Analysis of the Photothermal Engine Cycle in an AFM-IR Measurement of Photothermal Expansion

In the present work we develop a thermodynamic description of signal generation in a micromechanical measurement of the photothermal effect. In the photothermal effect, light absorbed by a material is converted into heat, leading to a local temperature increase and to sample expansion. Photothermal expansion is wavelength dependent and tracks the absorption coefficient of the material, making it a useful probe of the spectroscopic properties of a sample. The local measurement of the expansion can be performed with high spatial resolution by using a micromechanical detection scheme that relies on deflection of a cantilevered probe for atomic force microscopy in contact with the sample. The specific features of this detection mechanism rely on the complex interplay of multiple factors. In the present work we provide a new framework to analyze the factors involved in the detection mechanism, to account for the multiplicity of interactions that may affect the performance of the measurement. We collectively describe the thermomechanical processes that underlie light absorption and photothermal expansion in terms of a thermodynamic engine cycle. We discuss the general properties of this photothermal micromechanical engine, and we derive guidelines for performance optimization of the photothermal measurement. We conclude that the engine is intrinsically inefficient because most or all the energy derived from light absorption is dispersed as heat over one cycle. Nonetheless, we also show that efficiency is not a major consideration when assessed towards the main purpose of this mechanism, which is analytical rather than the conversion of heat into work

Growth of Halobacterium salinarum for purple membrane investigations

Poster presentation for the 4th International Workshop on Functional Nanostructured Materials (FuNaM-4), September 26th – 29th, 2023, Krakow, PolandGrants NCN 2018/31/B/NZ1/0134

Imaging and Spectroscopy of Domains of the Cellular Membrane by Photothermal-Induced Resonance

We use photothermal induced resonance (PTIR) imaging and spectroscopy, in resonant and non-resonant mode, to study the cytoplasmic membrane and surface of intact cells. Non-resonant PTIR images apparently provide rich details of the cell surface. However we show that non-resonant image contrast does not arise from the infrared absorption of surface molecules and is instead dominated by the mechanics of tip-sample contact. In contrast, spectra and images of the cellular surface can be selectively obtained by tuning the pulsing structure of the laser to restrict thermal wave penetration to the surface layer. Resonant PTIR images reveal surface structures and domains that range in size from about 20 nm to 1 um and are associated to the cytoplasmic membrane and its proximity. Resonant PTIR spectra of the cell surface are comparable to far-field IR spectra and provide the first selective measurement of the IR absorption spectrum of the cellular membrane of an intact cell. In resonant PTIR images, signal intensity, and therefore contrast, can be ascribed to a variety of factors, including mechanical, thermodynamic and spectroscopic properties of the cellular surface. While PTIR images are difficult to interpret in terms of spectroscopic absorption, they are easy to collect and provide unique contrast mechanisms without any exogenous labeling. As such they provide a new paradigm in cellular imaging and membrane biology and can be used to address a range of critical questions, from the nature of membrane lipid domains to viral membrane fusion

Understanding and controlling spatial resolution, sensitivity, and surface selectivity in resonant-mode photothermal-induced resonance spectroscopy

Photothermal-induced resonance (PTIR) is increasingly used in the measurement of infrared absorption spectra of submicrometer objects. The technique measures IR absorption spectra by relying on the photothermal effect induced by a rapid pulse of light and the excitation of the resonance spectrum of an AFM cantilever in contact with the sample. In this work, we assess the spatial resolution and depth response of PTIR in resonant mode while systematically varying the pulsing parameters of the excitation laser. We show that resolution is always much better than predicted by existing theoretical models. Higher frequency, longer pulse length, and shorter interval between pulses improve resolution, eventually providing values that are comparable to or even better than tip size. Pulsing parameters also affect the intensity of the signal and the surface selectivity in PTIR images, with higher frequencies providing increased surface selectivity. The observations confirm a difference in signal generation between resonant PTIR and other photothermal techniques that we ascribe to nonlinearity in the PTIR signal. In analogy with optical imaging, we show that PTIR takes advantage of such nonlinearity to perform photothermal measurements that are super-resolved when compared to the resolution allowed by the thermal wavelength. Finally, we show that by controlling the pulsing parameters of the laser we can devise high resolution surface sensitive measurements that do not rely on the use of optical enhancement effects

