This work describes a procedure for acquiring a spectrum of an analyte over an extended range of wavelengths and validating the wavelength and intensity assignments. To acquire a spectrum over an extended range of wavelengths with a spectrometer with a charge coupled device (CCD) array detector, it is necessary to acquire many partial spectra, each at a different angular position of the grating, and splice the partial spectra into a single extended spectrum. The splicing procedure exposes instrument dependent artifacts. It is demonstrated that by taking a spectrum of a reference irradiance source and making spectral correction, the artifacts exposed by the splicing are removed from the analyte spectrum. This is because the irradiance reference spectrum contains the same artifacts as the analyte spectrum. The artifacts exposed by the splicing depend on the wavelength of the splice; therefore it is important to measure the irradiance reference spectrum for the same range of wavelengths used to measure the spectrum of the analyte solution. In other words, there is no general spectral correction factor which is applicable to spectra taken for different range of wavelengths. The wavelength calibration is also carried out by splicing many partial spectra from a source like a krypton lamp. However the wavelength assignments are not sensitive to the splicing procedure and the same wavelength calibration can be used for spectra acquired over different extended wavelength ranges. The wavelength calibration checks the validity of the setting of the grating angular position, and the assignment of wavelengths to individual pixels on the CCD array detector. The procedure is illustrated by measuring the spectrum of an orange glass and the spectrum of a suspension of microalgae.

Measurements are presented of the absorbance, fluorescence emission, fluorescence quantum yield, and fluorescence lifetime of CdSe/ZnS nanocrystals, also known as quantum dots (QDs). The study included three groups of nanocrystals whose surfaces were either passivated with organic molecules, modified further with carboxyl groups, or conjugated with CD14 mouse anti-human antibodies. The surface modifications had observable effects on the optical properties of the nanocrystals. The oscillator strength (OS) of the band edge transition was about 1.0 for the nanocrystals emitting at 565 nm, 605 nm, and 655 nm. The OS could not be determined for QDs with emission at 700 nm and 800 nm. The fluorescence lifetimes varied from 26 ns for nanocrystals emitting near 600 nm to 150 ns for nanocrystals emitting near 800 nm. The quantum yield ranged between 0.4 and 0.9 for the nanocrystals in this study. A brightness index (BI) was used to evaluate the suitability of the nanocrystal labels for flow cytometer measurements. Most QD labels are at least as bright as fluorescein for applications in flow cytometer assays with 488 nm excitation. For optimal brightness the QDs should be excited with 405 nm light. We observed a strong dependence of the QD absorbance at 250 nm on the surface modification of the QD.

The number of techniques to measure number concentrations and size distributions of submicrometer particles has recently increased. Submicrometer particle standards are needed to improve the accuracy and reproducibility of these techniques. The number concentrations of fluorescently labeled polystyrene submicrometer sphere suspensions with nominal 100 nm, 200 nm and 500 nm diameters were measured using seven different techniques. Diameter values were also measured where possible. The diameter values were found to agree within 20%, but the number concentration values differed by as much as a factor of two. Accuracy and reproducibility related with the different techniques are discussed with the goal of using number concentration standards for instrument calibration. Three of the techniques were used to determine SI-traceable number concentration values, and the three independent values were averaged to give consensus values. This consensus approach is proposed as a protocol for certifying SI-traceable number concentration standards.

The National Institute of Standards and Technology (NIST), the National Institutes of Health (NIH) and other industry stakeholders have been working together to enable fluorescence intensities of flow cytometer calibration beads to be assigned quantitative equivalent reference fluorophore (ERF) values with high accuracy and precision. The ultimate goal of this effort is to accurately quantify the number of antibodies bound to individual living cells. The expansion of this effort to assign ERF values to more than 50 fluorescence channels and particles with diameters ranging from 10 μm down to 80 nm is reported here.