Biofilm formation is a primary cause of implant-associated infections in dental and orthopedic devices. Early interactions between bacteria and host cells at the implant surface critically influence subsequent infection or successful integration. Therefore, biocompatibility screening should evaluate both biofilm formation and cell adhesion. Adhesion Index, defined as the ratio of cell adhesion to biofilm adhesion, provides a non-dimensional number to optimize towards that end. In this work, the laser spallation technique is implemented to compare the effects of implant surface characteristics (rough and smooth titanium) and protein coatings (blood plasma and fibronectin) on bacterial biofilm (Streptococcus mutans, Staphylococcus aureus) and cell monolayer (MG-63) adhesion, to obtain quantitative adhesion measurements of each biological film. Over 18 different adhesion measurements are presented across surface roughness, biofilm type, cell type, and protein coating. Failure statistics are analyzed using two-parameter Weibull cumulative distribution functions to determine interfacial adhesion strengths and confidence intervals. Results show that MG-63 osteoblast-like cell adhesion strength increases with surface roughness from 143 MPa (95% CI: 114-176) on smooth to 292 MPa (95% CI: 267-306) on rough titanium. Fibronectin coating enhances cell adhesion on smooth titanium but reduces adhesion on rough surfaces relative to uncoated controls. Plasma coating yields similarly high cell adhesion strengths on both smooth and rough titanium, 268.7 MPa (95% CI: 248.8-289.3) and 269.4 MPa (95% CI: 249.7-289.6), respectively. In contrast, S. mutans and S. aureus exhibit minimal response to surface roughness. Plasma and fibronectin coatings reduce S. mutans adhesion on both smooth and rough surfaces, while S. aureus remains largely unaffected. Adhesion Index results indicate plasma-coated smooth titanium is optimal for dental applications, whereas rough titanium and plasma coating on both rough and smooth titanium are most favorable for orthopedic implants. These findings provide quantitative guidance for tailoring implant surface design to specific clinical applications.

Rapid and strategic cell placement is necessary for high throughput tissue fabrication. Current adhesive cell patterning systems rely on fluidic shear flow to remove cells outside of the patterned regions, but limitations in washing complexity and uniformity prevent adhesive patterns from being widely applied. Centrifugation is commonly used to study the adhesive strength of cells to various substrates; however, the approach has not been applied to selective cell adhesion systems to create highly organized cell patterns. This study shows centrifugation as a promising method to wash cellular patterns after selective binding of cells to the surface has taken place. After patterning H9C2 cells using biotin-streptavidin as a model adhesive patterning system and washing with centrifugation, there is a significant number of cells removed outside of the patterned areas of the substrate compared to the initial seeding, while there is not a significant number removed from the desired patterned areas. This method is effective in patterning multiple size and linear structures from line widths of 50-200 μm without compromising immediate cell viability below 80%. We also test this procedure on a variety of tube-forming cell lines (MPCs, HUVECs) on various tissue-like surface materials (collagen 1 and Matrigel) with no significant differences in their respective tube formation metrics when the cells were seeded directly on their unconjugated surface versus patterned and washed through centrifugation. This result demonstrates that our patterning and centrifugation system can be adapted to a variety of cell types and substrates to create patterns tailored to many biological applications.

Cell mechanics significantly influence cell-environment interactions and fundamental cellular functions such as growth, motility, and phagocytosis. Alveolar macrophages are a type of immune cell found in the alveoli of the lungs that are responsible for the removal of pathogens and help mediate inflammatory responses. Patients with severe lung inflammation are often treated with high levels of inhaled oxygen. This study investigates the effects of hyperoxia on the morphology and mechanical properties of alveolar macrophages. We test the hypothesis that structural changes due to hyperoxia exposure result in measurable differences in cell elasticity. Atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM) are used to measure changes in cell elasticity through force-indentation measurements and fluorescence imaging of cytoskeletal structure, respectively. Cells are harvested from C57BL/6 J mice, isolated, plated in glass-bottomed dishes, and immediately subjected to either hyperoxic (90

