In-depth: Advances in laser technology

Highlighting a range of lasers that are compact, affordable and easily integrated into mass spectrometers for use in clinical settings.

Mass spectrometry (MS), which is used to identify molecules within a sample by measuring the mass-to-charge ratio of ions, is employed across many fields of study, including biology, chemistry, physics and clinical medicine. As the technology continues to evolve, so will the applications that can benefit from this important tool.

One field that is truly seeing the benefits of MS innovation is in medical research and diagnostics. Currently, MS is used in a lab setting to analyse samples containing biomolecular species such as peptides and proteins in tissue, bacteria and cells. Information related to a sample’s composition of abundant and in some cases trace biomolecules can be obtained. Advances in micro-positioning equipment and large digital storage arrays have spurred advances in MS Imaging (MSI) which produces detailed maps of biomolecule distribution in tissue. However, new methodologies that allow direct analysis in real time within the sample’s native environment means samples no longer need to be sent to a lab and can provide medical personnel with fast answers during critical surgical procedures.

At the core of this new methodology are fast pulsed, mid-IR lasers that are capable of desorbing and/or ionising sample material with little to no sample preparation. Combined with other techniques for transporting sample material and improving ionisation efficiency, these lasers provide mass spectrometry equipment OEMs with a powerful component in a much smaller package and lower cost than previous alternatives.

The Evolution of Mass Spectrometry

To understand the progress towards real-time MS analyses, a brief background summary of how MS works using different desorption/ionisation techniques as they pertain to liquid and solid samples is helpful. Typically, sample material is desorbed into the gas phase and ionized in a one step process, then the subsequently produced ions are accelerated through an electric and/or magnetic field, affecting speed and trajectory. Ions will change trajectory at varying magnitudes depending on their mass-to-charge-ratio. The differences in mass-to-charge ratio allow a mass analyser to sort the ions, then produce a result that notes the relative abundance of the detected ions (based on the mass-to-charge ratio). Components of the sample are identified by correlating known masses of substances to the masses identified in the sample or through a fragmentation pattern.

There are many ways to desorb and ionise molecules. One of the traditional methods involves bombarding the sample with a highly energetic source, such as a corona discharge or a stream of highly charged ions. While such sources are highly efficient, they can easily destroy large, labile biomolecules such as peptides and proteins that are important to medical diagnostic testing. In some cases, fragmenting the molecules is the goal as such fragments can provide structural information if broken in a reproducible manner. However, there are many times when it is necessary to retain the structure of the molecules, which requires a different approach.

Two soft, ‘gentler’ desorption/ionisation techniques that allow the intact detection of biomolecules were awarded the 2002 Nobel prize in chemistry – the UV MALDI (ultraviolet matrix-assisted laser desorption and ionisation) technique and electrospray ionisation (ESI). UV MALDI uses a pulsed UV laser operating in the 330-360nm range that strikes a matrix of molecules with ultraviolet light to vaporise, desorb and ionise the molecules before introducing them into the mass spectrometer for analysis. The UV MALDI technique was one of the first to involve fast pulsed lasers for mass spectrometry. ESI involves dissolving sample material into an injectable liquid that forms an aerosol when subjected to a high voltage. Often, complex samples require separating out components before ESI using liquid chromatography due to the formation of multiple charges resulting in a complex mass spectra. However, major benefits to this technique include high ionisation efficiency and the ability to work at atmospheric pressure.

“With the UV MALDI technique, pulsed UV laser light alone can still be too energetic and fragment larger molecules. So, it was discovered that if you add a co-absorbing matrix in solution [a separate molecule added with a larger molecule like a protein] and allow them to air dry into a thin, crystalline film, you could desorb and ionize large biomolecules intact,” says Dr Mark Little, a consultant for Opotek, a manufacturer of tunable lasers for research and diagnostics.

Although the UV MALDI technique was a significant advancement that is still used today, it is not without its drawbacks. Samples must be mixed with a matrix material to be analysed, which complicates sample preparation to such a degree that expensive ‘matrix sprayers’ were developed to achieve more reproducible thin films. The matrix material also gets desorbed, ionised and detected which can interfere with the analysis of lower mass molecules. This process is further complicated because sample preparations are typically loaded into a high vacuum chamber through an interlock system and remain under vacuum to achieve the proper result.

