Wavelength and alignment tests for confocal spectral imaging systems

Abstract

Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference spectra within a particular system as well as between related and unrelated instruments. We also find evidence of instability that, if not diagnosed, could lead to inconsistent data. This variability confirms the need for diagnostic tools to provide a standardized, objective means of characterizing instability, evidence of misalignment, as well as performing calibration and validation functions. Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emit distinct, stable spectral features, in place of a sample. An MIDL characterization verifies the accuracy and consistency of a CSI system and validates acquisitions of biological samples. We examined a total of 10 CSI systems, all of which displayed spectral inconsistencies, enabling us to identify malfunctioning subsystems. Only one of the 10 instruments met its optimal performance expectations. We have found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrument errors and measure accuracy and reproducibility under normalized conditions. Using this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. It is evident that if CSI systems of a similar make and model were standardized to reveal the same spectral profile from a standard light source, then researchers could be confident that real-life experimental findings would be repeatable on any similar system.

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.…”