During epithelial-to-mesenchymal transition (EMT), cancer cells lose their cell-cell adhesion junctions as they become more metastatic, altering cell motility and focal adhesion disassembly associated with increased detachment from the primary tumor and a migratory response into nearby tissue and vasculature. Current in vitro strategies characterizing a cell's metastatic potential heavily favor quantifying the presence of cell adhesion biomarkers through biochemical analysis; however, mechanical cues such as adhesion and motility directly relate to cell metastatic potential without needing to first identify a cell specific biomarker for a particular type of cancer. This paper presents a comprehensive comparison of two functional metrics of cancer aggression, wound closure migration velocity and cell detachment from a culture surface, for three pairs of epithelial cancer cell lines (breast, endometrium, tongue tissue origins). It was found that one functional metric alone was not sufficient to categorize the cancer cell lines; instead, both metrics were necessary to identify functional trends and accurately place cells on the spectrum of metastasis. On average, cell lines with low metastatic potential (MCF-7, Ishikawa, and Cal-27) were more aggressive through wound closure migration compared to loss of cell adhesion. On the other hand, cell lines with high metastatic potential (MDA-MB-231, KLE, and SCC-25) were on average more aggressive through loss of cell adhesion compared to wound closure migration. This trend was true independent of the tissue type where the cells originated, indicating that there is a relationship between metastatic potential and the predominate type of cancer aggression. Our work presents one of the first combined studies relating cell metastatic potential to functional migration and adhesion metrics across cancer cell lines from selected tissue origins, without needing to identify tissue-specific biomarkers to achieve success. Using functional metrics provides powerful clinical relevancy for future predictive tools of cancer metastasis.

Biofilms consist of bacterial cells surrounded by a matrix of extracellular polymeric substance (EPS), which protects the colony from many countermeasures, including antibiotic treatments. Growth and formation of bacterial biofilms are affected by nutrients available in the environment. In the oral cavity, the presence of sucrose affects the growth of Streptococcus mutans that produce acids that erode enamel and form dental caries. Biofilm formation on dental implants commonly leads to severe infections and can restrict osseointegration necessary for the implant to be successful. This work determines the effect of sucrose concentration on biofilm EPS formation and adhesion of Streptococcus mutans, a common oral colonizer, to titanium substrates simulating common dental implants. Biofilm formation and profiles are visualized at high magnification with scanning electron microscopy (SEM). Large mounds and complex structures consisting of bacterial cells and EPS can be seen in biofilms at sucrose concentrations that are favorable for biofilm growth. The laser spallation technique is used to apply stress wave loading to the biofilm, causing the biofilm to delaminate at a critical tensile stress threshold. The critical tensile stress threshold is the adhesion strength. Because laser spallation applies the stress loading to the rear of the substrate, bulk adhesion properties of the biofilm can be determined despite the heterogenous composition and low cohesion strength of the biofilm. Statistical analysis reveals that adhesion strength of biofilms initially increase with increasing sucrose concentration and then decrease as sucrose concentration continues to increase. The adhesion strength of bacterial biofilms to the substrate in this study is compared to the adhesion of osteoblast-like cells to the same substrates published previously. When sucrose is present in the biofilm growth environment, S. mutans adhesion is higher than that of the osteoblast-like cells. Results of this study suggest sucrose-mediated S. mutans biofilms may outcompete osteoblasts in terms of adhesion during osseointegration, which could explain higher rates of peri-implant disease associated with high sugar diets. Further studies demonstrating adhesion differentials between biofilms and cells including co-cultures are needed and motivated by the present work.

Bacteria can proliferate orthopedic implants, resulting in infection rates as high as 5%. A consistent problem across implantology is the development of surfaces which successfully promote the adhesion and propagation of healthy fibroblast and osteoblast cells while deterring formation of bacterial biofilms. Selecting surface configurations which favor cell adhesion will lead to decreased infection rates. Progress in identifying appropriate surface configurations is hindered by the lack of quantitative adhesion techniques capable of comparing adhesion of cells and biofilms directly. Recent advancements in adhesion techniques have allowed for quantitatively measured adhesion strengths of both bacterial biofilms and cell monolayers using the laser spallation technique. The quantified stress-based adhesion values allow surface and environmental factors that modulate both bacterial and cell adhesion to implant surfaces to be evaluated. During implantation, blood propagates wound sites completely coating implant surfaces. Quantitatively determining the impact of preconditioning layers that accumulate on the implant surface on cell adhesion is vital to predict implant behavior. Previous work has demonstrated that these preconditioning layers either negatively or neutrally impact bacterial adhesion to titanium implant surfaces. This study focuses on the impact that blood plasma and fibronectin coatings have on the adhesion of osteoblastic (MG 63) cells and fibroblasts to the same titanium surfaces. Adhesion results indicate that preconditioning layers and increased surface roughness positively impact cell adhesion. Incorporating the increased adhesion values for cell adhesion into the Adhesion Index demonstrates that increased surface roughness, coupled with natural wound healing preconditioning of surfaces, yields positive biocompatibility.

