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Measurement of Scattering and Absorption Cross Sections of Dyed Microspheres.

Gaigalas AK, Choquette S, Zhang YZ - J Res Natl Inst Stand Technol (2013)

Bottom Line: Therefore A was first analyzed using values of the other parameters obtained from a fit to the absorbance due to scattering, A1-A, with the imaginary part neglected.The imaginary part obtained from the analysis of A was then used to reanalyze A1-A, and obtain better estimates of the other parameters.After a few iterations, consistent estimates were obtained of the scattering and absorption cross sections in the wavelength region 300 nm to 800 nm.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Standards and Technology, Gaithersburg, MD 20899.

ABSTRACT
Measurements of absorbance and fluorescence emission were carried out on aqueous suspensions of polystyrene (PS) microspheres with a diameter of 2.5 µm using a spectrophotometer with an integrating sphere detector. The apparatus and the principles of measurements were described in our earlier publications. Microspheres with and without green BODIPY(@) dye were measured. Placing the suspension inside an integrating sphere (IS) detector of the spectrophotometer yielded (after a correction for fluorescence emission) the absorbance (called A in the text) due to absorption by BODIPY(@) dye inside the microsphere. An estimate of the absorbance due to scattering alone was obtained by subtracting the corrected BODIPY(@) dye absorbance (A) from the measured absorbance of a suspension placed outside the IS detector (called A1 in the text). The absorption of the BODIPY(@) dye inside the microsphere was analyzed using an imaginary index of refraction parameterized with three Gaussian-Lorentz functions. The Kramer-Kronig relation was used to estimate the contribution of the BODIPY(@) dye to the real part of the microsphere index of refraction. The complex index of refraction, obtained from the analysis of A, was used to analyze the absorbance due to scattering ((A1 - A) in the text). In practice, the analysis of the scattering absorbance, A1-A, and the absorbance, A, was carried out in an iterative manner. It was assumed that A depended primarily on the imaginary part of the microsphere index of refraction with the other parameters playing a secondary role. Therefore A was first analyzed using values of the other parameters obtained from a fit to the absorbance due to scattering, A1-A, with the imaginary part neglected. The imaginary part obtained from the analysis of A was then used to reanalyze A1-A, and obtain better estimates of the other parameters. After a few iterations, consistent estimates were obtained of the scattering and absorption cross sections in the wavelength region 300 nm to 800 nm.

No MeSH data available.


The two traces show the absorbance of a 10 µmol/L solution of BODIPY@ dye in ethanol. The solid and dashed traces were obtained for samples outside (called A1 in the text) and inside (called A3 in the text) the integrating sphere (IS) detector respectively. The large decrease in the absorbance for the sample inside the IS detector is due to the emitted fluorescence. The IS detector cannot distinguish between transmitted and fluorescence photons, and interprets the fluorescence photons as transmitted photons.
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f4-jres.118.002: The two traces show the absorbance of a 10 µmol/L solution of BODIPY@ dye in ethanol. The solid and dashed traces were obtained for samples outside (called A1 in the text) and inside (called A3 in the text) the integrating sphere (IS) detector respectively. The large decrease in the absorbance for the sample inside the IS detector is due to the emitted fluorescence. The IS detector cannot distinguish between transmitted and fluorescence photons, and interprets the fluorescence photons as transmitted photons.

Mentions: Figure 4 shows the effect of fluorescence on absorbance measurements of a sample of 10 µmol/L green BODIPY® dye dissolved in ethanol and placed in holder 3. The solid and dashed traces in Fig. 4 show the absorbance measurements carried out in holder 1(outside IS) and holder 3 (inside IS) respectively. Measurements were also done for a sample of ethanol to subtract ethanol contribution. The absorbance measured in holder 1 is a good estimate (after subtracting the ethanol contribution) of the true BODIPY@ absorption. The reduction of the green BODIPY® dye absorbance observed in holder 3 was attributed to the fluorescence emitted by the excited green BODIPY® dye molecules. The spectrophotometer detector could not distinguish between transmitted and fluorescent photons and any fluorescence photon was interpreted by the instrument as a transmitted photon. A closer examination of the two traces in Fig. 4 also displayed a large difference in spectral shape. The spectral shape of the measurement in holder 3 was influenced by the spectral response of the detector. Most of the fluorescence photons were emitted around 570 nm while the wavelength of the transmitted photons varied in accordance with the spectrometer setting. Thus the measurements in holder 1 and holder 3 have a different effective detector spectral response. These considerations also apply to absorbance measurements of microspheres dyed with the green BODIPY® dye. The dashed trace in Fig. 5 shows the emitted fluorescence spectrum from a suspension of 2.5 µm dyed microspheres placed in a fluorimeter and excited with 555 nm light. For comparison, the solid trace in Fig. 5 shows the observed fluorescence emission when an ethanol solution of green BODIPY® dye was placed in a fluorimeter and excited with 555 nm light. A comparison of the two traces in Fig. 5 shows that the fluorescence emission from the microspheres is similar to the fluorescence emission from the dye solution except for a red shift of approximately 9 nm. Therefore it is reasonable to assume that the observed absorbance of 2.5 µm microsphere suspension, shown in Fig. 2, would be reduced by the emitted fluorescence and that the actual absorbance of the microsphere suspension is larger. In order to quantify the reduction of the measured absorbance due to fluorescence, it was assumed that the relative reduction in absorbance is proportionate to the quantum yield as indicated in Eq. (1).(1)A−A3A=mΦ


Measurement of Scattering and Absorption Cross Sections of Dyed Microspheres.

