Label free biosensors techniques and applications 1st Edition Matthew A. Cooper
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- 5. Label free biosensors techniques and applications 1st Edition Matthew A. Cooper Digital Instant Download Author(s): MatthewA. Cooper ISBN(s): 9780521884532, 0521884535 Edition: 1 File Details: PDF, 12.93 MB Year: 2009 Language: english
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- 8. LABEL-FREE BIOSENSORS Label-free biosensors use biological or chemical receptors to detect ana- lytes (molecules) in a sample. They give detailed information on the binding selectivity, affinity, and, in many cases, the stoichiometry, kinetics, and thermodynamics of an interaction. Although they can be powerful tools in the hands of a skilled user, there is often a lack of knowledge regarding the best way to utilize label-free assays to screen for biologically active molecules and to accurately and precisely char- acterize molecular recognition events. This book reviews both established and newer label-free techniques. It is intended to give both the expert user and the general reader insight into the field from expert opinion leaders and practitioners of these techniques. Chapters also contain worked examples that are written to guide the reader through the basics of experimental design, setup, assay development, and data analysis. Matthew A. Cooper is Founder and Managing Director of Cambridge Medical Innovations and Distinguished Australia Fellow at the Uni- versity of Queensland. Dr. Cooper has consulted widely for biosensor, biotechnology, and pharmaceutical companies in the United Kingdom, Europe, and the United States. He is a review panel member for Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, National Institutes of Health, National Institute of Allergy and Infectious Diseases, and Science Foun- dation Ireland and a Fellow of the Royal Society of Medicine.
- 10. Label-Free Biosensors TECHNIQUES AND APPLICATIONS Edited by Matthew A. Cooper The University of Queensland
- 11. CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo Cambridge University Press The Edinburgh Building, Cambridge CB2 8RU, UK First published in print format ISBN-13 978-0-521-88453-2 ISBN-13 978-0-521-71151-7 ISBN-13 978-0-511-50688-8 © Cambridge University Press 2009 2009 Information on this title: www.cambridge.org/9780521884532 This publication is in copyright. Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. Published in the United States of America by Cambridge University Press, New York www.cambridge.org paperback eBook (EBL) hardback
- 12. Contents Contributors page vii Preface ix 1 Label-free optical biosensors: An introduction 1 Brian T. Cunningham 2 Experimental design 29 Robert Karlsson 3 Extracting affinity constants from biosensor binding responses 48 Rebecca L. Rich and David G. Myszka 4 Extracting kinetic rate constants from binding responses 85 Rebecca L. Rich and David G. Myszka 5 Sensor surfaces and receptor deposition 110 Matthew A. Cooper 6 Macromolecular interactions 143 Francis Markey 7 Interactions with membranes and membrane receptors 159 Matthew A. Cooper 8 Application of SPR technology to pharmaceutical relevant drug-receptor interactions 179 Walter Huber 9 High-throughput analysis of biomolecular interactions and cellular responses with resonant waveguide grating biosensors 206 Ye Fang, Jack Fang, Elizabeth Tran, Xinying Xie, Michael Hallstrom, and Anthony G. Frutos 10 ITC-derived binding constants: Using microgram quantities of protein 223 Richard K. Brown, J. Michael Brandts, Ronan O’Brien, and William B. Peters 11 Electrical impedance technology applied to cell-based assays 251 Ryan P. McGuinness and Edward Verdonk Index 279 Color plates follow page 36 v
- 14. Contributors J. Michael Brandts Vice President MicroCal Northampton, MA Richard K. Brown, PhD President and CEO MicroCal Northampton, MA Matthew A. Cooper, PhD Managing Director Cambridge Medical Innovations Cambridge, UK Distinguished Australia Fellow Institute for Molecular Bioscience University of Queensland St. Lucia, Australia Brian T. Cunningham Associate Professor University of Illinois Department of Electrical and Computer Engineering Urbana, IL Jack Fang, PhD Strategic Analysis Manager Corning® Epic® System Corning, Inc. Corning, NY Ye Fang, PhD Research Manager Cellular Biophysics Corning, Inc. Corning, NY Anthony G. Frutos, PhD BioAssay Development Manager Science & Technology Corning, Inc. Corning, NY Michael Hallstrom Assistant Product Line Manager Corning® Epic® System Corning, Inc. Corning, NY Walter Huber, PhD Pharmaceutical Research Discovery Technology F. Hoffmann-La Roche Basel, Switzerland Robert Karlsson Research and Development Director, System and Applications Department GE Healthcare Uppsala, Sweden Francis Markey GE Healthcare Uppsala, Sweden vii
- 15. viii Contributors Ryan P. McGuinness Senior Scientist Drug Discovery MDS Analytical Technologies Sunnyvale, CA David G. Myszka, PhD Director Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, UT Ronan O’Brien, PhD Head of Applications Research MicroCal Northampton, MA William B. Peters, PhD Applications Scientist and Head of Training MicroCal Northampton, MA Rebecca L. Rich, PhD Senior Research Scientist Center for Biomolecular Interaction Analysis School of Medicine University of Utah Salt Lake City, UT Elizabeth Tran, PhD Senior Research Scientist Science & Technology Corning, Inc. Corning, NY Edward Verdonk, PhD Senior Scientist Drug Discovery MDS Analytical Technologies Sunnyvale, CA Xinying Xie Field Application Scientist Corning® Epic® System Corning, Inc. Houston, TX
- 16. Preface Over the past two decades the benefits of biosensor analysis have begun to be recognized in many areas of analytical science, research, and development, with analytical systems now used routinely as mainstream research tools in many laboratories in many fields. Simplistically, biosensors can be defined as devices that use biological or chemical receptors to detect analytes (molecules) in a sample. They give detailed information on the binding affinity and in many cases also the binding stoichiometry, thermodynamics, and kinetics of an interaction. Label-free biosensors, by definition, do not require the use of reporter elements (fluorescent, luminescent, radiometric, or colorimetric) to facilitate measure- ments. Instead, a receptor molecule is normally connected in some way to a transducer that produces an electrical signal in real time. Other techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), and mass spectrometry require neither reporter labels nor surface-bound receptors. In all cases detailed information on an interaction can be obtained during analysis while minimizing sample processing requirements. Unlike label- and reporter- based technologies that simply confirm the presence or absence of a detector molecule, label-free techniques can provide direct information on analyte bind- ing to target molecules typically in the form of mass addition or depletion from the surface of a sensor substrate or via changes in a physical bulk property (such as the heat capacity) of a sample. Until recently, label-free technologies have failed to gain widespread acceptance due to technical constraints, low throughput, high user expertise requirements, and cost. Whereas they have proved to be powerful tools in the hands of a skilled user, they have not always been readily adapted to everyday lab use in which simple-to-understand results are a prerequisite. Despite this limitation, the potential today for label-free approaches to complement or even displace other detection technologies has never been higher. This book covers established label-free technologies and emerging develop- ments in label-free detection systems, their underlying technology principles, and end-user case studies that reveal the power and limitations of such biosen- sors. The chapters are intended to give both the expert user and the general reader interested in the technologies and applications behind label-free biosensors an insight into the field from expert opinion leaders and practitioners. As such, most chapters contain one or more worked examples that guide the reader through ix
- 17. x Preface the basics of experimental design, setup, assay development, and data analysis. The book is heavily weighted toward applications using optical biosensors and surface plasmon resonance (SPR) instrumentation. This is primarily because of the overwhelming bias in the installed base of optical biosensors due to their early commercialization and uptake. Other label-free technologies conspicuous by their absence from this volume include analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and mass spectroscopy. These will be covered as part of a new cluster of titles on bioanalytical techniques and applications. Matt Cooper Cambridge, U.K. May 2008
- 20. 1 Label-free optical biosensors: An introduction Brian T. Cunningham INTRODUCTION 1 RATIONALE FOR LABEL-FREE DETECTION 3 OPTICAL BIOSENSORS 4 PERFORMANCE METRICS 7 Sensitivity 7 Assay sensitivity 8 Resolution (or limit of determination) 8 Ease of use 9 Sensor cost 10 Detection instrumentation 10 Throughput 11 REVIEW OF OPTICAL BIOSENSOR METHODS 12 Surface plasmon resonance 12 HOLOGRAM BIOSENSORS 14 Reflectometric interference spectroscopy 16 Dual polarization interferometry 18 PHOTONIC CRYSTAL BIOSENSOR 19 WHISPERING GALLERY MODE RESONATORS 22 ACTIVE RESONATORS: DFB LASER BIOSENSOR 25 CONCLUSIONS 25 REFERENCES 27 INTRODUCTION The ability to study interactions among biomolecules, to observe the activity of cells, and to detect analytes specifically from bodily fluids, manufacturing pro- cesses, or environmental samples are cornerstones of life science research, phar- maceutical discovery, medical diagnosis, and food/water safety assurance. These capabilities and many others are enabled by the ability to perform biochemical and cell-based assays that allow scientists to ask basic questions about whether 1
