Optical density and absorbance measurements

Absorbance measurements, often referred to as light absorbance, are so versatile and widespread that they are one of the first techniques many life science researchers encounter in a practical setting. Absorbance measurements are taken at a particular wavelength to determine the amount of light absorbed by a substance. Optical density is also widely used for many scientific applications. Whether it is determining concentrations of molecules like proteins or DNA, looking at enzyme kinetics for crucial reactions, or measuring something as fundamental as cell growth, you will find references to absorbance or optical density measurements. But how do they relate to each other?

In this blog, we examine the utility of absorbance and optical density measurements, including examples where microplate readers can help. We look at some widely used practical applications, consider some definitions, and shed some light on recommended usage.

What are absorbance and optical density?

Relationship of absorbance to transmitted light

One way to quantify absorbance is in terms of the light transmitted through a sample (Eqn 1).

Transmittance (T) is a direct measurement of how much light passes through a sample (Fig. 1).

Transmittance is often expressed as a percentage which is calculated from the ratio of the intensity of the incident light (I₀) to the intensity of the light transmitted (I):

A graphical representation of the relationship between absorbance and transmission is shown in Fig. 2.

This graphic shows a range of absorbance (A) values calculated for different transmission values using the following equation:

Note: While absorbance is a unitless quantity, it is quite common to see units of AU cited that are said to stand for arbitrary units or absorbance units. Absorbance is unitless and the use of units should be avoided. 

The Beer-Lambert law

How much light or the fraction of incident light absorbed by a solution at a particular wavelength is related to the thickness of the absorbing layer and the concentration of the absorbing species. These two relationships are expressed by the Beer-Lambert law¹:

where c is the concentration of the substance being measured, l is the path length, and ε is the absorption coefficient. 

The absorption coefficient is a measurement of how strongly a substance absorbs light at a specific wavelength and is essential for determining the concentration of a substance in solution. Equipped with the Beer-Lambert law, it is possible to work out the concentration of an absorbing substance if the absorption coefficient, pathlength and absorbance are known. As we will see later, this is very helpful for many practical applications in the life sciences, especially when dealing with precise measurement data.

An important factor in all absorbance or optical density measurements is the path length. In measurements with a cuvette, the path length is typically 1 cm. In microplates, however, the path length can vary from a few hundred micrometers to several millimeters. To compensate for this, BMG LABTECH offers automatic path length correction. This function automatically corrects the measured absorbance values to a 1 cm path length independent of the individual fill volume of each well. However, this method of path length correction is problematic when measuring OD₆₀₀. Light scattering by microbes occurs across a broad range of wavelengths including from 600 nm to 1000 nm. Because scattering interferes with absorbance measurements at 1000 nm, using a water peak-based path length correction can lead to inaccurate OD₆₀₀ values. Instead, a volume-based correction method, which accounts for well dimensions, is recommended. If plate dimensions and the used volume are available, the path length is calculated and applied by the software.

You can read more about absorbance and the Beer-Lambert law here.

Optical density values

Over the years, scientists have used the term optical density (OD) interchangeably with absorbance (e.g. Eqn 4) as follows:

However, optical density has also been used in other contexts which has sometimes led to confusion. For example, optical density is commonly used to refer to light scattering and OD measurement. The International Union of Pure and Applied Chemistry therefore discourages the use of optical density when absorbance is meant for reasons of clarity and consistency.2,3 In any other usage of optical density, it is recommended to clearly and precisely state what is being measured.

Optical density measurements at 600 nm (OD₆₀₀), which predominantly measure light scattering, are widely used to monitor microbial growth. This is a useful way to measure the growth of bacteria and yeast in culture and OD₆₀₀ is a widely used metric in microbiology and biotechnology for quantifying the growth of microbes.

OD measurements at 600 nm are crucial in various applications, including fermentation processes, antibiotic susceptibility testing, and the production of recombinant proteins. By providing insights into the physiological state of cultures, OD₆₀₀ measurements help optimize growth conditions and improve yields in industrial applications. The great popularity of the OD₆₀₀ method can also be explained by the fact that it is a non-destructive approach. It can be performed as often as needed during the growth of a culture without killing or harming the microbes.

You can read more about optical density and usage for the measurement of light scattering in the HowTo Note “How to optimize OD₆₀₀ measurements”.

