spores (washed and unwashed) fixed to a quartz substrate. Fluorescence spectra and QE of anthracene in ethanol was used as the standard. We measured the absorption and fluorescence signal of the spores as a function of the number of spores. The absorption was measured from 600 nm to 250 nm using the reflectance in an integrating sphere. The fluorescence spectra were measured using excitation wavelengths at 280, 360 and 400 nm at room temperature. The absorption cross sections for the unwashed spores were 1.3 × 10-8, 8 × 10-9, and 5 × 10-9 mm2/spore at 280, 360 and 400 nm, respectively. The fluorescence QE was 0.13 ± 0.03, 0.33 ± 0.12 and 0.43 ± 0.26 at 280, 360, and 400 nm, respectively. The QE decreased by a factor of 2, 4 and 4 at these same wavelengths after washing and redrying the spores.

© 2005 Optical Society of America

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References

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    [CrossRef] [PubMed]

Anal. Chem. (1)

J. T. Coburn, F. E. Lytle, and D. M. Huber, "Identification of Bacterial Pathogens by Laser Excited Fluorescence," Anal. Chem. 57, 1669-1673 (1985).
[CrossRef]

Anal. Chim. Acta (2)

J. Ho, " Future of biological aerosol detection," Anal. Chim. Acta 457, 125-148 (2002).
[CrossRef]

E. Yacoub-George, L. Meixner, W. Scheithauer, A. Koppi, S. Drost, H. Wolf, C. Danapel, and K. A. Feller, "Chemiluminescence multichannel immunosensor for biodetection," Anal. Chim. Acta 457, S 3-12 (2002).
[CrossRef]

Anal. Lett. (1)

R. F. Chen, "Fluorescence Quantum Yields of Tryptophan and Tyrosine," Anal. Lett. 1, 35-42 (1967).
[CrossRef]

Analyst (1)

A. T. R. Williams, S. A. Winfield, and J. N. Miller, "Relative fluorescence quantum yields using a computer controlled fluorescence spectrometer," Analyst 108, 1067 (1983).
[CrossRef]

Appl. Opt. (2)

G. W. Faris, R. A. Copeland, K. Mortelmans, and B. V. Bronk "Spectrally resolved absolute fluorescence cross sections for Bacillus spores," Appl. Opt. 36, 959-967 (1997).
[CrossRef]

J. Kunnil, B. Swartz, and L. Reinisch, "Changes in the Luminescence Between Dried and Wet Bacillus Spores," Appl. Opt. 43, 5404-5409 (2004).
[CrossRef] [PubMed]

Appl. Spectrosc. (2)

Biophys. J. (2)

L. Reinisch, W. P. Van de Merwe, and B. V. Bronk, "Comparative fluorescence spectra from bacteria and spores in different stages of growth," Biophys. J. 59, 161a (1991).

P. Marco, J. L. Terrance, E. W. Katherine, and J. M. Alexander, "The high-resolution architecture and structural dynamics of Bacillus spores," Biophys. J. 88, 603-608 (2004).

Environ. Sci. Technol. (1)

F. S. Ligler, G. P. Anderson, P. T. Davidson, R. J. Foch, J. T. Ives, K. D. King, G. Page, D. A. Stenger, and J. P. Whelan, "Remote sensing using an airborne biosensor," Environ. Sci. Technol. 32, 2461 (1998).
[CrossRef]

Eur. J. Biochem. (1)

C. Saavedra, C. Vasquez, and M. V. Encinas, "Structural studies of the Bst V1 restriction-modification proteins by fluorescence spectroscopy," Eur. J. Biochem. 263, 65-70 (1999).
[CrossRef] [PubMed]

Field Anal. Chem. Technol. (1)

G. A. Luoma, P. P. Cherrier, and L. A. Retfalvi, "Real-time warning of biological-agent attacks with the Canadian Integrated Biochemical Agent Detection System II (CIBADS II)," Field Anal. Chem. Technol. 3, 260-273 (1999).
[CrossRef]

IEEE. J. Quantum Electron. (1)

