You are finished with your super-arduous experiment. Now the fun part! To “read” your results with a microplate reader, but do you know which microplate reader to use? Or even that there are different types? Have no fear. I am here to help ye, scientific one! Read on for my pointers on picking the right microplate reader for your application.
What is a Microplate Reader?
Spectrophotometers have been used for decades in life science research to detect events in samples, but traditionally they have been limited to one sample at a time. However, with the introduction of microplate spectrophotometers (more commonly known as microplate readers) multiple samples can be read at once. Typically this is done with a 96-well plate containing numerous small-volume (50-200µL per well) samples.
Now when it comes to selecting the right microplate reader for you, there are other things to consider besides user interface capabilities and cost of the instrument. Even if the undergrads are only concerned about the former, and your PI the latter. Instead you must also consider the machine’s detection parameters, which can be done via 1) absorbance 2) fluorescence, or 3) luminescence.
1) Absorbance-based Microplate Readers.
Absorbance-based microplate readers use…you guessed it! Absorbance. Specifically, they measure the amount of ultraviolet or visible light absorbed by your sample. The exact wavelength used (and absorbed) of course depends on your sample.
This method is your “standard” plate reader and is most often used in colorimetric-based assays. Absorbance-based Microplate Readers, are moderately priced and are accurate for the reagents they detect. Common assays that use absorbance are MTT cell proliferation, cell viability, colorimetric ELISAs (enzyme-linked immunosorbent assays), nucleic acid (DNA, RNA) quantitation, and protein quantitation (e.g. Bradford assays).
2) Fluorescence-based Microplate Readers.
Fluorescence detection is a little more involved than just measuring absorption. Fluorescence detection works by:
- First: Illuminating a “fluorophore” in your sample over specific excitation wavelengths – the wavelength range needed to excite these fluorophores depends on the type of fluorophore used.
- Second: The energy from this excitation is then acquired by the sample and emitted as light (fluorescence) at different wavelengths – the wavelength range of the emission light is also fluorophore-dependent.
- Third: This emitted light is then collected and measured by an optical system (emission system).
Fluorescence-based microplate readers have a wider range of applications and are more sensitive than absorbance-based readers. Yay! Downside? These plate readers tend to be more expensive. Examples of fluorescence assays include FITC (fluorescein isothiocyanate)-based assays, fluorescence polarization (FP) assays, FRET (Förster resonance energy transfer) assays, or reporter gene assays such as GFP (green fluorescent protein).
3) Luminescence-based Microplate Readers.
Now, you might be wondering how luminescence differs from fluorescence? Well for one, they come from different critters. As I am sure you know, some living organisms glow, e.g. the firefly, the jellyfish, and certain bacteria. But did you know they do it in different ways? The jellyfish and bacteria use fluorescent proteins (e.g. GFP). These fluorescent proteins work by absorbing light at one wavelength and emitting light (fluorescing) at another. Conversely, bioluminescent glowing results from a chemical reaction; such is the case of the firefly.
So while both fluorescence and bioluminescence can cause a critter (or your sample!) to glow, the energy sources are different. Fluorescence is powered by light, and bioluminescence by chemical reactions.
Luminescence platforms are known for their high sensitivity, ability to offer low background, and a broad dynamic range for reporter gene assays and other applications. Also, because they read luminescence and not fluorescence, they do not require a light source for excitation so they are optically simpler than fluorescence readers.
The two most frequently used bioluminescent proteins in biological research is Luciferin-Luciferase and Aequorin – originally obtained, respectively, from the firefly and jellyfish. Common applications for luminescence include luciferase-based gene expression assays and ELISAs, ATP detection kits, as well as β-Glucuronidase (GUS) and β-Glucuronidase (β-Gal) reporter assays.
Single-mode vs Multi-mode
So now that you are familiar with the three major microplate platforms, you can decide which plate reader format is right for you. If you are only interested in only one particular platform, you can go with what is called a “single-mode” microplate reader that works by either absorbance, fluorescence intensity, OR luminescence detection. However, if your lab work is more diverse, you may prefer to purchase a “multi-mode” microplate reader, which can perform any of the three reading modes. These “multi-mode” microplate readers are great because they allow multiple assay types to be performed in one system. The tradeoff, of course, is that they are more expensive than the single-mode readers – duh. However, they tend to take up less space than three separate instruments, which is always good given how prime benchtop space is!
To summarize the three microplates’ major points, here is a handy-dandy table that I constructed just for you (I’m so nice!):
|Quick Comparison of Microplate Reader Platforms
|External Light Source Required||Yes||Yes||No|
|Sample Light Emission Required||No||Yes||Yes|
|Excitation Powered by||Light||Light||Chemical Reaction|