Understanding the relationship between molecular structure and function represents an important goal of undergraduate life sciences. Although evidence suggests that handling physical models supports gains in student understanding of structure–function relationships, such models have not been widely implemented in biochemistry classrooms. Three‐dimensional (3D) printing represents an emerging cost‐effective means of producing molecular models to help students investigate structure–function concepts. We developed three interactive learning modules with dynamic 3D printed models to help biochemistry students visualize biomolecular structures and address particular misconceptions. These modules targeted specific learning objectives related to DNA and RNA structure, transcription factor‐DNA interactions, and DNA supercoiling dynamics. We also designed accompanying assessments to gauge student learning. Students responded favorably to the modules and showed normalized learning gains of 49% with respect to their ability to understand and relate molecular structures to biochemical functions. By incorporating accurate 3D printed structures, these modules represent a novel advance in instructional design for biomolecular visualization. We provide instructors with the materials necessary to incorporate each module in the classroom, including instructions for acquiring and distributing the models, activities, and assessments. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):303–317, 2019.
Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom
ABSTRACT 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.
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- Award ID(s):
- 1845801
- NSF-PAR ID:
- 10217198
- Date Published:
- Journal Name:
- The Biophysicist
- Volume:
- 1
- Issue:
- 2
- ISSN:
- 2578-6970
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
Light microscopy provides a window into another world that is not visible to the unaided eye. Because of this and its importance in biological discoveries, the light microscope is an essential tool for scientific studies. It can also be used with a variety of easily obtained specimens to provide dramatic demonstrations of previously unknown features of common plants and animals. Thus, one way to interest young people in science is to start with an introduction to light microscopy. This is an especially effective strategy for individuals who attend less advantaged or under-resourced schools, as they may not have been previously exposed to scientific concepts in their classes. However, introducing light microscopy lessons in the classroom can be challenging because of the high cost of light microscopes, even those that are relatively basic, in addition to their usual large size. Efforts are underway by our laboratory in collaboration with the Biophysical Society (BPS) to introduce young people to light microscopy using small, easy-to-assemble wooden microscopes developed by Echo Laboratories. The microscopes are available online as low-cost kits ($10 each with shipping), each consisting of 19 parts printed onto an 81⁄2 x 11 inch sheet of light-weight wood (Fig. 1). After punching out the pieces, they can be assembled into a microscope with a moveable stage and a low-power lens, also provided in the kit (Fig. 2). Photos taken with a cell phone through the microscope lens can give magnifications of ~16-18x, or higher. At these magnifications, features of specimens that are not visible to the unaided eye can be easily observed, e.g., small hairs on the margins of leaves or lichens [1]. As a member of the BPS Education Committee, one of us (SAE) wrote a Lesson Plan on Light Microscopy specifically for use with the wooden microscopes. SAE was also able to obtain a gift of 500 wooden microscope kits for the BPS from Echo Laboratories and Chroma Technology Corp in 2016. The wooden microscope kits, together with the lesson plan, have provided the materials for our present outreach efforts. Rather than giving out the wooden microscope kits to individuals, the BPS asked the Education Committee to maximize the impact of the gift by distributing the microscopes with the Lesson Plan on Light Microscopy to teachers, e.g., through teachers’ workshops or outreach sessions. This strategy was devised to enable the Society to reach a larger number of young people than by giving the microscopes to individuals. The Education Committee first evaluated the microscopes as a tool to introduce students to scientific concepts by providing microscopes to a BPS member at the National University of Colombia who conducted a workshop on Sept 19-24, 2016 in Tumaco, Columbia. During the workshop, which involved 120 high school girls and 80 minority students, including Afro-Colombian and older students, the students built the wooden microscopes and examined specimens, and compared the microscopes to a conventional light microscope. Assembling the wooden microscopes was found to be a useful procedure that was similar to a scientific protocol, and encouraged young girls and older students to participate in science. This was especially promising in Colombia, where there are few women in science and little effort to increase women in STEM fields. Another area of outreach emerged recently when one of us, USP, an undergraduate student at Duke University, who was taught by SAE how to assemble the wooden microscopes and how to use the lesson plan, took three wooden microscopes on a visit to her family in Bangalore, India in summer 2018 [2]. There she organized and led three sessions in state