Calibration and validation of confocal spectral imaging systems

Abstract

Background: Confocal spectral imaging (CSI) microscopic systems currently on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, some CSI systems have been found to present inconsistent spectral profiles of reference spectra within a particular system and between related and unrelated instruments. This variability confirms that there is a need for a standardized, objective calibration and validation protocol. Methods: Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg ϩ , Ar ϩ , and inorganic fluorophores that emit distinct, stable, spectral features in place of a sample. We derived reference spectra from the MIDL data to accurately predict the spectral resolution, ratio of wavelength to wavelength, contrast, and aliasing parameters of any CSI system. We were also able to predict and confirm the influence of pinhole diameter on spectral profiles. Results: Using this simulation, we determined that there was good agreement between observed and theoretical expectations, thus enabling us to identify malfunctioning subsystems. We examined eight CSI systems and one non-

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

“…The pinhole aperture was opened in this protocol because we needed to maximize signal to noise by collecting thicker optical sections. As previous comprehensive studies have shown, the pinhole size clearly affects the spectral performance; therefore, all experiments were conducted in the exact same conditions (22,23). Our reflection/mirror standard of a chromium-coated coverslip allowed us to operate the CLSM intact using the illumination sources required for the fluorescence excitation of our fluorophores.…”

Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy

“…Additional factors for good images include the optimization of the confocal operational variables (i.e. objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration) of the confocal microscope and checking the CLSM to insure proper performance (36,37,(39)(40)(41)(42). We were able to effectively combine the spectra from two probes LT and AIF, which had different spectra, into an image that contained a specific probe (LT) for functional activity and a probe (AIF) to identify ovarian morphological features.…”

“…Depending on the size of the follicles, the following objectives were used: Leica Plan Apo 103 (0.4 NA) and Leica Plan Apo 203 (0.7 NA). A series of confocal microscopy performance tests were used to ensure that the confocal microscope was working correctly (36,37,(39)(40)(41)(42). A triple dichroic (TD) excitation filter was used for morphology and a 70/30 reflection dichroic was used for spectral analysis.…”

“…To acquire accurate spectra for comparison with the data derived from a confocal microscope, we used a PAR-ISS (Lightform, NJ) spectral imaging system (36,37). The system was connected with a C mount to a Nikon Optiphot 2 microscope.…”

“…Recently, spectroscopic imaging capacity has been incorporated into confocal microscopes from most confocal companies (28)(29)(30)(31)(32)(33)(34)(35)(36)(37). Although classical confocal systems acquire a single wavelength data point determined by the characteristics of a band pass filter, these spectrophotometer systems use a wavelength dispersive spectrometer to acquire a series of wavelength data points that cover the entire spectral range.…”