Pub Date : 2020-06-01DOI: 10.35459/tbp.2019.000117
J. W. Rupel, Sophia M. Sdao, Kadina Johnston, Ethan T. Nethery, K. Gabardi, Benjamin A. Ratliff, Z. Simmons, Jack T. Postlewaite, A. Kita, J. Rogers, M. Merrins
Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.
{"title":"Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom","authors":"J. W. Rupel, Sophia M. Sdao, Kadina Johnston, Ethan T. Nethery, K. Gabardi, Benjamin A. Ratliff, Z. Simmons, Jack T. Postlewaite, A. Kita, J. Rogers, M. Merrins","doi":"10.35459/tbp.2019.000117","DOIUrl":"https://doi.org/10.35459/tbp.2019.000117","url":null,"abstract":"\u0000 Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47229479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.35459/tbp.2019.000111
Leonel Malacrida, P. N. Hedde, Belén Torrado, E. Gratton
Transient barriers are fundamental to cell supramolecular organization and assembly. Discontinuities between spaces can be generated by a physical barrier but also by thermodynamic barriers achieved by phase separation of molecules. However, because of the transient nature and the lack of a visible barrier, the existence of phase separation is difficult to demonstrate experimentally. We describe an approach based on the 2-dimensional pair correlation function (2D-pCF) analysis of the spatial connectivity in a cell. The educational aim of the article is to present both a model suitable for explaining diffusion barrier measurements to a broad range of courses and examples of biological situations. If there are no barriers to diffusion, particles could diffuse equally in all directions. In this situation the pair correlation function introduced in this article is independent of the direction and is uniform in all directions. However, in the presence of obstacles, the shape of the 2D-pCF is distorted to reflect how the obstacle position and orientation change the flow of molecules. In the example shown in this article, measurements of diffusion of enhanced green fluorescent protein moving in live cells show the lack of connectivity at the nucleolus surface for shorter distances. We also observe a gradual increase in the connectivity for longer distances or times, presumably because of molecular trajectories around the nucleolus.
{"title":"Barriers to Diffusion in Cells: Visualization of Membraneless Particles in the Nucleus.","authors":"Leonel Malacrida, P. N. Hedde, Belén Torrado, E. Gratton","doi":"10.35459/tbp.2019.000111","DOIUrl":"https://doi.org/10.35459/tbp.2019.000111","url":null,"abstract":"Transient barriers are fundamental to cell supramolecular organization and assembly. Discontinuities between spaces can be generated by a physical barrier but also by thermodynamic barriers achieved by phase separation of molecules. However, because of the transient nature and the lack of a visible barrier, the existence of phase separation is difficult to demonstrate experimentally. We describe an approach based on the 2-dimensional pair correlation function (2D-pCF) analysis of the spatial connectivity in a cell. The educational aim of the article is to present both a model suitable for explaining diffusion barrier measurements to a broad range of courses and examples of biological situations. If there are no barriers to diffusion, particles could diffuse equally in all directions. In this situation the pair correlation function introduced in this article is independent of the direction and is uniform in all directions. However, in the presence of obstacles, the shape of the 2D-pCF is distorted to reflect how the obstacle position and orientation change the flow of molecules. In the example shown in this article, measurements of diffusion of enhanced green fluorescent protein moving in live cells show the lack of connectivity at the nucleolus surface for shorter distances. We also observe a gradual increase in the connectivity for longer distances or times, presumably because of molecular trajectories around the nucleolus.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84804800","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.35459/tbp.2019.000106
W. Thompson, A. Cattani, O. Lee, Xiang Ma, I. Tsvetkova, B. Dragnea
Self-organization is ubiquitous in biology, with viruses providing an excellent illustration of bioassemblies being much more than the sum of their parts. Following nature's lead, molecular self-assembly has emerged as a new synthetic strategy in the past 3 decades or so. Self-assembly approaches promise to generate complex supramolecular architectures having molecular weights of 0.5 to 100 MDa and collective properties determined by the interplay between structural organization and composition. However, biophysical methods specific to mesoscopic self-assembly, and presentations of the challenges they aim to overcome, remain underrepresented in the educational laboratory curriculum. We present here a simple but effective model for laboratory instruction that introduces students to the world of intermolecular forces and virus assembly, and to a cutting-edge technology, atomic force microscopy nanoindentation, which is able to measure the mechanical properties of single virus shells in vitro. In addition, the model illustrates the important idea that, at nanoscale, phenomena often have an inherent interdisciplinary character.