- 21. 2 Brian T. Cunningham one analyte interacts with another, how strong the binding affinity is between two proteins, whether a chemical compound will affect the proliferation rate of cancer cells, and the concentration of a biomarker for cancer within a patient’s blood sample. The development of technology to meet these requirements is challenging because biochemical analytes, which can include drug compounds with molecular weights below 500 Da, DNA oligomers, peptides, enzymes, anti- bodies, and viral particles, are exceedingly small and sometimes present within a test sample at concentrations in the fg/ml to pg/ml concentration range that simultaneously contains thousands of other molecules at concentrations orders of magnitude greater. Larger biochemical analytes – such as bacteria, spores, cells, and cell clusters – are less difficult to observe if they can be stained with a col- ored or fluorescent dye. However, such treatments generally result in the death of the specimen, thus preventing the ability to study a single population repeat- edly over a long time period. An additional challenge arises from the need to perform many thousands of individual measurements, as researchers working in pharmaceutical discovery and life science research seek to perform large numbers of assays in parallel as a means to understand how many molecular permutations affect the efficacy of a drug candidate and as researchers in the field of proteomics seek to characterize extremely complex protein interaction pathways at the heart of common human diseases. When assays must be performed in quantities in the tens of thousands to several millions, the cost of performing assays in terms of disposable labware, reagents, and scientist/technician time must be considered carefully – and the ability to translate instrument measurements into biologically relevant information efficiently is a key consideration for selection of a particular assay technology. Due to the difficulty in detecting biological analytes directly through their intrinsic physical properties (such as mass, size, electrical impedance, or dielectric permittivity), biological research has historically relied upon attachment of some sort of “label” to one or more of the molecules/viruses/cells being studied. The label is designed to be easily measured by its color or its ability to generate pho- tons at a particular wavelength and acts as a surrogate to indirectly indicate the presence of the analyte to which it has been attached. For example, many com- mercially available fluorescent dyes can be conjugated with DNA, proteins, or cells so that, when illuminated with a laser at the excitation wavelength of the fluor- ophore, light is emitted at a characteristic wavelength. Likewise, semiconductor quantum dots, fluorophore-embedded plastic beads, and metal nanoparticles can be functionalized with a surface that enables selective attachment to molecules or particular regions within a cell. In a similar fashion, a wide class of assays has been developed in which a molecule is labeled with a specific antibody that in turn can initiate an enzymatic reaction. The reaction generates a colored stain that can be visually observed. Though molecular, nanoparticle, enzyme, and radioactive labels have been central to implementing nearly all biochemical and cell-based assays, the labels themselves pose several potential problems. For example, though the detection of radioactive labels can be performed with tremendous sensitivity, their usage
- 22. Label-free optical biosensors: An introduction 3 requires specially outfitted “hot labs” and the generation of large quantities of contaminated reagents and labware that must be properly disposed. The exci- tation/emission efficiency of fluorophores is degraded by time and exposure to light (including room light and the laser excitation light) by photobleaching, reducing the ability of the technique to supply highly quantitative measure- ments and requiring that assays be read once only in an “end point” fashion so that kinetic information from an assay is lost. Quenching and self-quenching may also reduce the efficacy of fluorescent tags in an unpredictable manner. Nanoparticles, although as small as a few nanometers in diameter, are quite large when compared to biological molecules and cannot penetrate easily through cell membranes. In practice, label-based assays require a high degree of development to assure that the label does not block an important active site on the tagged molecule or modify the molecule’s conformation. Particularly for sandwich-type assays, intensive optimization of washing conditions, blocking conditions, and expo- sure protocols for a series of reagents must be performed. Labware for fluorescent assays must be constructed from materials that do not autofluoresce, as unin- tentional background fluorescence can overwhelm the signals being measured. The ultimate cost of using a label incorrectly may be the development of a drug whose side effects are not well characterized or an inaccurate assessment of the potency of a drug’s interaction with its targeted protein receptor. RATIONALE FOR LABEL-FREE DETECTION Due to the above considerations, there has been a drive to reduce assay cost and complexity while providing more quantitative information with high through- put. For example, direct measurement of the affinity binding constant between two proteins provides fundamental information regarding kinetics of the interac- tion, and the ability to quickly quantify binding kinetic parameters enables direct comparisons to be made between a wide variety of protein–protein pairs. Like- wise, a pharmaceutical company’s typical screening campaign to investigate how large libraries of chemical compounds interact with a target protein representing a disease’s biochemical pathway can involve several million assays performed over several months.1 Limiting assay complexity and associated advantages in assay development time, reagent usage, and accuracy of results is commercially important, as these factors directly affect the time and cost required to bring a new drug through initial discovery, characterization, and validation phases. In later phases of drug discovery, assays determine how chemical compounds and proteins interact with cells to learn how cell attachment, ion channel activation, proliferation, and apoptosis are modulated. Screening the interaction between large libraries of biochemical compounds and cell panels representing different tissues in the body or different groups of patients is used to predict the efficacy of a treatment and its likely side effects before trials are conducted with animals or people. Therefore, the ability to inexpensively perform large numbers of assays
- 23. 4 Brian T. Cunningham is central for pharmaceutical companies as they seek to provide innovative ther- apeutic compounds while limiting the time and cost required to bring them to market. There are now many methods that allow direct detection of biological ana- lytes without labels. Label-free detection generally involves a transducer capable of directly measuring some physical property of the chemical compound, DNA molecule, peptide, protein, virus, or cell. For example, all biochemical molecules and cells have finite mass, volume, viscoelasticity, dielectric permittivity, and conductivity that can be used to indicate their presence or absence using an appropriate sensor. The sensor functions as a transducer that can convert one of these physical properties (such as the mass of a substance deposited on the sen- sor’s active surface) into a quantifiable signal that can be gathered by an appropri- ate instrument (such as a current or voltage proportional to the deposited mass). Label-free detection removes experimental uncertainty induced by the effect of the label on molecular conformation, blocking of active binding epitopes, steric hindrance, inaccessibility of the labeling site, or the inability to find an appropri- ate label that functions equivalently for all molecules in an experiment. Label-free detection methods greatly simplify the time and effort required for assay devel- opment while removing experimental artifacts from quenching, shelf life, and background fluorescence. OPTICAL BIOSENSORS Though a biological analyte’s mass is a familiar intrinsic property for label-free detection, all molecules also have the capability to interact with electromag- netic fields that pass through them because they contain atomic nuclei and a variety of electrons in various orbital states. Most fundamentally, electrons within molecules experience a force when they are exposed to the oscillating electromagnetic fields associated with the propagation of light. Molecules with an abundance of free electrons will become polarized by exposure to the light’s electromagnetic field (one side of the molecule will be temporarily more nega- tively charged than the opposite side – resulting in the formation of an electric dipole), where the extent of the polarization may be different for any particular molecule depending on its size, shape, and orientation with respect to the elec- tric field. A constant known as the electric susceptibility, χe, quantifies the extent of a molecule’s “polarizability,” and molecules with greater χe are more easily polarized. When a polarizable molecule is placed in an electric field, the induced electrical dipole produces a secondary electric field such that the resulting electric field (i.e., the sum of the originally applied field and the secondary field) is of lower magnitude than the applied field. Because an electromagnetic field associ- ated with light is time-varying, the electrons within the molecule will experience a time-varying force so that electrons will oscillate within the molecule. Moving electrons, by definition, produce an electrical current, so the molecule actually experiences a “polarization current” as a result of this electron motion. The result
