Fluorescence has been documented at various points in history. In fact, Pliny described rubbed jellyfish slime as a type of torch way back in 79 AD. However, it wasn’t until 1852 Sir George Stokes was credited with officially describing fluorescence, noting the bright blue-white emission coming from fluorite after UV exposure.Following this, fluorescent microscopes were designed to study the natural fluorescence in some species of bacteria, plants, and animals. Albert Coons eventually founded the field of immunofluorescence in the 1940’s, after using antibodies labeled with fluorescence dyes for fixed tissues and cells. Since then, techniques using fluorescence skyrocketed, allowing scientists to discover, label, and determine the function and structure of genes, proteins, organelles, and more. Now, fluorescence measurements can be easily obtained through fluorescence microplate readers in the lab!
Absorbance vs Fluorescence Microplate Readers
Many scientists are familiar with absorbance, where the concentration of a solution is directly proportional to the absorbance of light in that solution. Particularly, absorbance is the quantity of light that is absorbed by a solution. Since its discovery, absorbance has been used to measure nucleic acids, protein concentration and expression, cell viability, and microbial growth, and more.
Contrarily, fluorescence is the elaborate phenomenon where scientists measure light emitted from a sample. To understand fluorescence, we have to reflect back to general chemistry. First, we know that samples exist in a low-energy ground state. When we want to measure fluorescence, UV or short wavelength visible light is aimed at the sample. This “excites” the sample, causing the sample to absorb some of this light energy and enter a high energy state. However, given how atoms favor low- energy states, the sample releases back this energy, emitting it as light. This energy is released in intermediate steps, so the amount of light emitted will be less than the amount of light absorbed. Meaning, the wavelengths in which scientists measure fluorescence will always be lower than the wavelength used to excite the samples.
Uses for fluorescence are similar to that of absorbance, where it is now a common technique for techniques related to nucleic acid and protein studies, cell viability, and growth. However, fluorescent alternatives are superior for two key reasons: specificity and sensitivity.
Scientists can measure fluorescence of targets by designing probes that attach directly to the molecule of interest. These probes are designed to absorb a specific wavelength and emit at a lower, longer wavelength. Furthermore, as a highly sensitive mode of detection, some fluorescence techniques have been capable of detecting as low as a single molecule in samples.
Uses for Fluorescence Microplate Readers
Methods optimizing fluorescence has skyrocketed throughout the years, with particular advancements in areas such as:
- Molecular Biology
- Gene Editing
- Protein Studies
- Lipid Studies
- Small Molecules
- Live Cell Cultures
- Microbiology
Fluorescence can be used as a verification tool to visually validate proper execution of experimental procedures, such as for positive controls for molecular transfections, and confirming detection of targets, such as in medical diagnostics. In addition, it can be used to add quantitative data, such as providing values for protein and enzyme activity assays, such as ELISAs and cell growth assays. Traditionally, many of these techniques were done in separate instruments, such thermocyclers, flow cytometers, or spectrophotometers. Now, fluorescence microplate readers are capable of handling multiple fluorescent applications in a contained system.
Common Applications
Molecular biologists are highly familiar with determining DNA and RNA concentrations by measuring absorbance at 260/280 nm. However, given absorbance measures light absorbed by the overall sample, contamination from salt, ethanol, and other impurities can affect the absorbance and cause inaccurate readings. Fluorescence microplate readers counter this disadvantage by targeting specific structures, such as single-stranded or double-stranded DNA or RNA, thereby providing more accurate concentration readings for the user.
In addition, fluorescence being highly specific allows for additional molecular methods, such as measuring gene expression via qPCR and sequencing DNA via Sanger sequencing or next- generation sequencing. These methods have also been adapted so that they can be completed in a multi-use fluorescence microplate reader!
Fluorescent immunostaining is also a common technique for protein applications, such as immunocytochemistry (ICC), immunohistochemistry (IHC), Western blot, enzyme linked immunosorbent assays (ELISA), and confocal microscopy. Because fluorescence is the process of measuring emitted light, it is common to use these techniques as visual methods for detection. However, technology has since advanced, allowing researchers to use fluorescence microplate readers to obtain more quantitative data, such as automated Western blots that can be read in a fluorescence microplate reader. Fluorescent versions of ELISAs have also shown advanced sensitivity in fluorescence microplate readers as well.
Convenience of Fluorescence Microplate Readers
The ability to design probes specific to target molecules allows research flexibility and accuracy in the molecules they can study. In addition to nucleic acids and proteins, scientists can design specific probes that attach to and fluoresce lipids, other organic compounds such as aldehydes, and inorganic compounds such as gaseous molecules. Being highly specific, many of these methods can be completely done with small sample sizes and live cultures within 96-well microplates.
The universality of 96-well microplates offers technical flexibility and simplicity for scientists, as they can culture specimens, extract and purify molecules, confirm detection, and obtain quantitative results all within the same plate. In particular, users can completely automate these steps in a simple, cohesive laboratory automation system. For example, microbes can be grown in a plate and DNA purified that is PCR ready. Or, proteins can be extracted, reagents added, and the plate washed in fast, accurate, and easy-to-manage automated ELISA workstation. All of this can be coordinated with automated laboratory scheduling software, with the fluorescence microplate reader being the key instrument.
Using a fluorescence microplate reader can give your lab valuable data. To find out more about the applications you can use with your microplate reader, contact Hudson Robotics today!