Gaigalas AK, Choquette S, Zhang YZ - J Res Natl Inst Stand Technol (2013)

The two traces show the absorbance of a 10 µmol/L solution of BODIPY@ dye in ethanol. The solid and dashed traces were obtained for samples outside (called A1 in the text) and inside (called A3 in the text) the integrating sphere (IS) detector respectively. The large decrease in the absorbance for the sample inside the IS detector is due to the emitted fluorescence. The IS detector cannot distinguish between transmitted and fluorescence photons, and interprets the fluorescence photons as transmitted photons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487309&req=5

f4-jres.118.002: The two traces show the absorbance of a 10 µmol/L solution of BODIPY@ dye in ethanol. The solid and dashed traces were obtained for samples outside (called A1 in the text) and inside (called A3 in the text) the integrating sphere (IS) detector respectively. The large decrease in the absorbance for the sample inside the IS detector is due to the emitted fluorescence. The IS detector cannot distinguish between transmitted and fluorescence photons, and interprets the fluorescence photons as transmitted photons.
Mentions: Figure 4 shows the effect of fluorescence on absorbance measurements of a sample of 10 µmol/L green BODIPY® dye dissolved in ethanol and placed in holder 3. The solid and dashed traces in Fig. 4 show the absorbance measurements carried out in holder 1(outside IS) and holder 3 (inside IS) respectively. Measurements were also done for a sample of ethanol to subtract ethanol contribution. The absorbance measured in holder 1 is a good estimate (after subtracting the ethanol contribution) of the true BODIPY@ absorption. The reduction of the green BODIPY® dye absorbance observed in holder 3 was attributed to the fluorescence emitted by the excited green BODIPY® dye molecules. The spectrophotometer detector could not distinguish between transmitted and fluorescent photons and any fluorescence photon was interpreted by the instrument as a transmitted photon. A closer examination of the two traces in Fig. 4 also displayed a large difference in spectral shape. The spectral shape of the measurement in holder 3 was influenced by the spectral response of the detector. Most of the fluorescence photons were emitted around 570 nm while the wavelength of the transmitted photons varied in accordance with the spectrometer setting. Thus the measurements in holder 1 and holder 3 have a different effective detector spectral response. These considerations also apply to absorbance measurements of microspheres dyed with the green BODIPY® dye. The dashed trace in Fig. 5 shows the emitted fluorescence spectrum from a suspension of 2.5 µm dyed microspheres placed in a fluorimeter and excited with 555 nm light. For comparison, the solid trace in Fig. 5 shows the observed fluorescence emission when an ethanol solution of green BODIPY® dye was placed in a fluorimeter and excited with 555 nm light. A comparison of the two traces in Fig. 5 shows that the fluorescence emission from the microspheres is similar to the fluorescence emission from the dye solution except for a red shift of approximately 9 nm. Therefore it is reasonable to assume that the observed absorbance of 2.5 µm microsphere suspension, shown in Fig. 2, would be reduced by the emitted fluorescence and that the actual absorbance of the microsphere suspension is larger. In order to quantify the reduction of the measured absorbance due to fluorescence, it was assumed that the relative reduction in absorbance is proportionate to the quantum yield as indicated in Eq. (1).(1)A−A3A=mΦ

Bottom Line: Therefore A was first analyzed using values of the other parameters obtained from a fit to the absorbance due to scattering, A1-A, with the imaginary part neglected.The imaginary part obtained from the analysis of A was then used to reanalyze A1-A, and obtain better estimates of the other parameters.After a few iterations, consistent estimates were obtained of the scattering and absorption cross sections in the wavelength region 300 nm to 800 nm.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Standards and Technology, Gaithersburg, MD 20899.

ABSTRACT
Measurements of absorbance and fluorescence emission were carried out on aqueous suspensions of polystyrene (PS) microspheres with a diameter of 2.5 µm using a spectrophotometer with an integrating sphere detector. The apparatus and the principles of measurements were described in our earlier publications. Microspheres with and without green BODIPY(@) dye were measured. Placing the suspension inside an integrating sphere (IS) detector of the spectrophotometer yielded (after a correction for fluorescence emission) the absorbance (called A in the text) due to absorption by BODIPY(@) dye inside the microsphere. An estimate of the absorbance due to scattering alone was obtained by subtracting the corrected BODIPY(@) dye absorbance (A) from the measured absorbance of a suspension placed outside the IS detector (called A1 in the text). The absorption of the BODIPY(@) dye inside the microsphere was analyzed using an imaginary index of refraction parameterized with three Gaussian-Lorentz functions. The Kramer-Kronig relation was used to estimate the contribution of the BODIPY(@) dye to the real part of the microsphere index of refraction. The complex index of refraction, obtained from the analysis of A, was used to analyze the absorbance due to scattering ((A1 - A) in the text). In practice, the analysis of the scattering absorbance, A1-A, and the absorbance, A, was carried out in an iterative manner. It was assumed that A depended primarily on the imaginary part of the microsphere index of refraction with the other parameters playing a secondary role. Therefore A was first analyzed using values of the other parameters obtained from a fit to the absorbance due to scattering, A1-A, with the imaginary part neglected. The imaginary part obtained from the analysis of A was then used to reanalyze A1-A, and obtain better estimates of the other parameters. After a few iterations, consistent estimates were obtained of the scattering and absorption cross sections in the wavelength region 300 nm to 800 nm.

No MeSH data available.