Top reading is usually more sensitive
Top reading usually provides better signal-to-noise ratios for solution-based assays such as DNA quantification or protein quantification. In general, reading fluorescence from the top is more sensitive than reading from the bottom. This is a result of the light being attenuated and scattered by the plastics of the well-bottom which can increase the background and decrease the efficiency of the measurement. Thus, the quality and thickness of the bottom clear plastic and the type of fluorophore are important considerations when designing your assay.
Microplate type is critical for bottom reading
Another factor to consider is of course the clear-bottomed plate type you are using – are you using an opaque-walled tissue-culture plate? Is the plate white or black? and, is the clear bottom made of either polystyrene or glass?
Answering the questions above is closely linked to the light wavelength of interest. If you are trying to detect visible light (350-900 nm) typically a plastic bottom will do. However, detecting below 350 nm requires a bottom made of UV-transparent material, for example quartz or modern COP/COC polymers. As a last resort, the more expensive optical glass bottom plates can be used allowing for detection from both, the top and bottom, as well as microscopic viewing of the cells with low background.
The key difference between white and black plates is their reflective properties. White plates reflect light and will maximize the light output signal. On the other hand, black plates absorb light and reduce background and crosstalk. Thus, white plates are commonly used for luminescent assays while black plates are used for fluorescent assays.
When does bottom reading provide better results?
- When working with adherent cells that grow at the bottom of the plate and either express, bind, or secrete a fluorophore at or close to their bottom attachment side only.
- Chemotaxis assays monitoring the migration of cells through a membrane. This is analysed typically by a combination of both, top and bottom reading.
- Certain cell-based assays (e.g. GeneBLAzer® or QBT™ Fatty Acid Uptake) cannot be read from the top as the optical properties of the buffers used in the assay interfere with the excitation or emission of the fluorophore.
If still in doubt which reading technology to use, you can program your microplate reader to run a protocol including both, top and bottom reading and compare the results to make your final choice.
GeneBLAzer® is a registered trademark of Invitrogen Corporation.
QBT™ Fatty Acid Uptake is a trademark of Molecular Devices.