{"title":"A Laboratory Model for Virus Particle Nanoindentation","authors":"W. Thompson, A. Cattani, O. Lee, Xiang Ma, I. Tsvetkova, B. Dragnea","doi":"10.35459/tbp.2019.000106","DOIUrl":"https://doi.org/10.35459/tbp.2019.000106","url":null,"abstract":"\u0000 Self-organization is ubiquitous in biology, with viruses providing an excellent illustration of bioassemblies being much more than the sum of their parts. Following nature's lead, molecular self-assembly has emerged as a new synthetic strategy in the past 3 decades or so. Self-assembly approaches promise to generate complex supramolecular architectures having molecular weights of 0.5 to 100 MDa and collective properties determined by the interplay between structural organization and composition. However, biophysical methods specific to mesoscopic self-assembly, and presentations of the challenges they aim to overcome, remain underrepresented in the educational laboratory curriculum. We present here a simple but effective model for laboratory instruction that introduces students to the world of intermolecular forces and virus assembly, and to a cutting-edge technology, atomic force microscopy nanoindentation, which is able to measure the mechanical properties of single virus shells in vitro. In addition, the model illustrates the important idea that, at nanoscale, phenomena often have an inherent interdisciplinary character.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46044545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.35459/tbp.2020.000150
D. Caballero, S. Kundu, R. Reis
The concepts and frameworks of soft matter physics and the laws of thermodynamics can be used to describe relevant developmental, physiologic, and pathologic events in which directed cell migration is involved, such as in cancer. Typically, this directionality has been associated with the presence of soluble long-range gradients of a chemoattractant, synergizing with many other guidance cues to direct the motion of cells. In particular, physical inputs have been shown to strongly influence cell locomotion. However, this type of cue has been less explored despite the importance in biology. In this paper, we describe recent in vitro works at the interface between physics and biology, showing how the motion of cells can be directed by using gradient-free environments with repeated local asymmetries. This rectification of cell migration, from random to directed, is a process reminiscent of the Feynman ratchet; therefore, this framework can be used to explain the mechanism behind directed cell motion.
{"title":"The Biophysics of Cell Migration: Biasing Cell Motion with Feynman Ratchets","authors":"D. Caballero, S. Kundu, R. Reis","doi":"10.35459/tbp.2020.000150","DOIUrl":"https://doi.org/10.35459/tbp.2020.000150","url":null,"abstract":"\u0000 The concepts and frameworks of soft matter physics and the laws of thermodynamics can be used to describe relevant developmental, physiologic, and pathologic events in which directed cell migration is involved, such as in cancer. Typically, this directionality has been associated with the presence of soluble long-range gradients of a chemoattractant, synergizing with many other guidance cues to direct the motion of cells. In particular, physical inputs have been shown to strongly influence cell locomotion. However, this type of cue has been less explored despite the importance in biology. In this paper, we describe recent in vitro works at the interface between physics and biology, showing how the motion of cells can be directed by using gradient-free environments with repeated local asymmetries. This rectification of cell migration, from random to directed, is a process reminiscent of the Feynman ratchet; therefore, this framework can be used to explain the mechanism behind directed cell motion.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42920462","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-02-07DOI: 10.20944/preprints202002.0097.v1
Kathy H. Le, Jared Adolf-Bryfogle, Jason Christopher Klima, Sergey Lyskov, Jason W. Labonte, S. Bertolani, S. Burman, Andrew Leaver-Fay, Brian D. Weitzner, Jack B. Maguire, R. Rangan, Matt A. Adrianowycz, Rebecca F. Alford, Aleexan Adal, Morgan L. Nance, Rhiju Das, Roland L. Dunbrack, W. Schief, B. Kuhlman, J. Siegel, Jeffrey J. Gray
Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of sixteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.