Despite the widespread use of the OD₆₀₀ method, it is important to know the suitability of your microorganism for this type of measurement. For example, this method may not be satisfactory for some bacteria that significantly transmit light (so called “optical density transparent” bacteria) that exhibit low levels of light scattering. Since the size of bacteria can influence optical density measurements, you also need to be aware of whether the size of your bacteria is changing during culture. In addition, some optical density measurements at 600 nm can be altered due to absorbance by bacteria in this region of the visible spectrum. This applies particularly to cyanobacteria and other pigment-containing bacteria.⁴

What are the limits of absorbance detection?

Absorbance measurements may not always be a linear relationship at high concentrations of the substance being measured (Fig. 3). In practice, measurements with an absorbance of 3-4 may be subject to greater error and be less accurate. Measurement of samples with an absorbance greater than 3.0 is therefore not recommended for reliable quantitative measurements. If readings are too high, it is advised to dilute samples and factor in the dilution factor to the final measurement. For the highest possible accuracy and precision, absorbance values between 0.1 and 1.0 are recommended which correspond to 90% and 10% of light transmission, respectively.

Applications of absorbance and optical density in microplate readers

Absorbance and optical density measurements can be used for many applications in the life sciences including protein quantification nucleic acid quantification, cell viability assays, microbial growth assays or other measurements. Microplate readers offer distinct advantages to researchers interested in making absorbance and optical density measurements for these types of assays. By offering higher throughput (96-, 384- and 1536-well plates), they enable considerable savings in time and resources (for additional benefits see Table 1). 

Table 1. Benefits of microplate readers for absorbance or optical density assays.

Protein quantification

Protein quantification assays that depend on absorbance measurements are often performed in the laboratory. In the application note Absorbance-based methods for protein quantification several examples of the most commonly used applications are given including measurement of the absorbance at 280 nm, use of the Bradford assay⁵ (Fig. 4), and other options like the Lowry or bicinchoninic (BCA) assays. Further tips on how to select the best method are described in the BMG LABTECH blog Protein measurement: find a suitable method. For some applications, only a limited sample volume is available. In these cases, BMG LABTECH offers the LVis plate, which enables absorbance measurements to be carried out in small sample volumes as low as 2 µl per measurement spot.

Nucleic acid quantification

Researchers are often faced with the need to quantify nucleic acids in samples. Many experiments rely on the use of known amounts of specific nucleic acids and success depends on being able to assess the levels of nucleic acids quickly with precision and accuracy. Another crucial parameter is DNA purity since impurities or contaminants may affect the successful implementation of subsequent experiments using purified DNA samples. Nucleic acids show natural absorbance at 260 nm (Fig. 5). Absorbance measurements at this specific wavelength can be used to determine DNA or RNA concentrations quickly. By taking absorbance measurements at other specific wavelengths (230, 280 and 340 nm) an assessment of nucleic acid purity can also be determined. Examples of this type of approach are given in the application note DNA purity ratio: evaluation of nucleic acid quality. The accuracy and precision of these measurements are validated by the measurement data shown. For these types of measurements, BMG LABTECH’s ultrafast spectrometer instantly captures an entire spectrum from 220 to 1000 nm in less than 1 second/well which eliminates the need for slower sequential measurements at each specific wavelength as is the case for conventional monochromators. The signature UV/vis spectrometer is available on all of BMG LABTECH’s single- and multi-mode microplate readers with absorbance detection. BMG LABTECH was the first microplate reader manufacturer to incorporate a UV/vis spectrometer for absorbance detection into its multi-mode instruments. In addition, the predefined templates available in the MARS data analysis software further simplify and accelerate the analysis of the data.

Absorbance measurements are also useful for following redox reactions. NADH/NAD⁺ and NADPH/NADP⁺ are cofactors used by many enzymes including those involved in energy metabolism, mitochondrial function, calcium homeostasis, oxidative stress, gene expression, immunological functions, aging and cell death. The reduction of NAD⁺ to NADH and NADP⁺ to NADPH can be monitored at 340 nm because the oxidized forms do not absorb light at this wavelength (Fig. 6). An example of this type of application for absorbance measurements is given in the application note ELISA assays and NADH/NADPH conversion detection.

ELISAs 

Enzyme-linked immunosorbent assays or ELISAs are an essential technique in today’s laboratory with many applications in the life sciences.⁶ They are often the method of choice to detect or measure specific biological molecules (analytes) for diagnostics, drug discovery or fundamental research. Absorbance measurements are used in many ELISA assays to quantify a specific protein in solution. An antibody immobilized on a microplate well is used to bind specifically to a protein of interest and binds this protein to the microplate. A second antibody that is also specific for the protein of interest binds to the captured protein and is recognized by a secondary enzyme-bearing antibody. The higher the amount of the protein of interest in the sample, the more enzyme will be bound to the microplate well. The conversion of a substrate by the enzyme bound to the secondary antibody into a chromophore is used to allow for measurement by absorbance and quantification of the amount of the specific protein present in the sample. An example of this type of application for absorbance measurements is given in the application note ELISA assays and NADH/NADPH conversion detection.

