How does a microplate reader measure absorbance?

Understanding How a Microplate Reader Measures Absorbance

A microplate reader, an essential tool in various scientific fields such as biology and chemistry, precisely measures the absorbance of samples in microplate wells. The fundamental principle behind measuring absorbance with a microplate reader involves the spectrophotometric analysis of samples. This detailed explanation outlines the steps and mechanisms involved in the process.

Key Components and Process

Light Source: The process begins with a light source within the microplate reader, typically a xenon flash lamp or a tungsten halogen lamp, which emits light across a broad spectrum of wavelengths.

Selection of Wavelength: Before the measurement, the desired wavelength is selected using a monochromator or filters. This selection is crucial because the absorbance reading will be specific to this particular wavelength, corresponding to the peak absorbance of the substance being measured.

Transmission Through the Sample: The selected light is then directed towards the wells of the microplate containing the samples. The amount of light passing through the sample (transmittance) varies depending on the concentration of the substance within the well.

Detection: Light that has passed through the sample is then captured by a detector. This detector measures the intensity of the transmitted light. The absorbance (A) is calculated based on the Lambert-Beer law, A = -log10(T), where T is the transmittance (ratio of the intensity of light passing through the sample to the intensity of the light before it enters the sample).

Analysis: Finally, the microplate reader's software analyzes the absorbance values, allowing researchers to infer the concentration of the analyte in each sample based on previously established calibration curves.

Application

Microplate readers are versatile instruments used in various assays, including enzyme-linked immunosorbent assays (ELISA), protein and nucleic acid quantitation, and cell viability tests. Their ability to quickly and accurately measure the absorbance of multiple samples simultaneously makes them invaluable in both research and diagnostic settings.