Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Spectroscopic imaging of arteries and atherosclerotic plaques

Fourier transform infrared (FTIR) spectroscopic imaging using a focal plane array detector has been used to study atherosclerotic arteries with a spatial resolution of 3-4 microm, i.e., at a level that is comparable with cellular dimensions. Such high spatial resolution is made possible using a micro-attenuated total reflection (ATR) germanium objective with a high refractive index and therefore high numerical aperture. This micro-ATR approach has enabled small structures within the vessel wall to be imaged for the first time by FTIR. Structures observed include the elastic lamellae of the tunica media and a heterogeneous distribution of small clusters of cholesterol esters within an atherosclerotic lesion, which may correspond to foam cells. A macro-ATR imaging method was also applied, which involves the use of a diamond macro-ATR accessory. This study of atherosclerosis is presented as an illustrative example of the wider potential of these ATR imaging approaches for cardiovascular medicine and biomedical applications.     

In situ monitoring of the nascent Pseudomonas fluorescens biofilm response to variations in the dissolved organic carbon level in low-nutrient water by attenuated total reflectance-Fourier transform infrared spectroscopy

Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent Pseudomonas fluorescens biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent P. fluorescens biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.     

In Situ Monitoring of the Nascent Pseudomonas fluorescens Biofilm Response to Variations in the Dissolved Organic Carbon Level in Low-Nutrient Water by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy

Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent Pseudomonas fluorescens biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent P. fluorescens biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.     

In situ assessment of the liver microcirculation in mechanically ventilated rats using sidestream dark-field imaging

Assessment of hepatic microcirculation by on-line visualization has been impossible for a long time. Sidestream dark-field (SDF) imaging is a relatively new method allowing direct visualization of both mucosal microcirculation and surface layers microcirculation of solid organs using hand-held probe for direct contact with target tissue. The aim of this study was to evaluate the feasibility of studying the rat hepatic microcirculation in situ by SDF imaging. The liver lobes were left in situ, and images were obtained using SDF imaging on the surface of the liver via upper midline laparotomy. Images were captured intermittently during 10-sec apnoea and recorded. The microvascular parameters were compared with previous validation studies. Clear high contrast SDF images were successfully obtained. Quantitative analysis revealed a mean FSD (functional sinusoidal density) of 402+/-15 cm/cm(2), a sinusoidal diameter of 10.2+/-0.5 microm and postsinusoidal venular diameter of 33.9+/-13 microm. SDF imaging is a suitable noninvasive method for accurate quantification of the basic microcirculatory parameters of the liver in situ without a need to exteriorize the liver lobes. This method seems to be applicable in animal studies with possibility to use SDF imaging also intraoperatively, providing unique opportunity to study liver microcirculation during various experimental and clinical settings.     

Rapid determination of trans-fatty acids in human adipose tissue: Comparison of attenuated total reflection infrared spectroscopy and gas chromatography

A rapid attenuated total reflection (ATR) infrared (IR) spectroscopy procedure was used for quantitating the levels of total trans-fatty acid methyl ester (FAME) derivatives in neat (without solvent) test samples isolated from human adipose tissue. This procedure requires no weighing of the laboratory sample. The single-beam spectrum of the trans-containing FAMEs was ‘ratioed' against that of a reference material having only cis double bonds in order to obtain a symmetric absorption band at 966 cm⁻¹ on a horizontal background. A single-reflection ATR diamond cell that requires only about 1 μl of neat FAMEs was used. The average level of trans-fatty acids in human adipose tissue found by ATR (3.07±0.27%) was generally higher than that obtained by gas chromatography (2.59±0.20%). Reasons for such a difference are discussed.     

In situ assessment of the brain microcirculation in mechanically-ventilated rabbits using sidestream dark-field (SDF) imaging

Assessment of the cerebral microcirculation by on-line visualization has been impossible for a long time. Sidestream dark-field (SDF) imaging is a relatively new method allowing direct visualization of cerebral surface layer microcirculation using hand-held probe for direct contact with target tissue. The aim of this study was to elucidate the feasibility of studying the cerebral microcirculation in situ by SDF imaging and to assess the basic cerebral microcirculatory parameters in mechanically ventilated rabbits. Images were obtained using SDF imaging from the surface of the brain via craniotomy. Clear high contrast SDF images were successfully obtained. Total small-vessel density was 14.6+/-1.8 mm/mm(2), total all-vessel density was 17.9+/-1.7 mm/mm(2), DeBacker score was 12.0+/-1.6 mm(-1) and microvascular flow index was 3.0+/-0.0. This method seems to be applicable in animal studies with possibility to use SDF imaging also intraoperatively, providing unique opportunity to study cerebral microcirculation during various experimental and clinical settings.     

