CloudSpec Technology
Our patented design enables absorption measurements free from optical scatter. CloudSpec™ spectra obey the Beer-Lambert law, so can be used to determine concentration of analytes, even in highly turbid media.
CloudSpec is easy to use for automated and rapid concentration analysis, taking ≈10 seconds per sample.
Scatter-Free Absorption (SFA)
CloudSpec measures true absorbance in the presence scattering, which would otherwise be confounded. Because traditional UV/Vis (strictly an extinction measurement) is the sum of scatter and absorption the scattering component often prevents absorption being used.
CloudSpec measures two spectra simultaneously and derives a third from those two:
· Regular UV-Vis (extinction) - measured directly
· Scatter-Free Absorption (absorption) - measured directly
· Optical scatter (scattering) - derived by subtracting absorption from extinction
Integrating Sphere Technology
To eliminate the effects of scattering CloudSpec uses a dual-pathway integrating-sphere cavity that enables both a transmission (regular UV/Vis) and a scattering measurement.
Using Marama's proprietary scatter-free analysis the data from these pathways is used to avoid collecting the scatter spectrum to an absorption spectrum that removes scatter and obeys Beer-Lambert (quantitative) principles.
CloudSpec’s Measurement Modes
CloudSpec creates three spectra simultaneously, making it a highly flexible formulation and analytical tool. The graphic below illustrates the information obtained by CloudSpec from RNA-loaded LNP samples. Click on the upper tabs to compare the different measurement modes for the same samples.
Scatter-free absorption spectra. RNA A260 = 0.5 O.D.
Concentration of RNA = 12.5µg/ml
Spectrum includes scatter. LNP+RNA A260 = 1.2 O.D.
Apparent RNA concentration = 30µg/ml
Error = 140% overestimate
Scatter contribution isolated from the extinction spectrum.
Ca. 60% of the traditional UV/Vis spectrum comes from scatter for the loaded LNPs.
CloudSpec Solves Some Important Problems
Accuracy
SFA allows you to accurately measure the true absorption spectrum of a scattering sample in a simple single measurement. The absorption value is converted to a concentration measurement by multiplication with the extinction coefficient.
It is impossible to accurately measure absorption of scattering samples using transmission UV/Vis because the scatter must be approximated from other information, e.g., extrapolation of scattering spectra from non-absorbing regions, if available.
Because scattering scales with the 6th power of the particle size and the inverse 4th power of the wavelength the fit must be very precise. SFA does not have this limitation.
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Noise
Due to the way SFA works it is able to overcome limitations of noise in multiple ways.
Firstly, in a subtraction of a fitted scattering function, the relative noise is amplified (a large number minus a large number), which is not the case with SFA. Secondly, SFA's dual pathways enable a larger effective pathlength, which increases sensitivity and reduces noise, even at low concentrations.
These factors mean that as scatter increases even small concentrations of material can be accurately measured, even when scattering >> absorption.
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FAQs
In traditional UV/Vis measurements samples need to be brilliantly clear and free from scatter. CloudSpec eliminates the effects of turbidity, even in highly cloudy liquids.
While it might seem impossible, samples as cloudy as milk can be measured. As long as the sample can be pipetted, with a suitable solvent as a blank, you can obtain a scatter-free absorption spectrum.
Like traditional UV/Vis there is an upper limit to the absorbance that can be measured. Samples with absorbances above this limit must be diluted by a suitable amount. For a known sample, e.g., RNA, this will have a typical target value – in RNA, for example, this is ideally around 1-10µg/ml. For CloudSpec to work there is an additional consideration - the sample must be exactly 1ml in volume.
Regular UV-Vis is, strictly, an extinction spectrum, which is the combined loss of transmitted light by the sum of absorption and scatter. In the absence of scatter an extinction spectrum is equivalent to an absorption spectrum. However, when scatter is present, the extinction spectrum equals absorption + scatter and it is not always possible to separate the two effects exactly or well.