How does the Fida instrument detect a signal?

The Fida instrument detects a fluorescent signal from the sample as it passes the detector. That signal can come from an external fluorescent label attached to the molecule, or from the molecule's own intrinsic fluorescence, which allows label-free measurement. Flow Induced Dispersion Analysis (FIDA), an efficient, in-solution method for protein characterisation, reads this fluorescence as the sample disperses under flow, and turns it into molecular size, binding and aggregation information.

What does FIDA use to excite and detect the signal?

FIDA excites the sample with an LED light source and amplifies the emitted fluorescence with a photomultiplier tube (PMT), which gives high sensitivity. Because it uses an LED rather than a laser, the sample is not heated during the measurement, so the native structure and behaviour of proteins and other biomolecules are preserved. This is one reason FIDA measurements reflect the molecule as it actually is in solution.

What detection wavelengths are available?

The Fida instrument offers several detection channels so that different fluorophores, and intrinsic fluorescence, can be followed without changing the sample. The channels are at 280 nm for intrinsic protein fluorescence and label-free work, and at 480 nm, 530 nm and 640 nm for common fluorescent labels. As a guide, 480 nm suits FITC, Alexa Fluor 488, Atto 488 and GFP; 530 nm suits mCherry, Alexa Fluor 568 and Atto 565; and 640 nm suits Cy5 and Alexa Fluor 647. Choosing the channel that matches the label, or using 280 nm for intrinsic fluorescence, is what lets FIDA measure a wide range of molecules with minimal sample modification.

Can FIDA detect a signal without a label?

Yes. Proteins emit intrinsic fluorescence from their aromatic amino acids, chiefly tryptophan, and FIDA can detect this directly at 280 nm, so many proteins can be sized and studied label-free. The label-free UV detector covers a hydrodynamic radius range from about 0.5 to 500 nm, and it has been used, for example, to size an E3 ligase at a hydrodynamic radius of 5.8 nm from its intrinsic fluorescence alone. Where a molecule has no useful intrinsic signal, or where work must be done in a complex matrix such as serum or lysate, a fluorescent label extends the same measurement. Source: Fidabio white paper Label-free biophysical assays using nanoliter sample volumes; label-free detection is also shown in the app note Reversible oligomerization and FIDA.

What readouts come from the detected signal?

From the same detected signal FIDA delivers three complementary readouts. The first is absolute molecular size, the hydrodynamic radius, obtained from Taylor dispersion and the Stokes-Einstein relation. The second is the Binding-Related Intensity Change (BRIC), which follows the change in fluorescence intensity when a binding event occurs. The third is Fida Lambda Dynamics, which measures the shift in the wavelength of the emitted fluorescence when binding changes the environment of the fluorophore. Together these mean a single detected signal reports size, binding and a shift in emission wavelength at once.

Common questions

Does FIDA use a laser?

No. FIDA uses LED excitation and PMT detection, not a laser, so the sample is not heated and its native state is preserved during the measurement.

How much signal does FIDA need?

As a rough guide, FIDA needs a labelled indicator at around 10 nM, or a protein at around 1 micromolar for label-free measurement using intrinsic fluorescence.

Can FIDA measure a protein without attaching a dye?

Yes. FIDA detects intrinsic protein fluorescence at 280 nm, so many proteins can be sized and characterised label-free, with a fluorescent label added only when the intrinsic signal is insufficient or the matrix demands it. Source: Label-free biophysical assays using nanoliter sample volumes.

Related resources

The white paper Label-free biophysical assays using nanoliter sample volumes shows label-free detection through intrinsic fluorescence across sizing, stability and particle work, and the app note Reversible oligomerization and FIDA is a worked label-free example.