Microbial growth

Measurements of the optical density at 600 nm or OD₆₀₀ are widely used to quantify the growth of microorganisms. These measurements are made in the absorbance mode of a microplate reader but in reality they measure the amount of light scattering from the microorganisms present in solution. At 600 nm most bacteria do not have significant absorbance but will mainly scatter light proportional to the number of organisms present. The application note High-throughput determination of bacterial growth kinetics using a BMG LABTECH microplate reader shows how optical density at 600 nm (OD₆₀₀) measurements allow the growth of bacterial mutants of Salmonella enterica serovar Typhimurium to be monitored in the presence of biocides (Fig. 7). In this case, kinetic measurements of bacterial growth were made at high throughput to assess the impact of different biocide agents on the different mutants used in the study. This type of analysis provides useful information on the possible impact of different biocidal interventions in environmental settings.A major advantage for the investigation of microbial growth in real-time is provided by readers that have an incubator function. The VANTAstar^®, the CLARIOstar^® Plus, the Omega series and NEPHELOstar^® Plus can be combined with the Atmospheric Control Unit (ACU) making them the preferred choice for different kinds of live cell assays including absorbance-based OD₆₀₀ bacterial growth assays. The ACU provides a complete solution to fully and independently regulate both O2 and CO2 gas levels within the microplate reader chamber. For example, the ACU has been successfully used to cultivate group B streptococcus in a CLARIOstar multimode reader for 12 h and monitor the growth rate in parallel with the OD600 method as shown in the application note “Expression of a stable green fluorescent protein mutant in group B Streptococcus: Growth, detection and monitoring with the CLARIOstar”.

Cell viability

Absorbance measurements at specific wavelengths also play a role in colorimetric assays used to assess cell viability.⁷ In this type of assay, absorbance-based methods depend on the ability of metabolically active cells to reduce substrates such as 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) or resazurin. The reduced forms of these substrates absorb at specific wavelengths and produce a signal that is directly proportional to the metabolic activity of the cells which is an indicator of cell viability. The application note Viability assays: a comparison of luminescence-, fluorescence-, and absorbance-based assays to determine viable cell counts describes the use of different detection options to assess cell viability including the use of absorbance assays with the Vybrant^® MTT Cell Proliferation Assay Kit (Fig. 8). You can read more about this type of colorimetric assay in the BMG LABTECH cell viability blog.

For cell viability studies in real-time, if necessary, with the help of the ACU, injectors provide additional support to simplify processes and reduce hands-on time. They can be used, for example, to add test substances with a potentially toxic effect during a kinetic study of cell viability over several hours or even days at a specific point in time. The Omega series, CLARIOstar Plus,  and PHERAstar^® FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection. The VANTAstar can be equipped with a modular injection unit for this purpose.

Conclusion

The demand for absorbance and optical density measurements will continue to rise as the life sciences continues to grow in the coming years. New opportunities in all areas of research, including clinical and environmental applications, will benefit from the inclusion of microplate readers as part of solutions for increased efficiencies and the optimal use of resources. Further advances in technology and innovation will increase accessibility to these versatile tools for absorbance and optical density measurements.

References

1. IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version: (2014) "Beer–Lambert law (or Beer–Lambert–Bouguer law)". doi:10.1351/goldbook.B00626
2. IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version: (2008) "Absorbance". doi:10.1351/goldbook.A00028
3. Glossary of terms used in photochemistry (IUPAC Recommendations 1996) Pure and Applied Chemistry 68, p. 2257. Source: tip 3 in OD600 measurement: 4 facts and tips to know for bacterial culture growth https://byonoy.com/journal/od600-4facts-4tips-bacterial-culture-growth/ Accessed October 31 2024.
4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 72:248-254. doi: 10.1006/abio.1976.9999.
5. Crowther, J.R. (1995). Chapter 2: Basic Principles of ELISA. ELISA: Theory and Practice. Humana Press. pp. 35–62. ISBN 0896032795.
6. Riss, T. L. (2013) Cell Viability Assays. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences. https://www.ncbi.nlm.nih.gov/books/NBK144065/