- 24. Label-free optical biosensors: An introduction 5 of the polarization current is that light travels more slowly through the molecule than it would through free space. Though free space has a permittivity defined by the constant ε0, (where ε0 = 8.85 × 10−12 F/m), the permittivity of a dielectric material containing molecules is given by ε = εr ε0, where εr is known as the rel- ative permittivity of the dielectric constant of the molecule. εr is directly related to the polarizability of the molecule because it is mathematically defined as εr = 1 + χe. Many people are more familiar with the term refractive index (n) to describe a dielectric material. In an ordinary dielectric material at optical wavelengths, n is defined as n = √ εr , so the refractive index is directly related to the polarizability of the molecules within a dielectric material. Generally, the refractive index is a quantity defined for a bulk material, while electric susceptibility and dielectric permittivity can apply to individual molecules such as those adsorbed to optical biosensor surfaces. The key behind optical biosensors’ ability to detect biological analytes is that biological molecules, including proteins, cells, and DNA, all have dielectric per- mittivity greater than that of air and water. Therefore, these materials all possess the intrinsic ability to reduce the propagation velocity of electromagnetic fields that pass through them. Optical biosensors are designed to translate changes in the propagation speed of light through a medium that contains biological mate- rial into a quantifiable signal proportional to the amount of biological material present on the sensor surface. In the design of optical biosensors, the detected biological material is often modeled as a thin film with a finite refractive index, although this is a simplification. Several studies have been performed to char- acterize the dielectric properties of representative molecular monolayer films.2–4 Therefore, if a biosensor transducer surface is covered with water, and if biolog- ical molecules can adsorb to the transducer surface, a small quantity of water molecules is displaced and replaced with a molecule that is more easily polarized by electromagnetic fields associated with light. Therefore, the design goal for all optical biosensors is to provide a transducer with some externally measurable characteristic that is modified by changes in dielectric permittivity on its surface. In this way, optical biosensors do not measure the mass of adsorbed material (as sometimes stated), although often the mass of deposited material is often related to the change in dielectric permittivity. As shown in several examples within this chapter, the majority of optical biosensors are measured by illuminating them with light (from a laser, light- emitting diode, or incandescent light bulb) and detecting changes in some char- acteristic of the light reflected from or transmitted through the sensor. This type of excitation/collection method is convenient because the sensor can be measured without the detection instrument making any direct physical contact with the sensor itself. Without requirements for electrical contacts, many optical biosensors can be measured simultaneously or sequentially by illuminating sen- sors at different locations on a single multisensor surface, and integration with liquid samples (which must be generally kept separate from electrical contacts to prevent short circuits) is greatly simplified. Many types of optical biosensors can be fabricated inexpensively on glass or plastic surfaces, while others require
- 25. 6 Brian T. Cunningham more complex definition of patterned regions using photolithography and etch- ing processes commonly used for integrated circuit manufacturing. Most optical biosensors are considered “passive” optical components from the standpoint that one measures changes in characteristics of light reflected/transmitted from them, and all the light is supplied externally. This is advantageous because the sensor itself consumes no power, and generally low illumination levels are required to produce measurable responses. More recently, “active” optical biosensors have been demonstrated that are capable of producing their own light output. We provide an example at the end of the chapter. The transducer alone, however, is not sufficiently intelligent to specifically identify material placed on its surface. For example, an optical biosensor cannot tell if an adsorbed layer of material consists of protein, plastic, or silicon oxide. To use a transducer as a biosensor, the surface of the transducer must have the ability to selectively attach specific material from a test sample while not allowing undesired material to attach. Selective detection capability is provided through the attachment of a layer of receptor molecules to the surface of the transducer. For example, when a single strand of DNA is attached to the sensor surface, the surface has the capability to preferentially bind a complementary strand of DNA from a test sample. Likewise, when a protein attaches to the sensor surface, the sensor has the capability to preferentially bind antibodies to that protein from a test sample. The material attached to the sensor surface is referred to as the receptor ligand. The detected material is called the analyte. Thus, a biosensor is the combination of a transducer that can generate a measurable signal from material that attaches to the transducer and a specific recognition surface coating con- taining a receptor ligand that can bind a targeted analyte from a test sample. For specific detection of biomolecules within complex mixtures that contain many potentially interfering molecules, means for “blocking” a surface with immobi- lized receptors to prevent nonspecific attachment of interferents is an important component of performing successful assays. The surface chemistry procedures used to attach receptor ligands to biosensor surfaces with covalent bond linkages (so they are not easily washed away) while maintaining the biological activity of the ligands is an extremely important element of a useful biosensor (optical or otherwise). This is outside the scope of this chapter. For most types of optical biosensors, a solid material medium confines an elec- tromagnetic wave in such a way that the wave has the opportunity to interact with a test sample. The electromagnetic wave may be in the form of a traveling wave or a standing wave, depending on the sensor configuration. For light to be guided by the sensor structure but concurrently interact with the external envi- ronment, the structure must be designed so that the light wave can extend from the sensor surface into the test sample. Electromagnetic fields bound to an opti- cal device that couple some energy to an external medium are called evanescent fields. The evanescent field intensity decays exponentially with distance from the transducer surface, with a decay length of approximately λ/2π, where λ is the wavelength of the light. When we consider light in the typical wavelength range for optical biosensors of λ = 600–900 nm, therefore, the evanescent field
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- 27. Mariolle was touched, and a little angry; he said to her: "Tell me what it was all about." She told him of the brutal conduct of the two painters immediately upon their arrival the night before, and then began to cry again, asking what she was to do, alone in the country and without friends or relatives, money or protection. Mariolle suddenly said to her: "Will you enter my service? You shall be well treated in my house, and when I return to Paris you will be free to do what you please." She looked him in the face with questioning eyes, and then quickly replied: "I will, Monsieur. "How much are you earning here?" "Sixty francs a month," she added, rather uneasily, "and I have my share of the pourboires besides; that makes it about seventy." "I will pay you a hundred." She repeated in astonishment: "A hundred francs a month?" "Yes. Is that enough?" "I should think that it was enough!" "All that you will have to do will be to wait on me, take care of my clothes and linen, and attend to my room." "It is a bargain, Monsieur." "When will you come?" "To-morrow, if you wish. After what has happened here I will go to the mayor and will leave whether they are willing or not." Mariolle took two louis from his pocket and handed them to her. "There's the money to bind our bargain." A look of joy flashed across her face and she said in a tone of decision: "I will be at your house before midday to-morrow, Monsieur."