W. F. Cheong and A. J. Welch, "A Review of the Optical Properties of Tissues," IEEE. J. Quantum Electron. 26, 2166-2185 (1990).
[CrossRef]

J. Aerosol Sci. (1)

S. Sarasanandarajah, J. Kunnil, E. Chacko, B. V. Bronk, and L. Reinisch, "Reversible changes in fluorescence of bacterial endospores found in aerosols due to hydration/drying," J. Aerosol Sci. 36, 689- 699 (2005).
[CrossRef]

J. Appl. Bacteriol. (1)

P. Setlow, "Mechanisms which contribute to the long-term survival of spores of Bacillus species," J. Appl. Bacteriol. 76, 49S-60S (1994).
[CrossRef]

J. Microbiol. Methods (1)

S. A. Glazier and H. H. Weetall, "Autofluorescence detection of Escherichia coli on silver membrane filters," J. Microbiol. Methods 20, 23-27 (1994).
[CrossRef]

J. Phys. Chem. (1)

W. R. Dawson and M. W. Windsor, "Fluorescence yields of aromatic compounds," J. Phys. Chem. 72, 3251-3260 (1968).
[CrossRef]

Laryngoscope (1)

J. A. Werkhaven, L. Reinisch, M. Sorrel, J. Tribble, and R. H. Ossoff, "Non-Invasive Optical Diagnosis of Bacteria Causing Otitis Media," Laryngoscope 104, 264-268 (1994).
[CrossRef] [PubMed]

Lasers Surg. Med. (1)

M. J. Sorrel, J. Tribble, L. Reinisch, J. A. Werkhaven, and R. H. Ossoff, "Bacteria Identification of Otitis Media with Fluorescence Spectroscopy," Lasers Surg. Med. 14, 155-163 (1994).
[CrossRef]

Opt. Express (1)

Photochem. Photobiol. (1)

S. Dhami, A. J. de Mello, G. Rumples, S. M. Bisshop, D. Philips, and A. Beeby, "Phthalocymine fluorescence at high concentration: dimmers or reabsorption effect?" Photochem. Photobiol. 61, 341-346 (1995).
[CrossRef]

PNAS (1)

A. J. Westphal, P. B. Price, T. J. Leighton, and K. E. Wheeler, "Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity," PNAS 100, 3461-3466 (2003).
[CrossRef] [PubMed]

Pure Appl. Chem. (1)

D. F. Eaton, "Reference materials for fluorescence measurement," Pure Appl. Chem. 60, 1107-1114 (1988).
[CrossRef]

Trans. Far. Trans. (1)

G. Weber and F. W. J. Teale, "Determination of the absolute quantum yield of fluorescent solutions," Trans. Far. Trans., 53, 646-655 (1957).
[CrossRef]

Other (10)

I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic molecules, 2nd ed. (Academic Press, New York and London, 1971).

T. M. Rossi and I. M. Warner, "Bacterial Identification using fluorescence spectroscopy," in Rapid Detection and Identification of Microorganisms, W. H. Nelson, ed. (Verlag Chemie, 1985), pp. 1-50.

B. C. Spector, L. Reinisch, D. Smith, and J. A. Werkhaven, "Noninvasive fluorescent identification of bacteria causing acute otitis media in a chinchilla model," Laryngoscope 110, 1119-1123 (2000).
[CrossRef] [PubMed]

A. Driks and P. Setlow, "Morphogenesis and properties of the bacterial spore," in Prokaryotic Development, Y. V. Brun and L. J. Shimkets, eds. (American Society for Microbiology, Washington D.C, 1999), pp. 191-218.

M. Paidhungat and P. Setlow, "Spore germination and outgrowth," in Bacillus subtilis and Its Relatives:from Genes to Cells, A. Hoch, R. Losick, and A. L. Shoneshein, eds. (J. Am. Soc. Microbiol., Washington DC, 2002), pp. 537 -548.

P. Setlow, in "Bacterial Stress Responses," G. Storz and R. Hengee-Aronis, eds. (Am. Soc. Micorbiol., Washington, DC, 2000), pp. 217-230.

