RMM Tutorial: Optical Spectroscopy Technologies

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Scientific Principles

Optical spectroscopy is an analytical tool that measures the interactions between light and the material being studied. Light scattering is a phenomenon in which the propagation of light is disturbed by its interaction with particles. There are a number of light scattering principles that may be utilized in rapid method technologies; therefore, it is appropriate to quickly review some of these principles in order to understand the scientific basis for the RMMs that will be discussed on this page.

Elastic Scattering

Elastic scattering is one of the specific forms of light scattering. In this process, the kinetic energy of the incident particles is conserved, and only their direction of propagation is modified. One form of elastic scattering is called Rayleigh scattering. When the particle size is much smaller than the wavelength of the incident light, the scattering is preferential to the shorter wavelength component of the light. An example may be found simply by looking up in the sky. Blue skies are produced as shorter wavelengths of the incoming visible light (blue and violet) are selectively scattered by small molecules of oxygen and nitrogen.

However, when the particle size is much larger than the wavelength of the incident light, all of the visible wavelengths are scattered more or less equally. This type of scattering is known as Mie scattering. An example of this type of scattering can also be observed when looking up in the sky, but this time, focus on the clouds. Because cloud droplets are larger than the incoming visible light, almost all of the light that enters clouds will be scattered, producing a white color. A similar effect occurs in mist or fog, when the lights from an oncoming vehicle appear like a white halo.

Whether or not these types of light scattering principles can be used in rapid methods will depend on the direction the light is scattered and the relationship between the light scattering and particle size. For example, in Rayleigh scattering, light is scattered in all directions and is not very sensitive to particle size. However, in Mie scattering, the scattered light is concentrated in a forward direction, and the scattered portion of the light is proportional to the particle size. This is why Mie scattering is used in many commercial particle detectors, and also forms the basis for using light scattering in environmental monitoring RMMs.

Instantaneous Active Air and Water Monitoring Using Intrinsic Fluorescence

A number of commercially available RMMs utilize Mie scattering for the real-time and continuous detection, sizing and enumeration of airborne microorganisms and total particles. For example, when an air sample is processed through a representative system, the detector will size and quantify particles from 0.5 to more than 10 µm in size. At the same time, a 405 nm laser that intersects the particle beam will cause biological material, such as microorganisms, to autofluoresce, due to the presence of NADH, riboflavin or dipicolinic acid. Fluorescing particles are recorded as biologic particles, and non-fluorescing particles are recorded as inert particles. The technology is highly sensitive, does not require any reagents or consumables, and provides instantaneous, simultaneous and continuous total particle and viable particle detection. Similar technologies using Mie scattering and intrinsic fluorescence have been developed for the testing of water samples, in which the instrumentation can be attached directly to water distribution loops.

Two PDA Journal of Pharmaceutical Science and Technology publications highlights the use of light scattering for real-time, active air monitoring. The first paper compares a Mie scattering RMM with an MAS-100™ and a CLiMET CI-450t in a cleanroom environment, and the second paper describes a case study in environmental monitoring during aseptic filling, intervention assessments, and glove integrity testing in manufacturing isolators (both of these papers can be downloaded from our References Page). Briefly, the data for the RMM and the CLiMET followed a similar trend of increasing counts for both the ≥ 0.5 μ m and the ≥ 5.0 μ m total particles when sampling progressed from the most controlled area (Grade A) to the least controlled area. Zero viable particle counts were observed for both the RMM and the MAS when sampling the Grade A location.

Next, continuous monitoring of three separate isolators for more than 16 hours and representing more than 28 m3 of air per isolator (under static conditions) yielded a mean viable particle count equal to zero (0) per cubic meter. No viable particles were detected during the manual transfer of sterilized components from transfer isolators into a filling isolator, and similar results were observed during an aseptic fill, a filling needle change-out procedure, and during disassembly, movement, and reassembly of a vibrating stopper bowl. During the continuous monitoring of a sample transfer port and a simulated mousehole, no viable particles were detected; however, when the sampling probe was inserted beyond the isolator-room interface, the RMM instantaneously detected and enumerated both viable and nonviable particles originating from the surrounding room. Finally, data from glove pinhole studies showed no viable particles being observed, although significant viable particles were immediately detected when the gloves were removed and a bare hand was allowed to introduce microorganisms into the isolator.

Inelastic Scattering

Inelastic scattering is different than elastic scattering, in that the kinetic energy of an incident particle is not conserved. In an inelastic scattering process, some of the energy of the incident particle is lost or gained. An example is Raman scattering, usually from a laser light in the visible, near infrared, or near ultraviolet range. When the laser interacts with a molecule, the energy of the laser photons is shifted up or down. This shift in energy provides information about the vibrations and rotations of the molecule. Because each molecule has its own unique Raman spectrum, or fingerprint, we can use Raman spectroscopic techniques to identify microorganisms.

One RMM technology that is currently under development utilizes Raman spectroscopy coupled with viability staining to provide a microbial identification without the need for microbial growth. A test sample is first retained on a supported film, stained with a viability reagent and then the surface is examined for microscopic particulates. A spectral signature is provided for each particle that gives a positive fluorescent signal (i.e., particles that pick up the viability stain), which are then statistically correlated with spectral signatures in a library composed of Raman signatures of known microorganisms. If there is a match, a microbial identification is provided. The technology is able to target a single cell for microbial identification, and, under the right conditions, enumeration, within minutes. The system’s capabilities allow for the analysis of air and liquid samples, and virtually anything that can be passed through the filtration membrane/support film.


Optical spectroscopic-based rapid microbiological methods represent the next generation in real-time or close to real-time detection and identification opportunities for the pharmaceutical and biopharmaceutical industries. These types of technologies offer an unprecedented advantage over conventional, growth-based methods for monitoring the state of microbiological control in manufacturing environments, and represents significant progress toward the acceptance of microbiology Process Analytical Technology (PAT) solutions that may one day, support the elimination of off-line or laboratory-based assays, and the parametric release of aseptically-filled products.