- 28. CHAPTER XII. CONSOLATION Elisabeth came to Montigny next day, attended by a countryman with her trunk on a wheelbarrow. Mariolle had made a generous settlement with one of his old women and got rid of her, and the newcomer took possession of a small room on the top floor adjoining that of the cook. She was quite different from what she had been at Marlotte, when she presented herself before her new master, less effusive, more respectful, more self-contained; she was now the servant of the gentleman to whom she had been almost an humble friend beneath the arbor of the inn. He told her in a few words what she would have to do. She listened attentively, went and took possession of her room, and then entered upon her new service. A week passed and brought no noticeable change in the state of Mariolle's feelings. The only difference was that he remained at home more than he had been accustomed to do, for he had nothing to attract him to Marlotte, and his house seemed less dismal to him than at first. The bitterness of his grief was subsiding a little, as all storms subside after a while; but in place of this aching wound there was arising in him a settled melancholy, one of those deep-seated sorrows that are like chronic and lingering maladies, and sometimes end in death. His former liveliness of mind and body, his mental activity, his interests in the pursuits that had served to occupy and amuse him hitherto were all dead, and their place had been taken by a universal disgust and an invincible torpor, that left him without even strength of will to get up and go out of doors. He no longer left his house, passing from the salon to the hammock and from the hammock to the salon, and his chief distraction consisted in watching the current of the Loing as it flowed by the terrace and the fisherman casting his net.
- 29. When the reserve of the first few days had begun to wear off, Elisabeth gradually grew a little bolder, and remarking with her keen feminine instinct the constant dejection of her employer, she would say to him when the other servant was not by: "Monsieur finds his time hang heavy on his hands?" He would answer resignedly: "Yes, pretty heavy." "Monsieur should go for a walk." "That would not do me any good." She quietly did many little unassuming things for his pleasure and comfort. Every morning when he came into his drawing-room, he found it filled with flowers and smelling as sweetly as a conservatory. Elisabeth must surely have enlisted all the boys in the village to bring her primroses, violets, and buttercups from the forest, as well as putting under contribution the small gardens where the peasant girls tended their few plants at evening. In his loneliness and distress he was grateful for her kind thoughtfulness and her unobtrusive desire to please him in these small ways. It also seemed to him that she was growing prettier, more refined in her appearance, and that she devoted more attention to the care of her person. One day when she was handing him a cup of tea, he noticed that her hands were no longer the hands of a servant, but of a lady, with well-trimmed, clean nails, quite irreproachable. On another occasion he observed that the shoes that she wore were almost elegant in shape and material. Then she had gone up to her room one afternoon and come down wearing a delightful little gray dress, quite simple and in perfect taste. "Hallo!" he exclaimed, as he saw her, "how dressy you are getting to be, Elisabeth!" She blushed up to the whites of her eyes. "What, I, Monsieur? Why, no. I dress a little better because I have more money." "Where did you buy that dress that you have on?" "I made it myself, Monsieur."
- 30. "You made it? When? I always see you busy at work about the house during the day." "Why, during my evenings, Monsieur." "But where did you get the stuff? and who cut it for you?" She told him that the shopkeeper at Montigny had brought her some samples from Fontainebleau, that she had made her selection from them, and paid for the goods out of the two louis that he had paid her as advanced wages. The cutting and fitting had not troubled her at all, for she and her mother had worked four years for a ready- made clothing house. He could not resist telling her: "It is very becoming to you. You look very pretty in it." And she had to blush again, this time to the roots of her hair. When she had left the room he said to himself: "I wonder if she is beginning to fall in love with me?" He reflected on it, hesitated, doubted, and finally came to the conclusion that after all it might be possible. He had been kind and compassionate toward her, had assisted her, and been almost her friend; there would be nothing very surprising in this little girl being smitten with the master, who had been so good to her. The idea did not strike him very disagreeably, moreover, for she was really very presentable, and retained nothing of the appearance of a servant about her. He experienced a flattering feeling of consolation, and his masculine vanity, that had been so cruelly wounded and trampled on and crushed by another woman, felt comforted. It was a compensation— trivial and unnoteworthy though it might be, it was a compensation —for when love comes to a man unsought, no matter whence it comes, it is because that man possesses the capacity of inspiring it. His unconscious selfishness was also gratified by it; it would occupy his attention and do him a little good, perhaps, to watch this young heart opening and beating for him. The thought never occurred to him of sending the child away, of rescuing her from the peril from which he himself was suffering so cruelly, of having more pity for her than others had showed toward him, for compassion is never an ingredient that enters into sentimental conquests.
- 31. So he continued his observations, and soon saw that he had not been mistaken. Petty details revealed it to him more clearly day by day. As she came near him one morning while waiting on him at table, he smelled on her clothing an odor of perfumery—villainous, cheap perfumery, from the village shopkeeper's, doubtless, or the druggist's—so he presented her with a bottle of Cyprus toilette-water that he had been in the habit of using for a long time, and of which he always carried a supply about with him. He also gave her fine soaps, tooth-washes, and rice-powder. He thus lent his assistance to the transformation that was becoming more apparent every day, watching it meantime with a pleased and curious eye. While remaining his faithful and respectful servant, she was thus becoming a woman in whom the coquettish instincts of her sex were artlessly developing themselves. He, on his part, was imperceptibly becoming attached to her. She inspired him at the same time with amusement and gratitude. He trifled with this dawning tenderness as one trifles in his hours of melancholy with anything that can divert his mind. He was conscious of no other emotion toward her than that undefined desire which impels every man toward a prepossessing woman, even if she be a pretty servant, or a peasant maiden with the form of a goddess—a sort of rustic Venus. He felt himself drawn to her more than all else by the womanliness that he now found in her. He felt the need of that—an undefined and irresistible need, bequeathed to him by that other one, the woman whom he loved, who had first awakened in him that invincible and mysterious fondness for the nature, the companionship, the contact of women, for the subtle aroma, ideal or sensual, that every beautiful creature, whether of the people or of the upper class, whether a lethargic, sensual native of the Orient with great black eyes, or a blue-eyed, keen-witted daughter of the North, inspires in men in whom still survives the immemorial attraction of femininity. These gentle, loving, and unceasing attentions that were felt rather than seen, wrapped his wound in a sort of soft, protecting envelope that shielded it to some extent from its recurrent attacks of
- 32. suffering, which did return, nevertheless, like flies to a raw sore. He was made especially impatient by the absence of all news, for his friends had religiously respected his request not to divulge his address. Now and then he would see Massival's or Lamarthe's name in the newspapers among those who had been present at some great dinner or ceremonial, and one day he saw Mme. de Burne's, who was mentioned as being one of the most elegant, the prettiest, and best dressed of the women who were at the ball at the Austrian embassy. It sent a trembling through him from head to foot. The name of the Comte de Bernhaus appeared a few lines further down, and that day Mariolle's jealousy returned and wrung his heart until night. The suspected liaison was no longer subject for doubt for him now. It was one of those imaginary convictions that are even more torturing than reality, for there is no getting rid of them and they leave a wound that hardly ever heals. No longer able to endure this state of ignorance and uncertainty, he determined to write to Lamarthe, who was sufficiently well acquainted with him to divine the wretchedness of his soul, and would be likely to afford him some clew as to the justice of his suspicions, even without being directly questioned on the subject. One evening, therefore, he sat down and by the light of his lamp concocted a long, artful letter, full of vague sadness and poetical allusions to the delights of early spring in the country and veiled requests for information. When he got his mail four days later he recognized at the very first glance the novelist's firm, upright handwriting. Lamarthe sent him a thousand items of news that were of great importance to his jealous eyes. Without laying more stress upon Mme. de Burne and Bernhaus than upon any other of the crowd of people whom he mentioned, he seemed to place them in the foreground by one of those tricks of style characteristic of him, which led the attention to just the point where he wished to lead it without revealing his design. The impression that this letter, taken as a whole, left upon Mariolle was that his suspicions were at least not destitute of foundation. His fears would be realized to-morrow, if
- 33. they had not been yesterday. His former mistress was always the same, leading the same busy, brilliant, fashionable life. He had been the subject of some talk after his disappearance, as the world always talks of people who have disappeared, with lukewarm curiosity. After the receipt of this letter he remained in his hammock until nightfall; then he could eat no dinner, and after that he could get no sleep; he was feverish through the night. The next morning he felt so tired, so discouraged, so disgusted with his weary, monotonous life, between the deep silent forest that was now dark with verdure on the one hand and the tiresome little stream that flowed beneath his windows on the other, that he did not leave his bed. When Elisabeth came to his room in response to the summons of his bell, she stood in the doorway pale with surprise and asked him: "Is Monsieur ill?" "Yes, a little." "Shall I send for the doctor?" "No. I am subject to these slight indispositions." "What can I do for Monsieur?" He ordered his bath to be got ready, a breakfast of eggs alone, and tea at intervals during the day. About one o'clock, however, he became so restless that he determined to get up. Elisabeth, whom he had rung for repeatedly during the morning with the fretful irresolution of a man who imagines himself ill and who had always come up to him with a deep desire of being of assistance, now, beholding him so nervous and restless, with a blush for her own boldness, offered to read to him. He asked her: "Do you read well?" "Yes, Monsieur; I gained all the prizes for reading when I was at school in the city, and I have read so many novels to mamma that I can't begin to remember the names of them."
