Quasi-continuous infrared matrix-assisted laser desorption electrospray ionization source coupled to a quadrupole time-of-flight mass spectrometer for direct analysis from well plates.

High-throughput screening (HTS) is a technique mostly used by pharmaceutical companies to rapidly screen multiple libraries of compounds to find drug hits with biological or pharmaceutical activity. Mass spectrometry (MS) has become a popular option for HTS given that it can simultaneously resolve hundreds to thousands of compounds without additional chemical derivatization. For this application, it is convenient to do direct analysis from well plates. Herein, we present the development of an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled directly to an Agilent 6545 for direct analysis from well plates. The source is coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer to take advantage of the high acquisition rates without sacrificing resolving power as required with Orbitrap or Fourier-transform ion cyclotron resonance (FTICR) instruments. The laser used for this source operates at 100 Hz, firing 1 pulse-per-burst, and delivers around 0.7 mJ per pulse. Continuously firing this laser for an extended duration makes it a quasi-continuous ionization source. Additionally, a metal capillary was constructed to extend the inlet of the mass spectrometer, increase desolvation of electrospray charged droplets, improve ion transmission, and increase sensitivity. Its efficiency was compared with the conventional dielectric glass capillary by measured signal and demonstrated that the metal capillary increased ionization efficiency due to its more uniformly distributed temperature gradient. Finally, we present the functionality of the source by analyzing tune mix directly from well plates. This source is a proof of concept for HTS applications using IR-MALDESI coupled to a different MS platform.

Transforming a Mid-infrared Laser Profile from Gaussian to a Top-Hat with a Diffractive Optical Element for Mass Spectrometry Imaging.

Many mass spectrometry imaging (MSI) applications such as infrared matrix-assisted electrospray ionization (IR-MALDESI) employ an infrared (IR) laser with a Gaussian profile where laser irradiance is highest in the center and decreases exponentially. To enable full ablation of a square region of interest, oversampling is often needed, which results in nonuniform ablation and leads to decreased image quality. A diffractive optical element (DOE) was integrated into the optical path to generate homogeneous intensity distributions while maintaining laser energy above the ablation threshold, to enable complete sample removal from laser pulses without oversampling. 2D and 3D imaging with the DOE inserted show clear and sharp ablation patterns with satisfactory biological signals gained. Further improvements will optimize the beam profile and generate a square top-hat laser beam for MSI application at higher spatial resolution.

Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

Improved spatial resolution of infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging using a reflective objective.

RATIONALE: The level of visual detail of a mass spectrometry image is dependent on the spatial resolution with which it is acquired, which is largely determined by the focal diameter in infrared laser ablation-based techniques. While the use of mid-IR light for mass spectrometry imaging (MSI) has advantages, it results in a relatively large focal diameter and spatial resolution. The continual advancement of infrared matrix-assisted electrospray ionization (IR-MALDESI) for MSI warranted novel methods to decrease laser ablation areas and thus improve spatial resolution. METHODS: In this work, a Schwarzschild-like reflective objective was incorporated into the novel NextGen IR-MALDESI source and characterized on both burn paper and mammalian tissue using an ice matrix. Ablation areas, mass spectra, and annotations obtained using the objective were compared against the current optical train on the NextGen system without modification. RESULTS: The effective resolution was determined to be 55 µm by decreasing the step size until oversampling was observed. Use of the objective improved the spatial resolution by a factor of three as compared against the focus lens. CONCLUSIONS: A Schwarzschild-like reflective objective was successfully incorporated into the NextGen source and characterized on mammalian tissue using an ice matrix. The corresponding improvement in spatial resolution facilitates the future expansion of IR-MALDESI applications to include those that require fine structural detail.

Characterization of a novel miniaturized burst-mode infrared laser system for IR-MALDESI mass spectrometry imaging.

Laser systems are widely used in mass spectrometry as sample probes and ionization sources. Mid-infrared lasers are particularly suitable for analysis of high water content samples such as animal and plant tissues, using water as a resonantly excited sacrificial matrix. Commercially available mid-IR lasers have historically been bulky and expensive due to cooling requirements. This work presents a novel air-cooled miniature mid-IR laser with adjustable burst-mode output and details an evaluation of its performance for mass spectrometry imaging. The miniature laser was found capable of generating sufficient energy for complete ablation of animal tissue in the context of an IR-MALDESI experiment with exogenously added ice matrix, yielding several hundred confident metabolite identifications. Graphical abstract The use of a novel miniature 2.94 µm burst-mode laser in IR-MALDESI allows for rapid and sensitive mass spectrometry imaging of a whole mouse.

IR-MALDESI Mass Spectrometry Imaging at 50 Micron Spatial Resolution.

High spatial resolution in mass spectrometry imaging (MSI) is crucial to understanding the biology dictated by molecular distributions in complex tissue systems. Here, we present MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) at 50 µm resolution. An adjustable iris, beam expander, and an aspherical focusing lens were used to reduce tissue ablation diameters for MSI at high resolution. The laser beam caustic was modeled using laser ablation paper to calculate relevant laser beam characteristics. The minimum laser spot diameter on the tissue was determined using tissue staining and microscopy. Finally, the newly constructed optical system was used to image hen ovarian tissue with and without oversampling, detailing tissue features at 50 µm resolution. Graphical Abstract ᅟ.

Versatile method for achieving 1% speckle contrast in large-venue laser projection displays using a stationary multimode optical fiber.

We propose a method based on quantitative theoretical analysis for achieving speckle contrast of 1% or less in images created by a full-frame laser projection display system. The method employs a stationary multimode optical fiber to achieve the effect of using a rapidly moving diffuser, but without moving the fiber or any other system component. When a suitably large projector lens is used, low-speckle illumination light delivered through the fiber acts in conjunction with wavelength diversity at the projection screen to achieve speckle contrast of 1% in viewed images. We describe in detail how the proposed method might be used with most types of high-power visible lasers being considered for large-venue displays. When used with visible laser diodes, the method may also be suitable for use in laser-based television.

Near-infrared photoinactivation of bacteria and fungi at physiologic temperatures.

We examined a laser system (870 and 930 nm), employing wavelengths that have exhibited cellular photodamage properties in optical traps. In vitro, with 1.5 cm diameter flat-top projections (power density of 5.66 W cm(-2)), at physiologic temperatures, we achieved photoinactivation of Staphylococcus aureus, Escherichia coli, Candida albicans and Trichophyton rubrum. Using nonlethal dosimetry, we measured a decrease in trans-membrane potentials (DeltaPsimt and DeltaPsip) and an increase in reactive oxygen species (ROS) generation in methicillin-resistant S. aureus (MRSA), C. albicans and human embryonic kidney cells. We postulate that these multiplexed wavelengths cause an optically mediated mechano-transduction of cellular redox pathways, decreasing DeltaPsi and increasing ROS. The cellular energetics of prokaryotic and fungal pathogens, along with mammalian cells, are affected in a similar manner when treated with these multiplexed wavelengths at the power densities employed. Following live porcine thermal tolerance skin experiments, we then performed human pilot studies, examining photodamage to MRSA in the nose and fungi in onychomycosis. No observable damage to the nares or the nail matrix was observed, yet photodamage to the pathogens was achieved at physiologic temperatures. The selective aspect of this near-infrared photodamage presents the possibility for its future utilization in human cutaneous antimicrobial therapy.