Adhesion of bacteria to oral implant surfaces can lead to oral infections, and the prevention of strong biofilm adherence to implant surfaces can assist in the prevention of these infections like peri-implantitis. In prior studies, single species biofilm adhesion has been quantitatively measured via the laser spallation technique. However, colonizing oral biofilms rarely consists of a single bacteria species. Multiple early colonizer species, including several strains of Streptococci, dominate initial oral biofilm formation. This study aims to characterize the adhesion of a multi-species oral biofilm consisting of S. oralis, S. sanguinis, and S. gordonii on titanium, a common implant material, using the laser spallation technique. Previous work has established these specific Streptococci strains as a multi-species periodontal biofilm model. This study is the first to provide a quantitative adhesion measurement of this multi-species model onto a dental implant surface. First, adhesion strength of the multi-species model is compared to adhesion strength of the single-species streptococci constituents. Fluorescent staining and imaging by fluorescent microscopy are used to identify individual bacteria species within the biofilm. The multi-species biofilm presented in this study provides a more representative model of in vivo early biofilms and provides a more accurate metric for understanding biocompatibility on implant surfaces.

Increasing antibiotic resistance in bacteria is a critical issue that often leads to infections or other morbidities. Mechanical properties of the bacterial cell wall, such as thickness or elastic modulus, may contribute to the ability of a bacterial cell to resist antibiotics. Techniques like atomic force microscopy (AFM) are used to quantify bacterial cell mechanical properties and image cell structures at nanoscale resolutions. An additional benefit of AFM is the ability to probe samples submerged in liquids, meaning that live bacteria can be imaged or evaluated in environments that more accurately simulate in vivo conditions as compared to other methods like electron microscopy. However, because AFM measurements are highly sensitive to small perturbations in the deflection of the tip of a sensor probe brought into contact with the specimen, immobilization of bacteria prior to measurement is essential for accurate measurements. Traditional chemical fixatives crosslink the molecules within the bacterial cell wall, which prevent the bacteria from locomotion. While effective for imaging, chemical crosslinkers are known to affect the measured stiffness of eukaryotic cells and also may affect the measured stiffness of the bacterial cell wall. Alternative immobilization methods include Cell-Tak™, an adhesive derived from marine mussels that does not interact with the bacterial wall and filters with known pore sizes which entrap bacteria. Previous studies have examined the effect of these immobilization methods on successful imaging of bacteria but have not addressed differences in measured modulus. This study compares the effects of immobilization methods including chemical fixatives, mechanical entrapment in filters, and Cell-Tak™ on the stiffness of the bacterial cell wall as measured by force spectroscopy.

Laser-induced spallation is a process in which a stress wave generated from a rapid, high-energy laser pulse initiates the ejection of surface material opposite the surface of laser impingement. Through knowledge of the stress-wave amplitude that causes film separation, the adhesion and interfacial properties of a film-on-substrate system are determined. Some advantages of the laser spallation technique are the noncontact loading, development of large stresses (on the order of GPa), and high strain rates, up to 108/s. The applicability to both relatively thick films, tens of microns, and thin films, tens of nm, make it a unique technique for a wide range of materials and applications. This review combines the available knowledge and experience in laser spallation, as a state-of-the-art measurement tool, in a comprehensive pedagogical publication for the first time. An historical review of adhesion measurement by the laser-induced spallation technique, from its inception in the 1970s through the present day, is provided. An overview of the technique together with the physics governing the laser-induced spallation process, including functions of the absorbing and confining materials, are also discussed. Special attention is given to applications of laser spallation as an adhesion quantification technique in metals, polymers, composites, ceramics, and biological films. A compendium of available experimental parameters is provided that summarizes key laser spallation experiments across these thin-film materials. This review concludes with a future outlook for the laser spallation technique, which approaches its semicentennial anniversary.

Biofilm formation is a significant problem in America, accounting for 17 million infections, and causing 550,000 deaths annually. An understanding of factors that contribute to strong biofilm surface adhesion at implant interfaces can guide the development of surfaces that prevent deleterious biofilms and promote osseointegration. The aim of this research is to develop a metric that quantifies the adhesion strength differential between a bacterial biofilm and an osteoblast-like cell monolayer to a medical implant-simulant surface. This metric will be used to quantify the biocompatible effect of implant surfaces on bacterial and cell adhesion. The laser spallation technique employs a high-amplitude short-duration stress wave to initiate spallation of biological films. Attenuation of laser energy results in failure statistics across increasing fluence values, which are calibrated via interferometry to obtain interface stress values. Several metrology challenges were overcome including how membrane tension may influence laser spallation testing and how to determine stress wave characteristics when surface roughness precludes in situ displacement measurements via interferometry. Experiments relating loading region within biofilm to centroid of biofilm revealed that location played no role in failure rate. A reflective panel was implemented to measure stress wave characteristics on smooth and rough titanium, which showed no difference in peak compressive wave amplitude. After overcoming these metrology challenges, the adhesion strength of Streptococcus mutans biofilms and MG 63 monolayers on smooth and rough titanium substrates is measured. An Adhesion Index is developed by obtaining the ratio of cell adhesion to biofilm adhesion. This nondimensionalized parameter represents the effect of surface modifications on increases or decreases in biocompatibility. An increase in Adhesion Index value is calculated for roughened titanium compared to smooth titanium. The increase in Adhesion Index values indicates that the increase in surface roughness has a more positive biological response from MG 63 than does S. mutans. In this work further experiments quantifying impact of various surface coating including blood plasma, and adhesion proteins found within the extracellular matrix to expand the Adhesion Index.

Purpose: Biofilm formation caused by infrequent contact lens case replacement and the ineffectiveness of multi-purpose solutions (MPS) on biofilm removal is associated with high rates of bacterial keratitis infections. This study demonstrated biofilm elimination from the contact lens case by microwave irradiation. Methods: Staphylococcus aureus biofilms indicative of 3-9 months of contact wear were cultured in contact lens cases and visualized with crystal violet (CV) staining. Biofilms in contact cases were then exposed to four treatment regimens: No treatment (n = 8), 45 s microwave irradiation (n = 8), tap water (n = 6), and MPS (n = 9). Bacterial survival was assessed by colony forming unit (CFU) assay using streak dilutions. Results: Visualization of the biofilms through CV staining revealed that biofilms coalesce between ribs of the contact case. In 5/8 cases no CFU were cultivated from the case after treatment with microwave irradiation. In tap water and MPS the first dilution averaged 6 +/- 2 and 31 +/- 13 CFUs per plate, respectively, while microwave irradiation averaged < 1 CFU per plate. In Dilution 2, the average reduced to 0.7 +/- 0.7 and 6 +/- 5 CFUs per plate for tap water and MPS, respectively, while microwave irradiation had 0 CFUs in Dilution 2. Conclusion: Biofilms that coalesce between the ribs of the contact case pose a threat because this area is difficult to thoroughly scrub and could act as a basis for infection through fouling of contact lenses. Of the four treatment regimens, microwave irradiation displayed the most consistent and highest rate of bacterial eradication. Tap water was less effective compared to microwave irradiation, and poses other harmful side effects, but greatly reduced CFU count compared to no treatment. MPS displayed the poorest bacterial eradication of the treatments. Thus, microwave irradiation is worth further investigation as a viable in-home disinfecting option.

Antibiotic resistance is the phenomenon in which bacteria is or becomes resistant to antibiotics. As it pertains to oral bacteria, current methods for elimination of their presence and reduction of infection is through mechanical means, such as brushing, flossing, or swishing with an alcoholic rinse. These methods loosen the bacteria from oral surfaces or attack bacteria through cell disruption by protein denaturation and lipid membrane dissolution. One strain of oral bacteria that can cause plaque accumulation, chewing difficulty, impaired speech, and dental caries is Streptococcus mutans (S. mutans). S. mutans is a group of Gram-positive, facultatively anaerobic, cocci bacteria that can contribute to a multitude of oral hygiene concerns, including loss of dental implants in cases of high concentration. Research suggests that modification of the cell wall may lead to new developments on how to regulate cell permeation, thereby, regulation of microbial resistance mechanisms. In this work, three S. mutans stains with altered peptidoglycan layers are studied through atomic force and confocal microscopy. Surface roughness differences due to molecular glycerol phosphate alterations are quantified and compared at two positions: near the pole of the cell and within the septal region. We show AFM images of bacteria scans, including the cell wall, and quantitative surface roughness measurements at the two difference regions for each strain investigated.

Objectives The aim of this study is to quantify the adhesion strength differential between an oral bacterial biofilm and an osteoblast-like cell monolayer to a dental implant-simulant surface and develop a metric that quantifies the biocompatible efficacy of implant surfaces. Methods High-amplitude short-duration stress waves generated by laser pulse absorption are used to spall bacteria and cells from titanium substrates. By carefully controlling laser fluence and calibration of laser fluence with applied stress, the adhesion difference between dental carry Streptococcus mutans biofilms and MG 63 osteoblast-like cell monolayers on smooth and rough titanium substrates is obtained. The Adhesion Index consists of a ratio of cell adhesion strength to biofilm adhesion strength obtaining a nondimensionalized parameter for biocompatibility assessments. Results Adhesion strength of 145±42 MPa is measured for MG 63 on smooth titanium, which increases to 288±24 MPa on roughened titanium. Adhesion strength for S. mutans on smooth titanium is 315±9 MPa and remained relatively constant at 332±9 MPa on roughened titanium. The Adhesion Index for smooth titanium is 0.46±0.12 which increased to 0.87±0.05 on roughened titanium. Significance The laser spallation technique provides a platform to examine the tradeoffs of adhesion modulators on both biofilm and cell adhesion. This tradeoff is characterized by the Adhesion Index, which is proposed to aid biocompatibility screening and could result in improved implantation outcomes. The Adhesion Index is implemented to determine surface factors that promote favorable adhesion of cells greater than biofilms. Here, an Adhesion Index >> 1 suggests favorable biocompatibility. Graphical Abstract Highlights Biofilm and cell monolayer adhesion are measured via the laser spallation technique Smooth and roughened dental implant-mimicking titanium surfaces are investigated Surface roughness increases cell adhesion but does not alter the adhesion of biofilms An Adhesion Index is developed to directly quantify the adhesive competition between bacteria and cells on an implant surface

Nicotine is a highly addictive compound present in tobacco, which causes the release of dopamine in different regions of the brain. Recent studies have shown that astrocytes express nicotinic acetylcholine receptors (nAChRs) and mediate calcium signaling. In this study, we examine the morphological and functional adaptations of astrocytes due to nicotine exposure. Utilizing a combination of fluorescence and atomic force microscopy, we show that nicotine-treated astrocytes exhibit time-dependent remodeling in the number and length of both proximal and fine processes. Blocking nAChR activity with an antagonist completely abolishes nicotine's influence on astrocyte morphology indicating that nicotine's action is mediated by these receptors. Functional studies show that 24-hr nicotine treatment induces higher levels of calcium activity in both the cell soma and the processes with a more substantial change observed in the processes. Nicotine does not induce reactive astrocytosis even at high concentrations (10 μM) as determined by cytokine release and glial fibrillary acidic protein expression. We designed tissue clearing experiments to test whether morphological changes occur in vivo using astrocyte specific Aldh1l1-tdTomato knock in mice. We find that nicotine induces a change in the volume of astrocytes in the prefrontal cortex, CA1 of the hippocampus, and the substantia nigra. These results indicate that nicotine directly alters the functional and morphological properties of astrocytes potentially contributing to the underlying mechanism of nicotine abuse.