- 34. He was curious to see how she would do, and he sent her into the studio to look among the books that he had packed up for the one that he liked best of all, "Manon Lescaut." When she returned she helped him to settle himself in bed, arranged two pillows behind his back, took a chair, and began to read. She read well, very well indeed, intelligently and with a pleasing accent that seemed a special gift. She evinced her interest in the story from the commencement and showed so much feeling as she advanced in it that he stopped her now and then to ask her a question and have a little conversation about the plot and the characters. Through the open windows, on the warm breeze loaded with the sweet odors of growing things, came the trills and roulades of the nightingales among the trees saluting their mates with their amorous ditties in this season of awakening love. The young girl, too, was moved beneath André's gaze as she followed with bright eyes the plot unwinding page by page. She answered the questions that he put to her with an innate appreciation of the things connected with tenderness and passion, an appreciation that was just, but, owing to the ignorance natural to her position, sometimes crude. He thought: "This girl would be very intelligent and bright if she had a little teaching." Her womanly charm had already begun to make itself felt in him, and really did him good that warm, still, spring afternoon, mingling strangely with that other charm, so powerful and so mysterious, of "Manon," the strangest conception of woman ever evoked by human ingenuity. When it became dark after this day of inactivity Mariolle sank into a kind of dreaming, dozing state, in which confused visions of Mme. de Burne and Elisabeth and the mistress of Des Grieux rose before his eyes. As he had not left his room since the day before and had taken no exercise to fatigue him he slept lightly and was disturbed by an unusual noise that he heard about the house.
- 35. Once or twice before he had thought that he heard faint sounds and footsteps at night coming from the ground floor, not directly underneath his room, but from the laundry and bath-room, small rooms that adjoined the kitchen. He had given the matter no attention, however. This evening, tired of lying in bed and knowing that he had a long period of wakefulness before him, he listened and distinguished something that sounded like the rustling of a woman's garments and the splashing of water. He decided that he would go and investigate, lighted a candle and looked at his watch; it was barely ten o'clock. He dressed himself, and having slipped a revolver into his pocket, made his way down the stairs on tiptoe with the stealthiness of a cat. When he reached the kitchen, he was surprised to see that there was a fire burning in the furnace. There was not a sound to be heard, but presently he was conscious of something stirring in the bath-room, a small, whitewashed apartment that opened off the kitchen and contained nothing but the tub. He went noiselessly to the door and threw it open with a quick movement; there, extended in the tub, he beheld the most beautiful form that he had ever seen in his life. It was Elisabeth. CHAPTER XIII. MARIOLLE COPIES MME DE BURNE When she appeared before him next morning bringing him his tea and toast, and their eyes met, she began to tremble so that the cup and sugar-bowl rattled on the salver. Mariolle went to her and relieved her of her burden and placed it on the table; then, as she
- 36. still kept her eyes fastened on the floor, he said to her: "Look at me, little one." She raised her eyes to him; they were full of tears. "You must not cry," he continued. As he held her in his arms, she murmured: "Oh! mon Dieu!" He knew that it was not regret, nor sorrow, nor remorse that had elicited from her those three agitated words, but happiness, true happiness. It gave him a strange, selfish feeling of delight, physical rather than moral, to feel this small person resting against his heart, to feel there at last the presence of a woman who loved him. He thanked her for it, as a wounded man lying by the roadside would thank a woman who had stopped to succor him; he thanked her with all his lacerated heart, and he pitied her a little, too, in the depths of his soul. As he watched her thus, pale and tearful, with eyes alight with love, he suddenly said to himself: "Why, she is beautiful! How quickly a woman changes, becomes what she ought to be, under the influence of the desires of her feelings and the necessities of her existence!" "Sit down," he said to her. He took her hands in his, her poor toiling hands that she had made white and pretty for his sake, and very gently, in carefully chosen phrases, he spoke to her of the attitude that they should maintain toward each other. She was no longer his servant, but she would preserve the appearance of being so for a while yet, so as not to create a scandal in the village. She would live with him as his housekeeper and would read to him frequently, and that would serve to account for the change in the situation. He would have her eat at his table after a little, as soon as she should be permanently installed in her position as his reader. When he had finished she simply replied: "No, Monsieur, I am your servant, and I will continue to be so. I do not wish to have people learn what has taken place and talk about it." He could not shake her determination, although he urged her strenuously, and when he had drunk his tea she carried away the salver while he followed her with a softened look.