{"title":"PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design.","authors":"Kathy H. Le, Jared Adolf-Bryfogle, Jason Christopher Klima, Sergey Lyskov, Jason W. Labonte, S. Bertolani, S. Burman, Andrew Leaver-Fay, Brian D. Weitzner, Jack B. Maguire, R. Rangan, Matt A. Adrianowycz, Rebecca F. Alford, Aleexan Adal, Morgan L. Nance, Rhiju Das, Roland L. Dunbrack, W. Schief, B. Kuhlman, J. Siegel, Jeffrey J. Gray","doi":"10.20944/preprints202002.0097.v1","DOIUrl":"https://doi.org/10.20944/preprints202002.0097.v1","url":null,"abstract":"Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of sixteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":"5 1","pages":"108-122"},"PeriodicalIF":0.0,"publicationDate":"2020-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76112021","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-01-01DOI: 10.35459/tbp.2019.000102
Carlos R. Baiz
Fourier transforms (FT) are universal in chemistry, physics, and biology. Despite FTs being a core component of multiple experimental techniques, undergraduate courses typically approach FTs from a mathematical perspective, leaving students with a lack of intuition on how an FT works. Here, I introduce interactive teaching tools for upper-level undergraduate courses and describe a practical lesson plan for FTs. The materials include a computer program to capture video from a webcam and display the original images side-by-side with the corresponding plot in the Fourier domain. Several patterns are included to be printed on paper and held up to the webcam as input. During the lesson, students are asked to predict the features observed in the FT and then place the patterns in front of the webcam to test their predictions. This interactive approach enables students with limited mathematical skills to achieve a certain level of intuition for how FTs translate patterns from real space into the corresponding Fourier space.
{"title":"Interactive Tools for Teaching Fourier Transforms","authors":"Carlos R. Baiz","doi":"10.35459/tbp.2019.000102","DOIUrl":"https://doi.org/10.35459/tbp.2019.000102","url":null,"abstract":"\u0000 Fourier transforms (FT) are universal in chemistry, physics, and biology. Despite FTs being a core component of multiple experimental techniques, undergraduate courses typically approach FTs from a mathematical perspective, leaving students with a lack of intuition on how an FT works. Here, I introduce interactive teaching tools for upper-level undergraduate courses and describe a practical lesson plan for FTs. The materials include a computer program to capture video from a webcam and display the original images side-by-side with the corresponding plot in the Fourier domain. Several patterns are included to be printed on paper and held up to the webcam as input. During the lesson, students are asked to predict the features observed in the FT and then place the patterns in front of the webcam to test their predictions. This interactive approach enables students with limited mathematical skills to achieve a certain level of intuition for how FTs translate patterns from real space into the corresponding Fourier space.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49659598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-01-01DOI: 10.35459/tbp.2019.000123
Tamar Schlick
Encouraging our high school and college students to gain experience and specialize in science, technology, engineering, and math (STEM) fields has never been more important. These fields are central to our countries’ health, wealth, and security. They also provide numerous opportunities for employment and stability for our young people. In contrast to doctors and lawyers, whose functions are familiar, being a scientist, mathematician, or engineer may be elusive to youngsters not exposed to these specialties at home. Counselors and parents encourage students to acquire technical knowledge in these fields through course and research experiences; thus we often see today hard-working young students with perfect high school and college grade point averages, two or three college majors, advanced placement certifications, and a bundle of research accomplishments and prizes. Some students today are better prepared because technology has awarded them new sets of tools to explore and incorporate in education and training. It has also offered them numerous ideas and incentives for creation and innovation. The technical experiences they are acquiring are prerequisites for STEM career paths. However, there are softer and more general professional aspects and skills that are as important for becoming successful leaders in these fields. Here are my suggestions.
{"title":"Eight Suggestions for Future Leaders of Science and Technology.","authors":"Tamar Schlick","doi":"10.35459/tbp.2019.000123","DOIUrl":"https://doi.org/10.35459/tbp.2019.000123","url":null,"abstract":"Encouraging our high school and college students to gain experience and specialize in science, technology, engineering, and math (STEM) fields has never been more important. These fields are central to our countries’ health, wealth, and security. They also provide numerous opportunities for employment and stability for our young people. In contrast to doctors and lawyers, whose functions are familiar, being a scientist, mathematician, or engineer may be elusive to youngsters not exposed to these specialties at home. Counselors and parents encourage students to acquire technical knowledge in these fields through course and research experiences; thus we often see today hard-working young students with perfect high school and college grade point averages, two or three college majors, advanced placement certifications, and a bundle of research accomplishments and prizes. Some students today are better prepared because technology has awarded them new sets of tools to explore and incorporate in education and training. It has also offered them numerous ideas and incentives for creation and innovation. The technical experiences they are acquiring are prerequisites for STEM career paths. However, there are softer and more general professional aspects and skills that are as important for becoming successful leaders in these fields. Here are my suggestions.","PeriodicalId":72403,"journal":{"name":"Biophysicist (Rockville, Md.)","volume":"1 1","pages":"1-5"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168634/pdf/nihms-1568096.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39054109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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