Application of infrared spectroscopy (attenuated total reflection) for monitoring enzymatic activity on substrate films

Infrared film analysis, a method based on infrared spectroscopy in the mode of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), is demonstrated as a novel analytical method for monitoring enzymatic activity on surface-attached substrate films in the mid infrared range (400-4000 cm(-1)). The ATR-FTIR technique is sensitive to molecules within a distance of approximately 1 microm from the ATR sampling unit surface (a 7 cm(2) hydrophobic ZnSe crystal). Applying a 0.2-0.3 microm thick film on the ATR unit surface, any chemical changes within this film as well as at the interface can be continuously monitored, even having an aqueous phase on top of the film. Infrared film analysis is considered especially useful for studying detergent enzymes, which act on surface bound films consisting of food component like vegetable oils (triacylglycerols) and carbohydrates (e.g. starch). Experimental data are presented for hydrolysis of a triacylglycerol film (triolein) by use of a triacylglycerol lipase (cutinase), and starch film degradation by use of an alpha-amylase.     

Covalent layer-by-layer assembly of hyperbranched polyether and polyethyleneimine: multilayer films providing possibilities for surface functionalization and local drug delivery

A convenient and simple route to multifunctional surface coatings via the alternating covalent layer-by-layer (LBL) assembly of p-nitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO(2)) and polyethylenimine (PEI) is described. The in situ chemical reaction between HBPO-NO(2) and PEI onto aminolyzed substrates was rapid and mild. Results from ellipsometry measurements, contact angle measurements, and ATR-FTIR spectra confirmed the successful LBL assembly of the building blocks, and the surface reactivity of the multilayer films with HBPO-NO(2) as the outmost layer was demonstrated by the immobilization of an amine-functionalized fluorophore. Furthermore, a biomimetic surface was achieved by surface functionalization of the multilayer films with extracellular matrix protein collagen to promote the adhesion and growth of cells. The studies on the drug loading and in vitro release behaviors of the multilayer films demonstrated their application potentials in local delivery of hydrophilic and hydrophobic therapeutic agents.     

On the production of glycogen by Pseudomonas fluorescens during biofilm development: an in situ study by attenuated total reflection-infrared with chemometrics

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor Pseudomonas fluorescens biofilms in situ, non-destructively, in real time, and under fully hydrated conditions. Changes accompanying the metabolic evolution of the sessile bacterial cells from the nascent biofilm monolayer to the beginning of the multi-layered structure in the presence of nutrients were identified via the ATR-FTIR fingerprints of the young biofilm on the ATR crystal. The ATR-FTIR spectra were analysed by classical methods (time evolution of integrated intensities and profile evolution of specific bands), and also by a multivariate curve resolution, Bayesian positive source separation, to extract the pure component spectra and their change of concentration over time occurring during biofilm settlement. This work showed clearly the overproduction of glycogen by sessile P. fluorescens, which had not previously been described by other research groups.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

Film-forming process and biocide assessment of high-molecular-weight chitosan as determined by combined ATR-FTIR spectroscopy and antimicrobial assays

This pioneering study reported about the film-forming properties of high-molecular-weight chitosan as followed in situ by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and has implications in fields such as biomedical, pharmaceutical, packaging, and coating applications. From the results, it was observed that immediately after dissolution in an acetic acid aqueous solution and subsequent casting over the ATR crystal, the formed carboxylate antimicrobial (-NH3+ -OOCH) species are not stable in the film formulation and become reduced over time; further assays confirmed previous research, which suggested that the presence and stability of these groups is strongly dependent, among other factors, on storage conditions. As-received chitosan and chitosan neutralized in NaOH films did not exhibit biocide performance towards Staphylococcus aureus. The antimicrobial tests were also found to strongly relate the presence of a sufficient quantity of these carboxylate groups to the chitosan activity as a biocide agent. Moreover, a novel methodology based on the use of a normalized infrared band centered at 1405 cm(-1) is proposed which can be correlated with the antimicrobial character of the biopolymer.     

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.     