- 37. When she was gone he reflected. "She is a woman," he thought, "and all women are equal when they are pleasing in our eyes. I have made my waitress my mistress. She is pretty, she will be charming! At all events she is younger and fresher than the mondaines and the cocottes. What difference does it make, after all? How many celebrated actresses have been daughters of concierges! And yet they are received as ladies, they are adored like heroines of romance, and princes bow before them as if they were queens. Is this to be accounted for on the score of their talent, which is often doubtful, or of their beauty, which is often questionable? Not at all. But a woman, in truth, always holds the place that she is able to create for herself by the illusion that she is capable of inspiring." He took a long walk that day, and although he still felt the same distress at the bottom of his heart and his legs were heavy under him, as if his suffering had loosened all the springs of his energy, there was a feeling of gladness within him like the song of a little bird. He was not so lonely, he felt himself less utterly abandoned; the forest appeared to him less silent and less void. He returned to his house with the glad thought that Elisabeth would come out to meet him with a smile upon her lips and a look of tenderness in her eyes. The life that he now led for about a month on the bank of the little stream was a real idyl. Mariolle was loved as perhaps very few men have ever been, as a child is loved by its mother, as the hunter is loved by his dog. He was all in all to her, her Heaven and earth, her charm and delight. He responded to all her ardent and artless womanly advances, giving her in a kiss her fill of ecstasy. In her eyes and in her soul, in her heart and in her flesh there was no object but him; her intoxication was like that of a young man who tastes wine for the first time. Surprised and delighted, he reveled in the bliss of this absolute self-surrender, and he felt that this was drinking of love at its fountain-head, at the very lips of nature. Nevertheless he continued to be sad, sad, and haunted by his deep, unyielding disenchantment. His little mistress was agreeable, but he
- 38. always felt the absence of another, and when he walked in the meadows or on the banks of the Loing and asked himself: "Why does this lingering care stay by me so?" such an intolerable feeling of desolation rose within him as the recollection of Paris crossed his mind that he had to return to the house so as not to be alone. Then he would swing in the hammock, while Elisabeth, seated on a camp-chair, would read to him. As he watched her and listened to her he would recall to mind conversations in the drawing-room of Michèle, in the days when he passed whole evenings alone with her. Then tears would start to his eyes, and such bitter regret would tear his heart that he felt that he must start at once for Paris or else leave the country forever. Elisabeth, seeing his gloom and melancholy, asked him: "Are you suffering? Your eyes are full of tears." "Give me a kiss, little one," he replied; "you could not understand." She kissed him, anxiously, with a foreboding of some tragedy that was beyond her knowledge. He, forgetting his woes for a moment beneath her caresses, thought: "Oh! for a woman who could be these two in one, who might have the affection of the one and the charm of the other! Why is it that we never encounter the object of our dreams, that we always meet with something that is only approximately like them?" He continued his vague reflections, soothed by the monotonous sound of the voice that fell unheeded on his ear, upon all the charms that had combined to seduce and vanquish him in the mistress whom he had abandoned. In the besetment of her memory, of her imaginary presence, by which he was haunted as a visionary by a phantom, he asked himself: "Am I condemned to carry her image with me to all eternity?" He again applied himself to taking long walks, to roaming through the thicknesses of the forest, with the vague hope that he might lose her somewhere, in the depths of a ravine, behind a rock, in a thicket, as a man who wishes to rid himself of an animal that he
- 39. does not care to kill sometimes takes it away a long distance so that it may not find its way home. In the course of one of these walks he one day came again to the spot where the beeches grew. It was now a gloomy forest, almost as black as night, with impenetrable foliage. He passed along beneath the immense, deep vault in the damp, sultry air, thinking regretfully of his earlier visit when the little half-opened leaves resembled a verdant, sunshiny mist, and as he was following a narrow path, he suddenly stopped in astonishment before two trees that had grown together. It was a sturdy beech embracing with two of its branches a tall, slender oak; and there could have been no picture of his love that would have appealed more forcibly and more touchingly to his imagination. Mariolle seated himself to contemplate them at his ease. To his diseased mind, as they stood there in their motionless strife, they became splendid and terrible symbols, telling to him, and to all who might pass that way, the everlasting story of his love. Then he went on his way again, sadder than before, and as he walked along, slowly and with eyes downcast, he all at once perceived, half hidden by the grass and stained by mud and rain, an old telegram that had been lost or thrown there by some wayfarer. He stopped. What was the message of joy or sorrow that the bit of blue paper that lay there at his feet had brought to some expectant soul? He could not help picking it up and opening it with a mingled feeling of curiosity and disgust. The words "Come—me—four o'clock—" were still legible; the names had been obliterated by the moisture. Memories, at once cruel and delightful, thronged upon his mind of all the messages that he had received from her, now to appoint the hour for a rendezvous, now to tell him that she could not come to him. Never had anything caused him such emotion, nor startled him so violently, nor so stopped his poor heart and then set it thumping again as had the sight of those messages, burning or freezing him as the case might be. The thought that he should never receive more of them filled him with unutterable sorrow.
- 40. Again he asked himself what her thoughts had been since he left her. Had she suffered, had she regretted the friend whom her coldness had driven from her, or had she merely experienced a feeling of wounded vanity and thought nothing more of his abandonment? His desire to learn the truth was so strong and so persistent that a strange and audacious, yet only half-formed resolve, came into his head. He took the road to Fontainebleau, and when he reached the city went to the telegraph office, his mind in a fluctuating state of unrest and indecision; but an irresistible force proceeding from his heart seemed to urge him on. With a trembling hand, then, he took from the desk a printed blank and beneath the name and address of Mme. de Burne wrote this dispatch: "I would so much like to know what you think of me! For my part I can forget nothing. ANDRÉ MARIOLLE." Then he went out, engaged a carriage, and returned to Montigny, disturbed in mind by what he had done and regretting it already. He had calculated that in case she condescended to answer him he would receive a letter from her two days later, but the fear and the hope that she might send him a dispatch kept him in his house all the following day. He was in his hammock under the lindens on the terrace, when, about three o'clock, Elisabeth came to tell him that there was a lady at the house who wanted to see him. The shock was so great that his breath failed him for a moment and his legs bent under him, and his heart beat violently as he went toward the house. And yet he could not dare hope that it was she. When he appeared at the drawing-room door Mme. de Burne arose from the sofa where she was sitting and came forward to shake hands with a rather reserved smile upon her face, with a slight constraint of manner and attitude, saying: "I came to see how you are, as your message did not give me much information on the subject."
- 41. He had become so pale that a flash of delight rose to her eyes, and his emotion was so great that he could not speak, could only hold his lips glued to the hand that she had given him. "Dieu! how kind of you!" he said at last. "No; but I do not forget my friends, and I was anxious about you." She looked him in the face with that rapid, searching woman's look that reads everything, fathoms one's thoughts to their very roots, and unmasks every artifice. She was satisfied, apparently, for her face brightened with a smile. "You have a pretty hermitage here," she continued. "Does happiness reside in it?" "No, Madame." "Is it possible? In this fine country, at the side of this beautiful forest, on the banks of this pretty stream? Why, you ought to be at rest and quite contented here." "I am not, Madame." "Why not, then?" "Because I cannot forget." "Is it indispensable to your happiness that you should forget something?" "Yes, Madame." "May one know what?" "You know." "And then?" "And then I am very wretched." She said to him with mingled fatuity and commiseration: "I thought that was the case when I received your telegram, and that was the reason that I came, with the resolve that I would go back again at once if I found that I had made a mistake." She was silent a moment and then went on: "Since I am not going back immediately,
- 42. may I go and look around your place? That little alley of lindens yonder has a very charming appearance: it looks as if it might be cooler out there than here in this drawing-room." They went out. She had on a mauve dress that harmonized so well with the verdure of the trees and the blue of the sky that she appeared to him like some amazing apparition, of an entirely new style of beauty and seductiveness. Her tall and willowy form, her bright, clean-cut features, the little blaze of blond hair beneath a hat that was mauve, like the dress, and lightly crowned by a long plume of ostrich-feathers rolled about it, her tapering arms with the two hands holding the closed sunshade crosswise before her, the loftiness of her carriage, and the directness of her step seemed to introduce into the humble little garden something exotic, something that was foreign to it. It was a figure from one of Watteau's pictures, or from some fairy-tale or dream, the imagination of a poet's or an artist's fancy, which had been seized by the whim of coming away to the country to show how beautiful it was. As Mariolle looked at her, all trembling with his newly lighted passion, he recalled to mind the two peasant women that he had seen in Montigny village. "Who is the little person who opened the door for me?" she inquired. "She is my servant." "She does not look like a waitress." "No; she is very good looking." "Where did you secure her?" "Quite near here; in an inn frequented by painters, where her innocence was in danger from the customers." "And you preserved it?" He blushed and replied: "Yes, I preserved it." "To your own advantage, perhaps." "Certainly, to my own advantage, for I would rather have a pretty face about me than an ugly one."