To address the limitations of the UV MALDI process, researchers have sought ways to remove the matrix requirement and analyse samples under atmosphere pressure. “After the invention of the UV MALDI process, longer wavelength lasers were tested in the mid-IR region,” according to Little. “It was discovered that IR MALDI offered enough energy to ionise the molecules without fragmentation, while eliminating the need to add a comatrix.” To accomplish this, a pure protein compound is deposited on an IR transparent substrate to avoid local heating effects and directly hit with a pulsed, mid-IR laser. If the sample has high absorption at the mid-IR laser wavelength, it acts as its own matrix. The molecules are desorbed and ionised without significant fragmentation. Unfortunately, larger biomolecule concentrations are needed due to the lower ionisation efficiency of using matrix-free, IR MALDI versus UV MALDI.

Atmosphere pressure (AP) UV MALDI sources have been developed to alleviate
the need to load samples into high vacuum but ionised sample material can be lost during transport into the mass spectrometer resulting in lower detection efficiency. Lower ionisation efficiency of IR MALDI combined with sample material loss during transport from AP sources required a new solution. The solution presented itself in the form of a novel hybrid technique where the efficient and soft (gentle) desorption of pulsed, mid-IR lasers could be combined with the efficient ionisation of atmosphere pressure ESI source. The results of this new technique have been presented under a variety of names (ELDI, LAESI, MALDESI) and commercial product attempts but a final, inexpensive MS source and ‘killer application’ remain elusive.

Enter Real-Time Medical Diagnostics

All biological samples such as tissue contain a large amount of water which is the medium under which most biological processes take place. Water has the highest mid-IR absorption around 3 microns and, therefore, mid-IR lasers operating in this region allow endogenous sample water to act as a light absorbing matrix.

The most common lasers operating in the 3 micron range include the Er:YAG and optical parametric oscillator (OPO) lasers. Due to electron to photon inefficiencies required large power supplies inherent in YAG products, Er:YAG lasers proved too large and cost prohibitive to be implemented in a compact MS source. First generation mid-IR OPO lasers were also bulky and expensive until the company decided to dedicate its research division into the creation of a ‘shoebox’ sized mid-IR OPO laser head for MS sources. While still requiring a ‘briefcase’ sized power supply with internal water-cooling, the company was successful in the creation of the Opolette 2940, a 2.94 micron OPO laser with a laser head footprint of 9.5 x 4.5 x 7.5 inches.

To shrink the footprint to the smallest possible size while keeping costs under control, the team removed the internal mechanisms for tunability since they are not required for mass spectrometry and integrated one of the smallest OPO ‘pump’ lasers commercially available. Considerable research also focused on testing numerous fibre optical interfaces for safe and easy delivery of laser light to the area of interest. The Opolette 2940 ships to end-users and OEM integrators without the need for installation by an engineer. The final cost of the system is less than half the cost of an Er:YAG laser and lower in cost than other competing OPO lasers of comparable specifications.

Future product development is focused on removal of the external ‘brief-case’ sized power supply and its internal water-cooling requirements. The goal is to increase the efficiency of the OPO ‘pump’ laser so that only a 24V power supply is needed. Internal water cooling will be replaced with a simple heatsink and fan. The end product in development would retain close to the same volume as the current generation. Other areas of advancement are concentrating on increasing the sample rate (repetition rate) of the laser to speed up analyses or provide more sample material for detection.

“With a compact, cost-effective mid-IR laser solution in hand, integration into a mass spectrometer equipped with an atmospheric pressure ESI source is sure to impact the medical field,” says Little. Biological tissue, for example, can now be analysed in real-time during a procedure at the doctor’s office or in surgery. In addition, with the advent of AI and sophisticated surgical robots, surgeries would gain an incredible diagnostic tool to power ‘doctor-less’ hospitals where the surgeon performs the operation from a different location.

“This type of diagnostic tool could be used for procedures such tumour removal because it can identify where the diseased tissue stops and the healthy tissue starts,” says Little. “Similar real-time sampling could be used to analyse anything from biological tissue to explosive material, in the moment, without shipping samples to a lab.”

The inherent focusability of lasers are also capable of sampling incredibly small size areas measured in tens of micrometres, a key benefit for MS Imaging. “With this fine level of selection criteria, you can take a complex sample and raster and move the laser to different areas and only remove the material from a very small area,” says Little.

“With a slice or cross section of a tissue sample, for example, you could desorb the material piece by piece, in sizes ranging from 10 microns to 100 microns, saving and analysing the data as you go. Then you move the laser to the next spot and repeat the process. This can produce a detailed image ‘map’ of the entire cross section,” adds Little.

As mass spectrometry uses become more widespread and advancements continue, particularly in the medical field, it will become increasingly important for equipment to be as compact and affordable as possible. OPO lasers help ensure the pinnacle of functionality in a compact design that helps to reduce the overall size and cost of mass spectrometers. In combination with improved ionisation techniques that allow the equipment to be used in atmospheric conditions, mass spectrometry is sure to impact medical diagnostics and research for years to come.

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