Plate readers & fluorometry
Excite the well at one wavelength and collect emitted light at a longer one. Sensitive down to picomolar fluorophore, but the readout is in arbitrary units and depends on the gain, the optics, the plate colour and the volume in the well.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Fluorescence
Excite the well at one wavelength and collect emitted light at a longer one. Sensitive down to picomolar fluorophore, but the readout is in arbitrary units and depends on the gain, the optics, the plate colour and the volume in the well.
How it differs from the standard method
- Use a black plate: clear and white plates let stray excitation light reach the detector and raise the background several-fold.
- Set the gain so the top standard reads 60–80% of full scale; clipped wells all report the same maximum number and cannot be quantified.
- Read from the bottom for adherent cells and from the top for solutions, and keep the emission band well away from the excitation band.
- Watch for the inner-filter effect and for compound autofluorescence, both of which bend or lift the curve at high concentration.
1One instrument, three different measurements
A microplate reader looks like a single machine but it makes three physically different measurements, and almost every mistake in plate-based work comes from treating them as interchangeable. In absorbance mode it shines light through the well from above to a detector below and reports how much was lost: the same measurement a cuvette spectrophotometer makes, with the depth of liquid as the path length.
Absorbance
Needs a clear, flat-bottom plate. Proportional to concentration and to liquid depth, so the well volume is part of the measurement.
Fluorescence
Needs a black plate to stop stray excitation light. Sensitive, but the signal depends on gain, optics, volume and the dye's environment.
Luminescence
Needs a white, opaque-walled plate. No excitation light means almost no background, which buys four or more logs of dynamic range.
Kinetic
Any of the three, repeated. The reader visits wells one at a time, so the read interval is limited by how fast the optics can travel.
2Choosing the wavelengths: filters or a monochromator
A fluorophore has an excitation spectrum and an emission spectrum separated by the Stokes shift. You want to excite near the peak of the first and collect near the peak of the second, while letting as little excitation light as possible reach the detector.
3Gain, saturation and what the detector can actually report
The photomultiplier's gain is a voltage setting, and its effect is steeply non-linear: a modest increase can multiply the signal several-fold. What it does not do is improve signal-to-noise, because it amplifies the detector's own dark current along with the signal.
4Three things about a well that a cuvette does not have
- Path length is whatever you pipetted
- A flat-bottom 96-well is about 0.32 cm², so 200 µL stands 0.62 cm deep, 100 µL 0.31 cm and 50 µL 0.16 cm. A plate absorbance is therefore not a cuvette OD, the conversion changes if someone pipettes 180 µL instead of 200, and at low volumes the meniscus is a large fraction of the depth. A round-bottom plate has no defined path at all.
- The inner-filter effect
- When the sample absorbs appreciably at the excitation wavelength, the far side of the well is less brightly illuminated than the near side, and emitted light is re-absorbed on its way out. Fluorescence then stops rising proportionally with concentration and the standard curve bends over. It is worse in a deeper well and worse with coloured or turbid samples, and it is the main reason you must never read a fluorescent sample above the top standard.
- The edge effect
- Wells around the outside of a plate evaporate faster and sit at a different temperature from the interior, so they read systematically differently, commonly 10–20% in a cell-based assay incubated for a day or more. If one replicate of each standard is on the edge and the other is not, the duplicates disagree and the whole curve tilts. Keep data in the interior and fill the outer ring with buffer or medium.
- Cross-talk
- Light from a bright well can reach its neighbours through the plastic. In luminescence this is why white plates have opaque walls: in a clear plate a well containing no cells at all, sitting next to a bright one, reads as though it had signal, and subtracting a blank from the other side of the plate does not remove something that depends on a well's neighbours.
5Kinetic reads: measuring a rate rather than a number
A kinetic read turns the plate reader into an instrument for measuring velocities. The quantity you want is almost always the initial rate, the slope while the substrate is still effectively unlimited, so you need several points on the straight part of the curve: two points are a difference, not a measurement, and they leave no residuals to show you that the line was not straight.
Key terms
Practise these as flashcards →Relative fluorescence or luminescence units: instrument-specific arbitrary numbers that depend on gain, optics, plate and volume. Only meaningful against something measured on the same plate.
The photomultiplier's amplification setting. It scales signal and detector noise together, so it sets where the signal sits in the detector's range rather than improving precision.
The gap between a fluorophore's excitation and emission peaks. A large shift makes it easy to separate the two bands and keeps the background low.
An optical filter passing a defined band, written as centre/width (485/20 = 475–495 nm). Efficient but fixed, unlike a tunable monochromator.
Loss of fluorescence because the sample itself absorbs the excitation or the emitted light, which bends a standard curve over at high concentration.
The depth of liquid: about 0.62 cm for 200 µL in a flat-bottom 96-well. It sets the absorbance and must be known to convert a plate reading into a concentration.
Systematic difference between the outer ring of wells and the interior, caused by evaporation and temperature gradients.
A measure of assay quality combining the separation of the positive and negative controls with their variability; above 0.5 is generally taken as screening-ready.
How long the detector counts photons for one well. Because luminescence is photon counting, precision improves as the square root of the counts collected.
When to use it
- Measuring the same thing in dozens or hundreds of samples at once: dose–response curves, viability and cytotoxicity assays, reporter assays, enzyme panels, growth curves.
- Following a reaction over time in many wells in parallel, where the quantity you want is a rate rather than an endpoint.
- Reading a fluorescent or luminescent reporter whose signal is far too dim to see down a microscope but easy to count in bulk.
- Quantifying nucleic acid or protein with a dye-based assay, where a fluorescent readout gives far more sensitivity and specificity than absorbance.
Compared with alternatives
A cuvette gives a defined 1 cm path and a scanning spectrum, so prefer it when the absolute absorbance or the shape of the spectrum matters rather than throughput.
An ELISA is one particular assay read on this instrument; the reader contributes the absorbance measurement, the plate layout and the standard curve.
Flow cytometry measures fluorescence per cell, so it separates a bright subpopulation from a uniformly dim one, something a well average can never do.
Microscope-based lifetime and FRET methods give spatial and sub-cellular information; a plate reader gives only the bulk signal of each well, but for hundreds of wells.
Most viability chemistries are designed around a plate reader, so the choice is which readout to use rather than which instrument.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Excite the well at one wavelength and collect emitted light at a longer one. Sensitive down to picomolar fluorophore, but the readout is in arbitrary units and depends on the gain, the optics, the plate colour and the volume in the well.
- Use a black plate: clear and white plates let stray excitation light reach the detector and raise the background several-fold.
- Set the gain so the top standard reads 60–80% of full scale; clipped wells all report the same maximum number and cannot be quantified.
- Read from the bottom for adherent cells and from the top for solutions, and keep the emission band well away from the excitation band.
- Watch for the inner-filter effect and for compound autofluorescence, both of which bend or lift the curve at high concentration.