- 43. "Is that the only feeling that she inspires in you?" "Perhaps it was she who inspired in me the irresistible desire of seeing you again, for every woman when she attracts my eyes, even if it is only for the duration of a second, carries my thoughts back to you." "That was a very pretty piece of special pleading! And does she love her preserver?" He blushed more deeply than before. Quick as lightning the thought flashed through his mind that jealousy is always efficacious as a stimulant to a woman's feelings, and decided him to tell only half a lie, so he answered, hesitatingly: "I don't know how that is; it may be so. She is very attentive to me." Rather pettishly, Mme. de Burne murmured: "And you?" He fastened upon her his eyes that were aflame with love, and replied: "Nothing could ever distract my thoughts from you." This was also a very shrewd answer, but the phrase seemed to her so much the expression of an indisputable truth, that she let it pass without noticing it. Could a woman such as she have any doubts about a thing like that? So she was satisfied, in fact, and had no further doubts upon the subject of Elisabeth. They took two canvas chairs and seated themselves in the shade of the lindens over the running stream. He asked her: "What did you think of me?" "That you must have been very wretched." "Was it through my fault or yours?" "Through the fault of us both." "And then?" "And then, knowing how beside yourself you were, I reflected that it would be best to give you a little time to cool down. So I waited." "What were you waiting for?"
- 44. "For a word from you. I received it, and here I am. Now we are going to talk like people of sense. So you love me still? I do not ask you this as a coquette—I ask it as your friend." "I love you still." "And what is it that you wish?" "How can I answer that? I am in your power." "Oh! my ideas are very clear, but I will not tell you them without first knowing what yours are. Tell me of yourself, of what has been passing in your heart and in your mind since you ran away from me." "I have been thinking of you; I have had no other occupation." He told her of his resolution to forget her, his flight, his coming to the great forest in which he had found nothing but her image, of his days filled with memories of her, and his long nights of consuming jealousy; he told her everything, with entire truthfulness, always excepting his love for Elisabeth, whose name he did not mention. She listened, well assured that he was not lying, convinced by her inner consciousness of her power over him, even more than by the sincerity of his manner, and delighted with her victory, glad that she was about to regain him, for she loved him still. Then he bemoaned himself over this situation that seemed to have no end, and warming up as he told of all that he had suffered after having carried it so long in his thoughts, he again reproached her, but without anger, without bitterness, in terms of impassioned poetry, with that impotency of loving of which she was the victim. He told her over and over: "Others have not the gift of pleasing; you have not the gift of loving." She interrupted him, speaking warmly, full of arguments and illustrations. "At least I have the gift of being faithful," she said. "Suppose I had adored you for ten months, and then fallen in love with another man, would you be less unhappy than you are?"
- 45. He exclaimed: "Is it, then, impossible for a woman to love only one man?" But she had her answer ready for him: "No one can keep on loving forever; all that one can do is to be constant. Do you believe that that exalted delirium of the senses can last for years? No, no. As for the most of those women who are addicted to passions, to violent caprices of greater or less duration, they simply transform life into a novel. Their heroes are different, the events and circumstances are unforeseen and constantly changing, the dénouement varies. I admit that for them it is amusing and diverting, for with every change they have a new set of emotions, but for him—when it is ended, that is the last of it. Do you understand me?" "Yes; what you say has some truth in it. But I do not see what you are getting at." "It is this: there is no passion that endures a very long time; by that I mean a burning, torturing passion like that from which you are suffering now. It is a crisis that I have made hard, very hard for you to bear—I know it, and I feel it—by—by the aridity of my tenderness and the paralysis of my emotional nature. This crisis will pass away, however, for it cannot last forever." "And then?" he asked with anxiety. "Then I think that to a woman who is as reasonable and calm as I am you can make yourself a lover who will be pleasing in every way, for you have a great deal of tact. On the other hand you would make a terrible husband. But there is no such thing as a good husband, there never can be." He was surprised and a little offended. "Why," he asked, "do you wish to keep a lover that you do not love?" She answered, impetuously: "I do love him, my friend, after my fashion. I do not love ardently, but I love." "You require above everything else to be loved and to have your lovers make a show of their love."
- 46. "It is true. That is what I like. But beyond that my heart requires a companion apart from the others. My vainglorious passion for public homage does not interfere with my capacity for being faithful and devoted; it does not destroy my belief that I have something of myself that I could bestow upon a lover that no other man should have: my loyal affection, the sincere attachment of my heart, the entire and secret trustfulness of my soul; in exchange for which I should receive from him, together with all the tenderness of a lover, the sensation, so sweet and so rare, of not being entirely alone upon the earth. That is not love from the way you look at it, but it is not entirely valueless, either." He bent over toward her, trembling with emotion, and stammered: "Will you let me be that man?" "Yes, after a little, when you are more yourself. In the meantime, resign yourself to a little suffering once in a while, for my sake. Since you have to suffer in any event, isn't it better to endure it at my side rather than somewhere far from me?" Her smile seemed to say to him: "Why can you not have confidence in me?" and as she eyed him there, his whole frame quivering with passion, she experienced through every fiber of her being a feeling of satisfied well-being that made her happy in her way, in the way that the bird of prey is happy when he sees his quarry lying fascinated beneath him and awaiting the fatal talons. "When do you return to Paris?" she asked. "Why—to-morrow!" "To-morrow be it. You will come and dine with me?" "Yes, Madame." "And now I must be going," said she, looking at the watch set in the handle of her parasol. "Oh! why so soon?" "Because I must catch the five o'clock train. I have company to dinner to-day, several persons: the Princess de Malten, Bernhaus,
- 47. Lamarthe, Massival, De Maltry, and a stranger, M. de Charlaine, the explorer, who is just back from upper Cambodia, after a wonderful journey. He is all the talk just now." Mariolle's spirits fell; it hurt him to hear these names mentioned one after the other, as if he had been stung by so many wasps. They were poison to him. "Will you go now?" he said, "and we can drive through the forest and see something of it." "I shall be very glad to. First give me a cup of tea and some toast." When the tea was served, Elisabeth was not to be found. The cook said that she had gone out to make some purchases. This did not surprise Mme. de Burne, for what had she to fear now from this servant? Then they got into the landau that was standing before the door, and Mariolle made the coachman take them to the station by a roundabout way which took them past the Gorge-aux-Loups. As they rolled along beneath the shade of the great trees where the nightingales were singing, she was seized by the ineffable sensation that the mysterious and all-powerful charm of nature impresses on the heart of man. "Dieu!" she said, "how beautiful it is, how calm and restful!" He accompanied her to the station, and as they were about to part she said to him: "I shall see you to-morrow at eight o'clock, then?" "To-morrow at eight o'clock, Madame." She, radiant with happiness, went her way, and he returned to his house in the landau, happy and contented, but uneasy withal, for he knew that this was not the end. Why should he resist? He felt that he could not. She held him by a charm that he could not understand, that was stronger than all. Flight would not deliver him, would not sever him from her, but would be an intolerable privation, while if he could only succeed in showing a little resignation, he would obtain from her at least as
- 48. much as she had promised, for she was a woman who always kept her word. The horses trotted along under the trees and he reflected that not once during that interview had she put up her lips to him for a kiss. She was ever the same; nothing in her would ever change and he would always, perhaps, have to suffer at her hands in just that same way. The remembrance of the bitter hours that he had already passed, with the intolerable certainty that he would never succeed in rousing her to passion, laid heavy on his heart, and gave him a clear foresight of struggles to come and of similar distress in the future. Still, he was content to suffer everything rather than lose her again, resigned even to that everlasting, ever unappeased desire that rioted in his veins and burned into his flesh. The raging thoughts that had so often possessed him on his way back alone from Auteuil were now setting in again. They began to agitate his frame as the landau rolled smoothly along in the cool shadows of the great trees, when all at once the thought of Elisabeth awaiting him there at his door, she, too, young and fresh and pretty, her heart full of love and her mouth full of kisses, brought peace to his soul. Presently he would be holding her in his arms, and, closing his eyes and deceiving himself as men deceive others, confounding in the intoxication of the embrace her whom he loved and her by whom he was loved, he would possess them both at once. Even now it was certain that he had a liking for her, that grateful attachment of soul and body that always pervades the human animal as the result of love inspired and pleasure shared in common. This child whom he had made his own, would she not be to his dry and wasting love the little spring that bubbles up at the evening halting place, the promise of the cool draught that sustains our energy as wearily we traverse the burning desert? When he regained the house, however, the girl had not come in. He was frightened and uneasy and said to the other servant: "You are sure that she went out?" "Yes, Monsieur."