I. D. Campbell and R. A. Dwek, Biological Spectroscopy (Benjamin/Cummings Publishing Co, Menlo Park, CA, 1984).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum Press, New York, 1999).

J. R. Stephens, "Measurements of the ultraviolet fluorescence cross sections and spectra of Bacillus anthracis simulants," (Los Alamos National Laboratory, Los Alamos, NM, 1999).

J. Kunnil, S. Sarasanandarajah, E. Chacko, B. Swartz, and L. Reinisch, " Identification of Bacillus Spores Using Clustering of Principal Components of Fluorescence Data," (2005).

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Figures (9)

Fig. 1.
Fig. 1.

Schematic of the dry spores mounted to the quartz slide and the optical geometry used in the fluorometer.

Fig. 2.
Fig. 2.

(A) Absorption spectrum of a 14 μM anthracene solution in ethanol. (B) Fluorescence spectrum of anthracene in ethanol with excitation at 360 nm.

Fig. 3.
Fig. 3.

The integrated fluorescence intensity (counts) of anthracene in ethanol is shown versus absorption (O.D) at excitation wavelength 320 (open triangles) and 360 nm (filled triangles). The lines are linear least squares fits. The slope of the line is proportional to the QE of anthracene in ethanol at the excitation wavelength selected.

Fig. 4.
Fig. 4.

Response curve of the fluorometer. The slope of the integrated fluorescence intensity (counts) versus absorption (O.D) is plotted against the excitation wavelength from 320 to 360 nm. The straight line is a linear least squares fit of the data. The response curve is extrapolated to be constant (dashed lines) at shorter and longer wavelengths.

Fig. 5.
Fig. 5.

Representative absorption and fluorescence spectra of unwashed B. globigii (i) spores: (A) Absorption measured from 250–600 nm and (B) the fluorescence measured at excitation wavelengths 280, 360 and 400 nm with emission ranges 300–570, 380–700 and 420–700 nm respectively.

Fig. 6.
Fig. 6.

(A) The absorption (O.D) of B. globigii (i) spores as function of number spores at 280 nm (diamonds), 360 nm (squares) and 400 nm (triangles). (B) The integrated fluorescence intensity (counts) of B. globigii (i) spores is shown versus the sample absorption (O.D). The lines are linear least squares fits to the data and restricted to pass through the origin. Diamonds are excitation at 280 nm, squares are excitation at 360 nm and the triangles are excitation at 400 nm.

Fig. 7.
Fig. 7.

Representative absorption and fluorescence spectra of twice washed and redried B. globigii (i) spores: (A) Absorption is measured from 250–600 nm. The dotted line is, shown for comparison, the spore absorption before washing. It was scaled to be similar to the after washing curve. The gray line is the difference in the absorption before minus after washing. (B) The fluorescence measured at excitation wavelengths 280, 360 and 400 nm with emission ranges 300–570, 380–700 and 420–700 nm respectively.

Fig. 8.
Fig. 8.

The integrated fluorescence intensity (counts) of B. globigii (i) spores is shown versus the sample absorption (O.D). The lines are linear least squares fits to the data and restricted to pass through the origin. Diamonds are excitation at 280 nm, squares are excitation at 360 nm and the triangles are excitation at 400 nm.

Fig. 9.
Fig. 9.

Comparison of fluorescence between washed (squares) and unwashed (triangles) B. globigii (i) spores at 400 nm excitation wavelength. The integrated fluorescence intensity (counts) of B. globigii (i) spores is shown versus absorption (O.D). The lines are linear least squares fits.

Tables (1)

Tables Icon

Table 1. Absorption cross sections and QE of the Bacillus globigii samples investigated.

Equations (3)

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N f = I 0 ( 1 10 D ) QE
N f 2.303 ( εcl ) I 0 QE
QE sample = QE s tan dard [ Slope sample Slope s tan dard ] [ n 1 2 n 2 2 ] g ( λ 1 , λ 2 )

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