run, under-resourced government schools, involving classes of ~25-40 students each. This was very successful – the students enjoyed learning about the microscopes and building them, and the science teachers were interested in expanding the sessions to other government schools. USP taught the teachers how to assemble and use the microscopes and gave the teachers the microscopes and lesson plan, which is also available to the public at the BPS web site. She also met with a founder of the organization, Whitefield Rising, which is working to improve teaching in government schools, and taught her and several volunteers how to assemble the microscopes and conduct the sessions. The Whitefield Rising members have been able to conduct nine further sessions in Bangalore over the past ~18 months (Fig. 3), using microscope kits provided to them by the BPS. USP has continued to work with members of the Whitefield Rising group during her summer and winter breaks on visits to Bangalore. Recently she has been working with another volunteer group that has expanded the outreach efforts to New Delhi. The light microscopy outreach that our laboratory is conducting in India in collaboration with the BPS is having a positive impact because we have been able to develop a partnership with volunteers in Bangalore and New Delhi. The overall goal is to enhance science education globally, especially in less advantaged schools, by providing a low-cost microscope that can be used to introduce students to scientific concepts.more » « less
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Summary Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent
in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescentin situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetricin situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level. -
null (Ed.)We utilize a cost-effective frequency-domain fluorescence lifetime imaging microscope to measure the phase lifetime of mTFP1 in mTFP1-mVenus fluorescence resonance energy transfer (FRET) constructs relevant to the VinTS molecular tension probe. Our data were collected at 15 modulation frequencies ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τ D = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τ D ) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τ ϕ D A , was directly related to the amplitude-weighted lifetime τ a v e D A inferred from the known FRET efficiency ( E FRET true ) of the constructs. A linear fit to a plot of ( ω · τ ϕ D A ) v s . ( ω · τ a v e D A ) yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R 2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent E FRET app based on the measured phase lifetime and E FRET true for frequencies such that (ω · τ D ) < 1.1. We had previously reported a similar relationship between E FRET app and E FRET true at 42 MHz. Our current results provide additional evidence in support of this observation, but further investigation is still required to fully characterize these results. A direct relationship between τ ϕ D A and τ a v e D A has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.more » « less
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null (Ed.)Abstract There are many applications throughout the military and commercial industries whose thermal profiles are dominated by intermittent and/or periodic pulsed thermal loads. Typical thermal solutions for transient applications focus on providing sufficient continuous cooling to address the peak thermal loads as if operating under steady-state conditions. Such a conservative approach guarantees satisfying the thermal challenge but can result in significant cooling overdesign, thus increasing the size, weight, and cost of the system. Confluent trends of increasing system complexity, component miniaturization, and increasing power density demands are further exacerbating the divergence of the optimal transient and steady-state solutions. Therefore, there needs to be a fundamental shift in the way thermal and packaging engineers approach design to focus on time domain heat transfer design and solutions. Due to the application-dependent nature of transient thermal solutions, it is essential to use a codesign approach such that the thermal and packaging engineers collaborate during the design phase with application and/or electronics engineers to ensure the solution meets the requirements. This paper will provide an overview of the types of transients to consider—from the transients that occur during switching at the chip surface all the way to the system-level transients which transfer heat to air. The paper will cover numerous ways of managing transient heat including phase change materials (PCMs), heat exchangers, advanced controls, and capacitance-based packaging. Moreover, synergies exist between approaches to include application of PCMs to increase thermal capacitance or active control mechanisms that are adapted and optimized for the time constants and needs of the specific application. It is the intent of this transient thermal management review to describe a wide range of areas in which transient thermal management for electronics is a factor of significance and to illustrate which specific implementations of transient thermal solutions are being explored for each area. The paper focuses on the needs and benefits of fundamentally shifting away from a steady-state thermal design mentality to one focused on transient thermal design through application-specific, codesigned approaches.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.35459/tbp.2019.000117