- 49. Thereupon he also went out in the hope of finding her. When he had taken a few steps and was about to turn into the long street that runs up the valley, he beheld before him the old, low church, surmounted by its square tower, seated upon a little knoll and watching the houses of its small village as a hen watches over her chicks. A presentiment that she was there impelled him to enter. Who can tell the strange glimpses of the truth that a woman's heart is capable of perceiving? What had she thought, how much had she understood? Where could she have fled for refuge but there, if the shadow of the truth had passed before her eyes? The church was very dark, for night was closing in. The dim lamp, hanging from its chain, suggested in the tabernacle the ideal presence of the divine Consoler. With hushed footsteps Mariolle passed up along the lines of benches. When he reached the choir he saw a woman on her knees, her face hidden in her hands. He approached, recognized her, and touched her on the shoulder. They were alone. She gave a great start as she turned her head. She was weeping. "What is the matter?" he said. She murmured: "I see it all. You came here because she had caused you to suffer. She came to take you away." He spoke in broken accents, touched by the grief that he in turn had caused: "You are mistaken, little one. I am going back to Paris, indeed, but I shall take you with me." She repeated, incredulously: "It can't be true, it can't be true." "I swear to you that it is true." "When?" "To-morrow." She began again to sob and groan: "My God! My God!" Then he raised her to her feet and led her down the hill through the thick blackness of the night, but when they came to the river-bank
- 50. he made her sit down upon the grass and placed himself beside her. He heard the beating of her heart and her quick breathing, and clasping her to his heart, troubled by his remorse, he whispered to her gentle words that he had never used before. Softened by pity and burning with desire, every word that he uttered was true; he did not endeavor to deceive her, and surprised himself at what he said and what he felt, he wondered how it was that, thrilling yet with the presence of that other one whose slave he was always to be, he could tremble thus with longing and emotion while consoling this love-stricken heart. He promised that he would love her,—he did not say simply "love"—, that he would give her a nice little house near his own and pretty furniture to put in it and a servant to wait on her. She was reassured as she listened to him, and gradually grew calmer, for she could not believe that he was capable of deceiving her, and besides his tone and manner told her that he was sincere. Convinced at length and dazzled by the vision of being a lady, by the prospect—so undreamed of by the poor girl, the servant of the inn—of becoming the "good friend" of such a rich, nice gentleman, she was carried away in a whirl of pride, covetousness, and gratitude that mingled with her fondness for André. Throwing her arms about his neck and covering his face with kisses, she stammered: "Oh! I love you so! You are all in all to me!" He was touched and returned her caresses. "Darling! My little darling!" he murmured. Already she had almost forgotten the appearance of the stranger who but now had caused her so much sorrow. There must have been some vague feeling of doubt floating in her mind, however, for presently she asked him in a tremulous voice: "Really and truly, you will love me as you love me now?" And unhesitatingly he replied: "I will love you as I love you now."
- 51. THE OLIVE GROVE AND OTHER TALES THE OLIVE GROVE When the 'longshoremen of Garandou, a little port of Provence, situated in the bay of Pisca, between Marseilles and Toulon, perceived the boat of the Abbé Vilbois entering the harbor, they went down to the beach to help him pull her ashore. The priest was alone in the boat. In spite of his fifty-eight years, he rowed with all the energy of a real sailor. He had placed his hat on the bench beside him, his sleeves were rolled up, disclosing his powerful arms, his cassock was open at the neck and turned over his knees, and he wore a round hat of heavy, white canvas. His whole appearance bespoke an odd and strenuous priest of southern climes, better fitted for adventures than for clerical duties. He rowed with strong and measured strokes, as if to show the southern sailors how the men of the north handle the oars, and from time to time he turned around to look at the landing point. The skiff struck the beach and slid far up, the bow plowing through the sand; then it stopped abruptly. The five men watching for the abbé drew near, jovial and smiling. "Well!" said one, with the strong accent of Provence, "have you been successful, Monsieur le Curé?" The abbé drew in the oars, removed his canvas head-covering, put on his hat, pulled down his sleeves, and buttoned his coat. Then having assumed the usual appearance of a village priest, he replied
- 52. proudly: "Yes, I have caught three red-snappers, two eels, and five sunfish." The fishermen gathered around the boat to examine, with the air of experts, the dead fish, the fat red-snappers, the flat-headed eels, those hideous sea-serpents, and the violet sunfish, streaked with bright orange-colored stripes. Said one: "I'll carry them up to your house, Monsieur le Curé." "Thank you, my friend." Having shaken hands all around, the priest started homeward, followed by the man with the fish; the others took charge of the boat. The Abbé Vilbois walked along slowly with an air of dignity. The exertion of rowing had brought beads of perspiration to his brow and he uncovered his head each time that he passed through the shade of an olive grove. The warm evening air, freshened by a slight breeze from the sea, cooled his high forehead covered with short, white hair, a forehead far more suggestive of an officer than of a priest. The village appeared, built on a hill rising from a large valley which descended toward the sea. It was a summer evening. The dazzling sun, traveling toward the ragged crests of the distant hills, outlined on the white, dusty road the figure of the priest, the shadow of whose three-cornered hat bobbed merrily over the fields, sometimes apparently climbing the trunks of the olive-trees, only to fall immediately to the ground and creep among them. With every step he took, he raised a cloud of fine, white dust, the invisible powder which, in summer, covers the roads of Provence; it clung to the edge of his cassock turning it grayish white. Completely refreshed, his hands deep in his pockets, he strode along slowly and ponderously, like a mountaineer. His eyes were fixed on the distant village where he had lived twenty years, and where he hoped to die.
- 53. Its church—his church—rose above the houses clustered around it; the square turrets of gray stone, of unequal proportions and quaint design, stood outlined against the beautiful southern valley; and their architecture suggested the fortifications of some old château rather than the steeples of a place of worship. The abbé was happy; for he had caught three red-snappers, two eels, and five sunfish. It would enable him to triumph again over his flock, which respected him, no doubt, because he was one of the most powerful men of the place, despite his years. These little innocent vanities were his greatest pleasures. He was a fine marksman; sometimes he practiced with his neighbor, a retired army provost who kept a tobacco shop; he could also swim better than anyone along the coast. In his day he had been a well-known society man, the Baron de Vilbois, but had entered the priesthood after an unfortunate love- affair. Being the scion of an old family of Picardy, devout and royalistic, whose sons for centuries had entered the army, the magistracy, or the Church, his first thought was to follow his mother's advice and become a priest. But he yielded to his father's suggestion that he should study law in Paris and seek some high office. While he was completing his studies his father was carried off by pneumonia; his mother, who was greatly affected by the loss, died soon afterward. He came into a fortune, and consequently gave up the idea of following a profession to live a life of idleness. He was handsome and intelligent, but somewhat prejudiced by the traditions and principles which he had inherited, along with his muscular frame, from a long line of ancestors. Society gladly welcomed him and he enjoyed himself after the fashion of a well-to-do and seriously inclined young man. But it happened that a friend introduced him to a young actress, a pupil of the Conservatoire, who was appearing with great success at the Odéon. It was a case of love at first sight.
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