Real-time technique for improving molecular imaging and guiding drug delivery in large blood vessels: in vitro and ex vivo results

Ultrasound-based molecular imaging employs targeted microbubbles to image vascular pathology. This approach also has the potential to monitor molecularly targeted microbubble-based drug delivery. We present an image-guided drug delivery technique that uses multiple pulses to translate, image, and cavitate microbubbles in real time. This technique can be applied to both imaging of pathology in large arteries (sizes and flow comparable to those in humans) and guiding localized drug delivery in blood vessels. The microbubble translation (or pushing) efficacy of this technique was compared in a variety of flow media: saline, viscous saline (4 cp), and bovine blood. It was observed that the performance of this approach was marginally better (by 6, 4, and 2 dB) in viscous saline than in bovine blood with varying levels of hematocrit (40%, 30%, and 10%). The drug delivery efficacy of this technique was evaluated by in vitro and ex vivo experiments. High-intensity pulses mediated fluorophore (DiI) deposition on endothelial cells (in vitro) without causing cell destruction. Ex vivo fluorophore delivery experiments conducted on swine carotids of 2 and 5 mm cross-section diameter demonstrated a high degree of correspondence in spatial localization of the fluorophore delivery between the ultrasound and composite fluorescence microscopy images of the arterial cross sections.     

Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10(5) cells per cm(2), while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.     

Glioma-derived mutations in isocitrate dehydrogenase 2 beneficial to traditional chemotherapy

Sonoporation is a promising drug delivery technique with great potential in medicine. However, its applications have been limited mostly by the lack of understanding its underlying biophysical mechanism, partly due to the inadequacy of the existing models for coupling with highly sensitive imaging techniques to directly observe the actual precursor events of cell–microbubble interaction under low intensity ultrasound. Here, we introduce a new in vitro method utilizing capillary-microgripping system and micro-transducer to achieve maximum level of experimental flexibility for capturing real time highly magnified images of cell–microbubble interaction, hitherto unseen in this context. Insonation of isolated single cells and microbubbles parallel with high speed microphotography and fluorescence microscopy allowed us to identify dynamic responses of cell-membrane/microbubble in correlation with sonoporation. Our results showed that bubble motion and linear oscillation in close contact with the cell membrane can cause local deformation and transient porosity in the cell membrane without rupturing it. This method can also be used as an in situ gene/drug delivery system of targeted cells for non-invasive clinical applications.     

Chiral separation of Frovatriptan isomers by HPLC using amylose based chiral stationary phase

A stereospecific HPLC method for separation of Frovatriptan enantiomers in bulk drug and pharmaceutical formulations was developed and validated on a normal-phase amylose derivertized chiral column. The effects of the organic modifiers namely 2-propanol, ethanol and diethyl amine (DEA) in the mobile phase were optimized to obtain the best enantiomeric separation. Calibration curves were linear over the range of 200-6150 ng/mL, with a regression coefficient (R(2)) of 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) were 65 ng/mL and 200 ng/mL, respectively. The method was accurate and precise and suitable for the intended purpose. Analysis results were compared with the results obtained by using a validated chiral CE method and found to be in very good agreement. This method can be successfully applied to the enantiomeric purity analysis of Frovatriptan in pharmaceutical bulk drug samples and formulations.     

Experimental ATR device for real-time FTIR imaging of living cells using brilliant synchrotron radiation sources

In this contribution we present the design of an original Attenuated Total Reflection (ATR)-based device designed for an IR microscope coupled to a FPA detector and optimized for in-vivo cell imaging. The optical element has been designed to perform real time experiments of cell biochemical processes. The device includes a manually removable Ge-crystal that guarantees an ease manipulation during the cell culture and a large flat surface to support the cell growth and the required change of the culture wells. This layout will allow performing sequential ATR IR imaging with the crystal immersed in the culture wells, minimizing contributions due to water vapors in the optical system. Using existing brilliant synchrotron radiation sources this ATR device may collect images at the surface of the Ge crystal at a sub-cellular spatial resolution with a penetration depth of the evanescent wave inside the sample of ~ 500 nm within few seconds. A brief summary of the cellular components that should be detected with such optical device is also presented.     

Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.     

Rapid analysis of disease state in liquid human serum combining infrared spectroscopy and “digital drying”

In recent years, the diagnosis of brain tumors has been investigated with attenuated total reflection‐Fourier transform infrared (ATR‐FTIR) spectroscopy on dried human serum samples to eliminate spectral interferences of the water component, with promising results. This research evaluates ATR‐FTIR on both liquid and air‐dried samples to investigate “digital drying” as an alternative approach for the analysis of spectra obtained from liquid samples. Digital drying approaches, consisting of water subtraction and least‐squares method, have demonstrated a greater random forest (RF) classification performance than the air‐dried spectra approach when discriminating cancer vs control samples, reaching sensitivity values higher than 93.0% and specificity values higher than 83.0%. Moreover, quantum cascade laser infrared (QCL‐IR) based spectroscopic imaging is utilized on liquid samples to assess the implications of a deep‐penetration light source on disease classification. The RF classification of QCL‐IR data has provided sensitivity and specificity amounting to 85.1% and 75.3% respectively.