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"The start is what stops most people."
Wise words from former coach Mike Ditka, and a concise and accurate summary of our daily tasks. But the question is then: Where to start? The Square One cuvette holder from Ocean Insight captures all the usual functionalities you've come to expect from a cuvette system, along with some unique hallmarks.
Your basic transmission measurement for absorbance or otherwise can be done as usual using the two ports in parallel, though the third port on the unit faces a mirror for added fluorescence intensity. When you attach your LED or other excitation source to this port, the signal will travel through your sample to excite fluorescence, then bounce off the mirror, and travel once more through the sample for a second-pass excitation. Tests have shown a 30% increase in fluorescence signal using this mirror versus a standard single-pass configuration, depending on the nature of the sample and optics.
To further maximize fluorescence signal, or to incite secondary response from the sample, a second light source may be installed such that the first is 90° to the collection leg and the second is 270° to the collection. If using a clone of the first source, you may expect an additional 20% signal gain versus a single source using the backside mirror. If working with a mixture of fluorophores with differing excitations, for example, using a different secondary excitation provides the freedom to toggle which sample is fluorescing without changing fiber connections or removing the sample. A rough calculation of fluorescence efficiency can be made by using two spectrometers rather than two light sources, with one spectrometer in transmissive mode at 180° to the light source, and the other in fluorescent mode at 90° to the source.
Using an attenuator to map both the absorbance and fluorescence as a function of input intensity can provide some preliminary figures around fluorophore efficiency. When you have a need for more than a standard cuvette holder, for a holder that provides repeatability and adaptability, and you're wondering where to start, the answer is simple: Square One. https://oceanoptics.com/product/square-one-cuvette-holder/
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