Characterization of intact eukaryotic cells with subcellular spatial resolution by photothermal-induced resonance infrared spectroscopy and imaging

Photothermal-Induced Resonance (PTIR) spectroscopy and imaging with infrared light has seen increasing application in molecular spectroscopy of biological samples. The appeal of the technique lies in its capability to provide information about IR light absorption at a spatial resolution better than allowed by light diffraction, typically below 100 nm. In the present work we test the capability of the technique to perform measurements with subcellular resolution on intact eukaryotic cells, without drying or fixing. We demonstrate the possibility to obtain PTIR images and spectra from the nucleus and multiple organelles with high resolution. We obtain particularly strong signal from bands typically assigned to acyl lipids and proteins. We also show that while a stronger signal is obtained from some subcellular structures, other large subcellular components provide a weaker or undetectable PTIR response. The mechanism that underlies such variability in response is presently unclear. We propose and discuss different possibilities, addressing thermomechanical, geometrical and electrical properties of the sample and the presence of cellular water, from which the difference in response may arise

Is AFM-IR a photothermal technique?

Atomic Force Microscopy - Infrared (AFM-IR) has emerged as a useful technique for measuring absorption spectra with spatial resolution better than the optical diffraction limit. The technique relies on the movement of a probe for atomic force microscopy for detecting the local expansion of a material caused by the photothermal effect. While AFM-IR is seeing increased application to a wider range of samples, reports have also appeared in the literature that are inconsistent with an interpretation of the AFM-IR response simply in terms of photothermal expansion. The present article addresses the issue by critically evaluating existing experimental observations. It is concluded that observed discrepancies arise from the contribution of non-photothermal effects to the signal, which affect both intensity and spatial resolution of the measurement

Identification and characterization of stemlike cells in human esophageal adenocarcinoma and normal epithelial cell lines

ObjectiveRecent studies have suggested that human solid tumors may contain subpopulations of cancer stem cells with the capacity for self-renewal and the potential to initiate and maintain tumor growth. The aim of this study was to use human esophageal cell lines to identify and characterize putative esophageal cancer stem cell populations.MethodsTo enrich stemlike cells, Het-1A (derived from immortalized normal esophageal epithelium), OE33, and JH-EsoAd1 (each derived from primary esophageal adenocarcinomas) were cultured using serum-free media to form spheres. A comprehensive analysis of parent and spheroid cells was performed by flow cytometry, Western blot analysis, immunohistochemistry and polymerase chain reaction array to study cancer stem cell-related genes, colony formation assays to assess clonogenicity, xenotransplantation to assess tumorigenicity, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays to assess chemosensitivity to 5-fluorouracil and Cisplatin.ResultsFor all cell lines, clonogenicity, tumorigenicity, and chemoresistance to 5-fluorouracil and Cisplatin were significantly higher than for spheroid cells compared with parent cells. Spheroids exhibited an increased frequency of cells expressing integrin α6bri/CD71dim, and Achaete-scute complex homolog 2 messenger RNA and protein were also significantly overexpressed in spheroid cells compared with parent cells.ConclusionsThe higher clonogenicity, tumorigenicity, and drug resistance exhibited by spheroids derived from Het-1A, OE33, and JH-EsoAd1 reflects an enrichment of stemlike cell populations within each esophageal cell line. Esophageal cells enriched for integrin α6bri/CD71dim and/or overexpressing Achaete-scute complex homolog 2 would appear to represent at least a subpopulation of stemlike cells in Het-1A, OE33, and JH-EsoAd1

Three-dimensional mid-infrared tomographic imaging of endogenous and exogenous molecules in a single intact cell with subcellular resolution

Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell