Introduction 

Biofilms are composed of living, reproducing microorganisms such as bacteria that form a colony or community. These living systems possess a social structure that offers both protection and promotes growth. Researchers are still working to fully comprehend this structure.¹. A biofilm is a thin layer of tiny organisms that attach to a surface, forming what can be described as a “slimy adhesive.” This biofilm both inhibits and promotes the growth of bacteria. It forms when bacteria adhere to a surface and produce a structure made of extracellular polymeric substances (EPS).² Microbial communities known as biofilms are ubiquitous in the environment, appearing as dental plaque on teeth, in water systems, air-conditioning units, food processing facilities, and various healthcare surfaces. Within biofilms, bacteria can interact, survive, nourish themselves, and proliferate. Bacteria inside biofilms are approximately 200 times more resistant to antibiotics or cleaning agents. As long as they remain viable, they continue to pose a threat to patients. The bacteria will keep multiplying until the biofilm is disrupted or free bacteria are released, potentially spreading to other surfaces via hands, gloves, cleaning cloths, or other materials. Pathogenic bacteria from genera such as Bacillus, Streptococcus, Staphylococcus, as well as Shigella, Escherichia coli, and Enterobacter aerogenes These microorganisms are classified as pathogenic, indicating their ability to induce illnesses. As they accumulate on firm surfaces, they form a slender, adhesive coating referred to as a biofilm. This protective layer facilitates the bacteria’s growth and proliferation, enhancing their survival and spread.³

In nature, most bacteria are not found as isolated, suspended cells. Rather, they exist in aggregates (clusters of bacterial cells) attached to surfaces. These aggregates, known as biofilms, consist of microorganisms adhering to each other on a surface. Microorganisms attach to solid surfaces and create a self-generated matrix of extracellular polysaccharides.⁴ This biofilm provides significant protection for the bacteria, serving as a crucial survival mechanism. The biofilm acts as a barrier, shielding microorganisms from antibiotics, sanitizing agents, and other external threats. Both harmless and harmful bacteria can form biofilms. The development of biofilms in processing environments is particularly concerning when it involves pathogenic bacteria, as it enhances their chances of survival and increases contamination risks. Consequently, it is vital to eliminate and prevent biofilm formation in facilities.⁵

Biofilm Formation⁶

There are typically five processes involved in the production of biofilms (see Figure 1 below):

Biofilm formation occurs in several stages:

Primary adhesion: The process begins when free-swimming bacteria attach to a surface.

Permanent bonding: As the biofilm grows through cellular multiplication, the extracellular polymeric substance (EPS) matrix produced by the cells aids in their adhesion to surfaces and each other. The EPS strengthens the connection between bacterial cells and the substrate. These bonds become more robust over time, resulting in irreversible attachment. 3. Development: The biofilm evolves into a structured, resilient formation that exhibits increased resistance to harmful chemicals and biocides.

Formation of microcolonies: Bacteria colonize preconditioned surfaces. This phase, which is still reversible, may progress to the irreversible stage of biofilm growth, followed by the actual establishment of the biofilm.

Maturation and dispersal: Biofilm maturation during which cells grow, cell density increases, and cells synthesize and release signaling molecules allowing them to sense and communicate with each other

Dispersal or detachment phase: where the cells depart in large numbers to become planktonic cells again. Bacterial cells are released into the surrounding environment.

Figure 1: Representation of biofilm formation process in five steps7   Click here to view Figure

Imaging methods play a vital role across many scientific disciplines, particularly in biological and medical research, where they are essential for examining and understanding complex objects and processes. These techniques allow for non-invasive, three-dimensional analysis of specimens at various spatial scales.⁸ The range of sizes examined spans from nanometers (for macromolecules) to micrometers (for cellular structures, microbial clusters, and biofilms) and even centimeters (for microbial mats, tissues, organs, and the human body). The journey into this microscopic realm began with basic tools such as magnifying glasses and simple optical microscopes. The first light microscope was used by Robert Hooke in 1665, shortly followed by Antonie van Leeuwenhoek, who became a pioneer in microbiology by examining samples from his mouth, specifically oral biofilms. For over a century, conventional light microscopy and its variations have been the primary methods for observing and studying biological materials. At one point, it was believed that light microscopy had reached its maximum developmental potential.⁹

Recently, numerous cutting-edge methods have been either created or modified to examine, leading to a better understanding of their physiological characteristics, structural features, and compositional elements. This article explores both traditional and modern methods used to analyze biofilm biomass, viability, structure, composition, and physiological properties.¹⁰ These methods include:

Microscopic analyses

Light microscope

Light microscopy (LM) is an important imaging technique for visually identifying biofilms and provides valuable prognostic information. Its simplicity and cost-effectiveness make it suitable for quantifying biofilm biomass. However, LM has limitations in magnification and resolution, which prevent it from capturing the finer details of extracellular polymeric substance (EPS) structure or the morphology of biofilm cells. Despite these limitations, LM can be effectively combined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in correlative studies.¹¹

Transmission electron microscope (TEM) 

Electrons are used to create highly magnified and detailed images of cells and their components, including proteins and nucleic acids. Negative staining techniques in transmission electron microscopy (TEM) allow for direct visualization of cellular structures. Because photons and electrons have limited penetration into cells, thin sections of cellular material are treated with stabilizing and staining agents such as osmic acid, permanganate, uranium, lanthanum, or lead salts. These staining compounds contain elements with high atomic weights, which enhances contrast by increasing electron scattering from the sample.¹²

Scanning electron microscope (SEM) 

Researchers apply heavy metals such as gold to create 3D representations of cell samples. Scanning electron microscopy (SEM) generates images by detecting electrons emitted from these metal-coated samples. While SEM is similar to transmission electron microscopy (TEM), it has distinct differences: it does not require infiltration, resin embedment, polymerization, or staining with lead citrate and uranyl acetate staining.¹³ However, SEM does involve the use of additional substances like gold. SEM uses accelerated electrons as a light source, collecting information about surface topography based on changes in energy signals. The most commonly utilized signal in SEM comes from secondary electrons, which can reveal minute structures with a resolution as fine as 0.5 nm. The high level of detail achieved is due to electrons having wavelengths that can be up to 100,000 times shorter than those of photons in visible light.¹⁴ It is important to note that although SEM images lack vertical resolution, they have a significant depth of field, giving them a three-dimensional appearance. SEM has been effectively used to examine biofilm structures, as its extensive depth of focus allows for excellent visualization of the spatial organization within the biofilm.

Confocal laser scanning microscopy (CLSM) 

An advanced imaging technique called confocal laser scanning microscopy (CLSM) allows researchers to investigate biofilms that form on transparent surfaces of flow cells. It facilitates three-dimensional (3D) analysis of the morphology and physiology of biofilms. CLSM is particularly valuable for visualizing dense samples, such as biofilms, and for observing microorganisms that are embedded deep within these structures. This method is crucial for investigating biofilm architecture and offers significant potential for real-time imaging of fully hydrated, living specimens.¹⁵ A fluorescent method improves the spatial resolution of optical microscopy, enabling nanoscale imaging capabilities. This “super-resolution optical microscopy” provides both qualitative and quantitative data about biofilms. CLSM uniquely allows for the measurement of biofilm growth rates and cell behaviors, including attachment, detachment, and re-attachment, as biofilms develop and form high-diffusion areas in situ.¹⁶

Atomic force microscopy (AFM)

Atomic Force Microscopy has unique characteristics that make it an effective tool for obtaining authentic 3-D surface topography. This method presents specific challenges and techniques associated with imaging biofilm formation. Researchers have successfully gathered high-resolution qualitative and quantitative data on the structure of biofilms.¹⁷ Unlike Scanning Electron Microscopy (SEM), AFM enables the evaluation of biomass height and surface corrosion. In recent years, AFM has rapidly gained popularity for studying biofilms on various substrate surfaces and bacterial species, as evidenced by numerous examples in review articles. Its high sensitivity and resolution in topography analysis have been extensively used to examine individual cells and biofilms on surfaces that come into contact with food.¹⁸

AFM-based infrared spectroscopy (AFM-IR)

Molecular species measurement in laboratory and industrial settings often utilizes infrared spectroscopy. This technique enables the analysis of samples in their natural states, such as solids and liquids, allowing for the collection of data on organic compounds within biofilms. When combined with atomic force microscopy (AFM), the AFM-infrared (AFM-IR) method can investigate the molecular composition of individual bacterial cells, providing valuable insights into their behavior during biofilm formation.¹⁹

In AFM-IR, the thermal expansion of the sample at the atomic scale is measured using an AFM tip that interacts with radiation from pulsed infrared light. The sample is mounted on a zinc selenide (ZnSe) prism, which is transparent to infrared light, and is illuminated by a specially designed internal reflection laser. The absorption of light at specific wavelengths causes the sample to expand thermally, which leads to oscillations in the cantilever. By analyzing the cantilever’s response and applying a fast Fourier transform (FFT) to the original oscillation signal in the time domain, researchers can obtain the infrared spectrum.²⁰

Fluorescent in situ hybridization (FISH)

Fluorescence In Situ Hybridization (FISH) is a laboratory technique that integrates molecular biology, fluorescence microscopy, and histology to examine cellular structures at a microscopic level. It allows for the visualization of microorganisms within tissue samples, enabling the identification and quantification of their abundance, position, and metabolic state. This method uses fluorescently tagged probes that specifically bind to microbial ribosomal RNA (rRNA), which facilitates the detection of bacteria and fungi at the genus or species level. For instance, probes can be designed to target only Staphylococcus aureus or to identify all types of microorganisms. FISH can also be applied to study fluorescently labeled bacteria in biofilms.²¹ The probes are coupled with fluorescent dyes such as FITC or Rhodamine, or with enzymes like horseradish peroxidase, to hybridize with the 16S rRNA of microorganisms. One advantage of using horseradish peroxidase-conjugated probes is that they do not kill biofilm microorganisms. Since the ribosome count is correlated with growth activity, FISH can estimate bacterial growth rates within biofilms. Probes need to be developed to label conserved regions of specific species.²²

FISH assesses the ribosome content of individual cells to determine microbial activity. Successful antimicrobial treatment of biofilms results in reduced fluorescence signals compared to active biofilms. The effectiveness of antimicrobial agents is measured by the decrease in the FISH-positive fraction relative to the total biofilm mass, which can be quantified through digital image analysis. Therefore, FISH is an ideal method for examining biofilms, evaluating antimicrobial compounds against them, and developing new anti-biofilm materials.²³

AFM-based Raman spectroscopy

Raman spectroscopy is an additional form of label-free spectroscopic analysis. In fact, Raman and IR spectroscopy complement each other in chemical analysis, both utilizing different approaches to examine molecular vibrations. This allows Raman spectroscopy to overcome certain difficulties associated with IR spectrum identification. Raman spectroscopy is a powerful tool for examining biological systems, showcasing its versatility in analyzing various states of matter, including solids, liquids, gels, and various mixtures in different environments, such as aqueous settings. This analytical method can be used without causing harm or making changes to the specimen, making it especially valuable for non-invasive investigations.²⁴

 The technique employs the measurement of Raman scattering to determine chemical composition. A major challenge in Raman spectroscopy is the weak Raman signal, which is often obscured by strong background noise. The integration of Raman spectroscopy with atomic force microscopy (AFM) provides more than just an extra chemical analysis tool to complement high-resolution AFM images. By coating AFM tips with conductive metals, such as gold or silver nanoparticles, it is possible to enhance Raman signals by up to 108 times in specific regions between the tip and the sample surface. This combination merges AFM’s nanoscale topographic resolution with Raman spectroscopy’s chemical fingerprint identification capabilities. The integration of Raman spectroscopy and AFM is facilitated by the stable and robust nature of Raman spectral detection equipment, which consists of a limited number of standard and modular components. Consequently, TERS instruments are easily commercialized.²⁵

ATP bioluminescence 

The enzyme luciferase plays a crucial role in ATP bioluminescence by producing measurable light in the presence of ATP, which can be detected using a luminometer. This method is advantageous because it provides quick results, typically within 20 seconds. However, it faces challenges when applied to biofilms, especially mature ones. The main issue is the limited movement of ATP within these biofilms, which results in low ATP readings. As a consequence, when a mature biofilm contains a high number of microorganisms, the low ATP levels may lead to an underestimation of the actual microbial population.²⁶

Mass spectrometry

Mass spectrometry (MS) is a powerful technique used to identify and analyze extracellular polymeric substances (EPS) and other large biomolecules found in complex biological structures like biofilms. This method provides a detailed examination of the chemical components involved in the development of biofilms. The two main types of mass spectrometry are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). A time-of-flight (TOF) mass spectrometer analyzes mass by examining desorbed ions in a vacuum chamber.²⁷ The combination of these techniques is known as MALDI-TOF. In this process, a laser is used to vaporize and ionize the sample. The resulting ions then travel through an electric field along the MALDI-TOF device’s column towards the TOF detector. The TOF measurements are based on the mass-to-charge ratio of the molecules, with ions moving faster when this ratio is lower. MALDI techniques have various applications, including bacterial identification, monitoring bacterial protein expression in response to antimicrobials (such as surface proteins and exoenzymes like β-lactamase), and measuring bacterial growth.²⁸

Quantitative real‑time PCR analysis 

Bacteria growing in biofilms often exhibit different gene expression patterns compared to their planktonic counterparts. Quantitative reverse transcriptase real-time PCR (qRT-PCR) is a reliable method for measuring RNA transcript levels of specific genes in biofilm-associated bacteria. The technique’s wide dynamic range makes it particularly valuable for validating gene expression data from microarray studies.²⁹ Additionally, qRT-PCR’s sensitivity enables gene expression quantification in biofilm samples with limited biological material, such as those obtained through laser capture microdissection microscopy (LCMM). The most commonly used qRT-PCR techniques are the SYBR Green and dual-labeled probe (Taqman) methods. Both approaches involve converting mRNA to complementary DNA (cDNA) through reverse transcription, followed by PCR amplification of the resulting cDNA. This paper outlines the crucial steps for performing qRT-PCR, including: (1) designing primers, (2) evaluating primer and probe efficacy, (3) executing qRT-PCR using the Corbett Rotor-Gene platform, and (4) extracting and analyzing data.³⁰

Bio Finder

Traditional microbiological culture techniques for identifying biofilms on food-contact surfaces can be quite time-consuming. However, the food industry requires faster methods to confirm microbial contamination, assess environmental hygiene, and implement immediate corrective measures. BioFinder provides a solution by rapidly detecting biofilms within seconds or minutes of application. This substance interacts with catalase, an enzyme present in nearly all living cells and biofilms. When biofilms are detected, a distinct color change occurs, accompanied by the formation of small bubbles. Additionally, BioFinder can be used to verify the effectiveness of sanitation practices in production facilities and to examine critical inspection areas right before disinfection.³¹

Table 1: Different advanced and Imaginary techniques and their applications:

Conclusion

Research into biofilms has uncovered their intricate nature and widespread influence across various settings, including healthcare and food processing industries. Biofilm development involves a complex, multi-stage process that results in resilient microbial communities, which are notoriously difficult to eliminate. Our comprehension of biofilm structure, composition, and behavior has been greatly enhanced by cutting-edge imaging and analytical methods. These methods, which include microscopic examination, spectroscopic analysis, and molecular biology tools, provide crucial information about biofilm properties and potential avenues for creating more efficient control strategies. As research in this area advances, the integration of various analytical approaches is anticipated to provide a more thorough understanding and creative solutions for handling biofilms across different settings. The field of biomembrane research has made significant strides through the application of innovative analytical techniques that improve our understanding of membrane composition and structure. Atomic Force Microscopy (AFM) delivers high-resolution imaging of biomembranes, enabling the measurement of their physical properties and dynamics at the nanoscale.³² Cryogenic Electron Microscopy (Cryo-EM) has similarly facilitated the determination of membrane protein structures, further expanding our knowledge of biomembrane architecture.³³ Single-Molecule Localization Microscopy (SMLM) provides unparalleled resolution for visualizing membrane proteins and lipids, offering insights into their interactions and dynamics.³⁴ Moreover, Neutron Scattering serves as a powerful tool for examining membrane organization and protein-lipid interactions, revealing crucial information about membrane behavior at the molecular level.³⁵ Grasping the functions of membrane proteins, which are essential to various cellular processes, is crucial for comprehending the overall role of biomembranes.³⁶ Collectively, these techniques establish a comprehensive framework for exploring biomembrane research.

Acknowledgement

We wish to express our sincere thanks and gratitude to Dr. D. Y. Patil ACS College, Pimpri, Pune, for providing the necessary facilities to carry out this research. It is appropriate to express gratitude to the Dr. D. Y. Patil Biotechnology & Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra, India for providing crucial administrative assistance. The authors are thankful to Ms. Deepali Akolkar for her assistance in statistical analysis.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other sources

 Not Applicable

Author Contributions

Sanchita Choubey: The major contributor to manuscript writing, reviewed the writing

Jyoti Deshpande approved the final manuscript.

Reference 

1. Liu X., Yao H., Zhao X., & Ge C. Biofilm formation and control of foodborne pathogenic bacteria. Molecules. 2023; 28(6): 2432. https://doi.org/10.3390/molecules28062432.
   CrossRef
2. Li W., Liu M., Siddique M.S., Graham N., & Yu W. Contribution of bacterial extracellular polymeric substances (EPS) in surface water purification. Environmental Pollution. 2021; 280: 116998. https://doi.org/10.1016/j.envpol.2021.116998.
   CrossRef
3. Zhao A., Sun J., & Liu Y. Understanding bacterial biofilms: From definition to treatment strategies. Frontiers in Cellular and Infection Microbiology. 2023; 13: 1137947. https://doi.org/10.3389/fcimb.2023.1137947.
   CrossRef
4. Dinh D., Awad T.S., Baker P., Howell P.L., & Hatton B.D. Non-eluting, surface-bound enzymes disrupt surface attachment of bacteria by continuous biofilm polysaccharide degradation. Biomaterials. 2018; 167: 168–176. https://doi.org/10.1016/j.biomaterials.2018.03.016.
   CrossRef
5. Mgomi F.C., Yang Y.R., Cheng G., & Yang Z.Q. Lactic acid bacteria biofilms and their antimicrobial potential against pathogenic microorganisms. Biofilm. 2023; 5: 100118. https://doi.org/10.1016/j.bioflm.2023.100118.
   CrossRef
   
6. Kahraman H. Biofilm formation in bacteria. International Journal of Biochemistry & Physiology. 2022; 7(1). https://doi.org/10.23880/ijbp-16000200.
   CrossRef
7. Stages of biofilm formation | QualiTru. (n.d.). QualiTru Sampling Systems. https://qualitru.com/five-main-phases-of-biofilm-development/.
8. Ren S., Lu X., Hu Z., & Liu J. Light and scanning electron microscopy observations on morphological diversity of Rosaceae stigma and its taxonomic significance. Microscopy Research and Technique. 2022; 85(8): 2866–2879. https://doi.org/10.1002/jemt.24135.
   CrossRef
9. Almatroudi A. Investigating biofilms: Advanced methods for comprehending microbial behavior and antibiotic resistance. Frontiers in Bioscience (Landmark Edition). 2024; 29(4): 133. https://doi.org/10.31083/j.fbl2904133.
   CrossRef
10. McCutcheon J. & Southam G. Advanced biofilm staining techniques for TEM and SEM in geomicrobiology: Implications for visualizing EPS architecture, mineral nucleation, and microfossil generation. Chemical Geology. 2018; 498: 115–127. https://doi.org/10.1016/j.chemgeo.2018.09.016.
    CrossRef
11. Choi M.-L. Transmission electron microscope (TEM) v1. Zappylab. 2022. https://doi.org/10.17504/protocols.io.ewov1n3zpgr2/v1.
    CrossRef
12. Seifert P. Modified Hiraoka TEM grid staining apparatus and technique using 3D printed materials and gadolinium triacetate tetrahydrate, a nonradioactive uranyl acetate substitute. Journal of Histotechnology. 2017; 40(4): 130–135. https://doi.org/10.1080/01478885.2017.1361117.
    CrossRef
13. Huang A. Sample preconditioning before scanning electron microscopy (SEM) v1. Zappylab. 2022. https://doi.org/10.17504/protocols.io.36wgqj465vk5/v1.
    CrossRef
14. Relucenti M., Familiari G., Donfrancesco O., Taurino M., Li X., Chen R., Artini M., Papa R., & Selan L. Microscopy methods for biofilm imaging: Focus on SEM and VP-SEM pros and cons. Biology. 2021; 10(1): 51. https://doi.org/10.3390/biology10010051.
    CrossRef
15. Agarwal H., Jain N., Pippal B., & Gurnani B. Capturing the micro-communities: Insights into biogenesis and architecture of bacterial biofilms. BBA Advances. 2025; 7: 100133. https://doi.org/10.1016/j.bbadva.2024.100133.
    CrossRef
16. Main K.H.S., Provan J.I., Haynes P.J., Wells G., Hartley J.A., & Pyne A.L.B. Atomic force microscopy—a tool for structural and translational DNA research. APL Bioengineering. 2021; 5(3): 031504. https://doi.org/10.1063/5.0054294.
    CrossRef
17. Cattò C. & Cappitelli F. Testing anti-biofilm polymeric surfaces: Where to start? International Journal of Molecular Sciences. 2019; 20(15): 3794. https://doi.org/10.3390/ijms20153794.
    CrossRef
18. Mathurin J., Wagner M., Dazzi A., Dartois E., Bazin D., & Deniset-Besseau A. Photothermal AFM-IR spectroscopy and imaging: Status, challenges, and trends. Journal of Applied Physics. 2022; 131(1): 010901. https://doi.org/10.1063/5.0063902.
    CrossRef
19. Duan H., Strohmeier B.R., & Fang Y. Insights into thermal degradation of hot melt pressure-sensitive adhesive (PSA) with atomic force microscopy – infrared spectroscopy (AFM-IR) analysis. Microscopy and Microanalysis. 2022; 28(S1): 2490–2491. https://doi.org/10.1017/s1431927622009527.
    CrossRef
20. Smajkan A. Applications of fluorescent in situ hybridization (FISH). Bioengineering Studies. 2021; 2(2): 12–20. https://doi.org/10.37868/bes.v2i2.id192.
    CrossRef
21. Ramos J.M. Fluorescent in situ hybridization (FISH). Methods in Molecular Biology (Clifton, N.J.). 2021; 2422: 179–189. https://doi.org/10.1007/978-1-0716-1948-3_12.
    CrossRef
22. Lopez C. RNA fluorescent in situ hybridization (FISH) v1. Zappylab. 2024. https://doi.org/10.17504/ protocols.io.14egn63dpl5d/v1.
    CrossRef
23. Chandra A., Kumar V., Garnaik U.C., Dada R., Qamar I., Goel V.K., & Agarwal S. Unveiling the molecular secrets: A comprehensive review of Raman spectroscopy in biological research. ACS Omega. 2024; 9(51): 50049–50063. https://doi.org/10.1021/acsomega.4c00591.
    CrossRef
24. Foti A., Venkatesan S., Lebental B., Zucchi G., & Ossikovski R. Comparing commercial metal-coated AFM tips and home-made bulk gold tips for tip-enhanced Raman spectroscopy of polymer functionalized multiwalled carbon nanotubes. Nanomaterials. 2022; 12(3): https://doi.org/10.3390/nano12030451.
    CrossRef
25. Baeza N. & Mercade E. Relationship between membrane vesicles, extracellular ATP and biofilm formation in Antarctic gram-negative bacteria. Microbial Ecology. 2020; 81(3): 645–656. https://doi.org/10.1007/s00248-020-01614-6.
    CrossRef
26. Van Hoogstraten S.W.G., Kuik C., Arts J.J.C., & Cillero-Pastor B. Molecular imaging of bacterial biofilms—a systematic review. Critical Reviews in Microbiology. 2024; 50(6): 971–992. https://doi.org/10.1080/1040841X.2023.2223704.
    CrossRef
27. Quantitative Real-Time PCR. Springer New York. 2020. https://doi.org/10.1007/978-1-4939-9833-3.
    CrossRef
28. Tkatch T. Quantitative real-time PCR v1. Zappylab. 2023. https://doi.org/10.17504/protocols.io. e6nvwd4o2lmk/v1.
    CrossRef
29. Detección de Biofilms – Biofinder – Biofilm Remove. Biofilm Detection, Biofilm Remove. 2021. https://biofilmremove.com/en/detection/.
30. Gerle C. Essay on biomembrane structure. The Journal of Membrane Biology. 2019; 252(2-3): 115–130. https://doi.org/10.1007/s00232-019-00061-w.
    CrossRef
31. Nygaard R., Kim J., & Mancia F. Cryo-electron microscopy analysis of small membrane proteins. Current Opinion in Structural Biology. 2020; 64: 26–33. https://doi.org/10.1016/j.sbi.2020.05.009.
    CrossRef
32. Zhang O., Lew M.D. Single-molecule orientation-localization microscopy: Applications and approaches. Quarterly Reviews of Biophysics. 2024; 57:e17. https://doi.org/10.1017/ S0033583524000167.
    CrossRef
33. Heller W.T. Small-angle neutron scattering for studying lipid bilayer membranes. Biomolecules. 2022; 12(11):1591. https://doi.org/10.3390/biom12111591.
    CrossRef
34. Jelokhani-Niaraki M. Membrane proteins: Structure, function and motion. International Journal of Molecular Sciences. 2022; 24(1): 468. https://doi.org/10.3390/ijms24010468.
    CrossRef