Tens of millions of Americans receive medical devices and procedures annually at a cost of $170 billion. Unfortunately, over half of all hospital acquired infections result from a permanent or temporary implanted device procedure. Although deleterious bacteria adherent to medically implanted devices are major contributors to this problem, biocompatibility standards lack consideration for these adherence properties. A biocompatibility assessment that considers both the adhesion of invasive bacteria and host cells is needed to advance patient care. The quantitative determination of the effect of implant surface characteristics on bacterial adhesion, as well as our bodies natural cell adhesion, will lead to the development of more successful medical implants. The laser spallation technique has been used previously for measurements of biofilm and cell adhesion. Laser spallation has distinct advantages as it results in quantified adhesion strength through a non-contact high strain rate force. The non-contact force does not disturb or alter the living films pre-testing, which ensures accurate adhesion measurements. This mechanical test is implemented alongside one dimensional wave analysis and an interface adhesion strength is measured for the biomaterial-implant mimicking surface interfaces. In this study, use of laser spallation on a titanium surface mimicking a dental implant device is explored. Streptococcus mutans, an oral gram-positive facultative anaerobe, is chosen because it is a common oral microbe and earlier colonizer of dental biofilms. In as much as titanium dental implants require osseous-integration, MG 63, selected for its numerous osteoblastic traits that are typical of an immature osteoblast, is optimal for initial adhesion studies. Bacteria and cells are cultured on titanium substrates that have undergone large grit sandblasting to mimic the roughness of dental implant surfaces. We demonstrate a quantitative evaluation of adhesion strength of an oral biofilm on dental implant mimicking surfaces through the adaptation of the laser spallation technique. The adhesion values for both invasive bacteria and constructive cells are compared to determine the biocompatibility for dental implant surfaces, by obtaining a unitless ratio referred to as the Adhesion Index. The Adhesion Index can be used to determine the biocompatibility of other medical implant devices and optimize future implant designs.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe "pokes" the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

We investigate laser-induced acoustic wave propagation through smooth and roughened titanium-coated glass substrates. Acoustic waves are generated in a controlled manner via the laser spallation technique. Surface displacements are measured during stress wave loading by the alignment of a Michelson-type interferometer. A reflective coverslip panel facilitates capture of surface displacements during loading of as-received smooth and roughened specimens. Through interferometric experiments, we extract the substrate stress profile at each laser fluence (energy per area). The shape and amplitude of the substrate stress profile are analyzed at each laser fluence. Peak substrate stress is averaged and compared between smooth specimens with the reflective panel and rough specimens with the reflective panel. The reflective panel is necessary because the surface roughness of the rough specimens precludes in situ interferometry. Through these experiments, we determine that the surface roughness employed has no significant effect on substrate stress propagation and smooth substrates are an appropriate surrogate to determine stress wave loading amplitude of roughened surfaces less than 1.2 μm average roughness (Ra). No significant difference was observed when comparing the average peak amplitude and loading slope in the stress wave profile for the smooth and rough configurations at each fluence.

Integral to the production of safe and biocompatible medical devices is to determine the interfacial properties that affect or control strong biofilm adhesion. The laser spallation technique has recently emerged as an advantageous method to quantify biofilm adhesion across candidate biomedical surfaces. However, there is a possibility that membrane tension is a factor that contributes to the stress required to separate biofilm and substrate. In that case, the stress amplitude, controlled by laser fluence, that initiates biofilm rupture would vary systematically with location on the biofilm. Film rupture, also known as spallation, occurs when film material is ejected during stress wave loading. In order to determine effects of membrane tension, we present a protocol that measures spall size with increasing laser fluence (variable fluence) and with respect to distance from the biofilm centroid (iso-fluence). Streptococcus mutans biofilms on titanium substrates serves as our model system. A total of 185 biofilm loading locations are analyzed in this study. We demonstrate that biofilm spall size increases monotonically with laser fluence and apply our procedure to failure of non-biological films. In iso-fluence experiments, no correlation is found between biofilm spall size and loading location, thus providing evidence that membrane tension does not play a dominant role in biofilm adhesion measurements. We recommend our procedure as a straightforward method to determine membrane effects in the measurement of adhesion of biological films on substrate surfaces via the laser spallation technique. Graphical Abstract