Inexpensive, Open Source Epifluorescence Microscopes - Journal of

May 13, 2016 - Second, we describe how to build a similar microscope using supplies that are available at most hardware stores or online. We demonstra...
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Inexpensive, Open Source Epifluorescence Microscopes Chris Stewart and John Giannini* Biology Department, St. Olaf College, 1520 St. Olaf Avenue, Northfield, Minnesota 55057, United States S Supporting Information *

ABSTRACT: In an effort to help lower the costs of fluorescence microscopy and expand the use of this valuable technique in the classroom, teaching lab, and hopefully beyond, we provide two different open source designs for inexpensive epifluorescence microscopes. First, we explain how to 3D print the parts for a simple adapter that can be mounted onto a conventional compound light microscope with a removable head to convert it for epifluorescence and bright-field viewing. Second, we describe how to build a similar microscope using supplies that are available at most hardware stores or online. We demonstrate the capabilities of our designs using Tetrahymena thermophila cells that were stained with two common fluorophores (Rhodamine B or Acridine Orange) or tagged with a fluorescent protein. We further explain how these microscopes can be used to teach basic principles of photochemistry, biochemistry, and histochemistry, as well as cellular and molecular biology. In the spirit of making these designs open and accessible to all, we have named them “the OPN Scope”, and we include instructions on how to 3D print or build these microscopes (along with the underlying computer design files) as Supporting Information, so that others can access, use, or modify them as needed. Ultimately, we hope that these designs can provide new opportunities for expanding scientific education and research, especially in schools or regions that may lack the funding for more sophisticated scientific equipment. KEYWORDS: General Public, Laboratory Instruction, Hands-On Learning/Manipulatives, Laboratory Equipment/Apparatus

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INTRODUCTION A powerful analytical tool for biochemists and molecular biologists alike, the epifluorescence microscope enables investigators to visualize cells and their internal compartments, monitor the corresponding chemical environments in those locations, and follow the production and distribution of proteins and other molecules within a living cell.1,2 For example, by using various fluorophores, researchers can identify near instantaneous changes in intracellular biochemistry, such as alterations in pH, ATP, or GTP levels; the production and distribution of cAMP, Ca2+, or other second messengers; and interactions between or modifications of different proteins.3−5 Moreover, with the development of green and other fluorescent proteins, scientists can now “tag” different macromolecular structures to identify their location inside a living cell and further use these tags as miniature biosensors to monitor the internal chemical environment of a cell in vivo.6−8 As such, epifluorescence microscopes can provide valuable information about and insights into the underlying chemistry at work in biological organisms. An epifluorescence microscope works by transmitting a specific wavelength of light onto a specimen, exciting electrons in the sample.1,2 When those electrons fall back down to their natural energy state, they release light energy at a slightly longer wavelength (i.e., one at a lower energy and a different color), which is called fluorescence.6,9 For example, in a now common application involving Green Fluorescent Protein (GFP), an intense white light is first sent through an excitation filter, © XXXX American Chemical Society and Division of Chemical Education, Inc.

which screens out all but a narrow band in the blue spectrum (Figure 1, left). Then, a dichroic mirror (which allows only certain wavelengths to pass) reflects that blue light down onto

Figure 1. Viewing green fluorescent protein (GFP) with an epifluorescence microscope. Received: December 8, 2015 Revised: April 21, 2016

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the specimen, exciting electrons in the GFP sample.1,4 As these electrons fall back down to their natural state, they release light energy at a longer wavelength, which is seen as a green glow. That light then passes up through the dichroic mirror (which is designed to let this specific wavelength pass) and can be seen through the eyepiece (Figure 1, right). In the process, the objective and ocular lenses of the microscope help to focus the excitation light onto the specimen and the emission light for the viewer to see.2,6 While the basic concept behind epifluorescence microscopy is fairly straightforward, the cost of the necessary equipment (and the skill often needed to operate and maintain it) can place these instruments out of reach for many educators, researchers, and other professionals. Consequently, numerous teams have offered several innovative designs for these instruments,10−16 including some very practical and low-cost devices that can be used to educate students about the underlying photochemical principles giving rise to this powerful analytical technique.17−19 In this spirit, we set out to develop a simple and inexpensive epifluorescence microscope that would also allow for brightfield viewing. We further wanted our design to be compact and durable, so that it would store or carry easily and be able to withstand a certain degree of rough handling (a quality that is sometimes lacking in certain scientific equipment). With these goals in mind, we designed two relatively inexpensive epifluorescence microscopes that are also capable of brightfield viewing, and we describe these models below. First, we explain how to 3D print the parts for a simple adapter, which can be easily assembled and then mounted on a conventional compound light microscope that contains a removable headpiece (Figure 2A). However, because not

who may lack the funds for more expensive equipment. Thus, in the spirit of making these designs open and accessible to all, we have named them the 3D-printed and PVC versions of “the OPN Scope”, and we provide instructions on how to make these instruments (as well as the underlying computer files) as Supporting Information, so that others can use or modify them to fit their educational or research needs.

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MATERIALS AND METHODS The 3D-printed version of the OPN Scope consists of six parts: (i) an outer shell; (ii) a drawer that fits into this shell and holds a fluorescence filter cube or filter set; (iii) a handle for the drawer; (iv) a tube that holds the eyepiece from the underlying microscope or a digital camera; (v) a tube that holds the light source; and (vi) a mounting adapter that fits a conventional light microscope, whose head can be removed (Figure 3A). In

Figure 3. Schematics of (A) the OPN Scope and (B) the OPN Drawer. Components are numbered as listed in the text.

addition, because new filter cubes are rather expensive and the supply of used cubes is often limited (both in quantity and variety), we designed a special drawer (called “the OPN Drawer”) that fits into the OPN Scope and can hold the parts for many fluorescence filter sets currently on the market (whether new or used). As such, the OPN Drawer consists of five 3D-printed parts: (i) the drawer itself; (ii) a corresponding handle; (iii) a holder for the excitation filter; (iv) a holder for the dichroic mirror, and (v) a holder for the emission filter (Figure 3B). For detailed instructions on how to 3D print the parts for the OPN Scope and OPN Drawer, please see the Supporting Information (S1). We have also included as Supporting Information the underlying Computer Aided Design (CAD) files (S2) and related STereoLithographic (STL) files (S3) for these parts, so that readers can print or modify them as needed.

Figure 2. OPN Scope. (A) 3D-printed version mounted on an Olympus CH-2 microscope. (B) PVC version.

everyone has access to 3D printers or even compound microscopes, we also describe how to build an epifluorescence microscope using materials available at most hardware stores or online, such as PVC board, PVC pipe, and used microscope optics (Figure 2B). Our primary hope behind this project was (and continues to be) that these designs might help to expand the use of fluorescence microscopy in the classroom and teaching lab, making the science behind the techniqueand, more importantly, the scientific exploration that can be conducted with itmore accessible to students and teachers alike. We also thought that such a device might be especially useful to those B

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HAZARDS Before 3D printing the parts for the OPN Scope, working with any hand or power tools to make the corresponding PVC version, or using any chemicals to prepare T. thermophila cells for fluorescent viewing, readers should review the Supporting Information (S1, S4, and S5) for a discussion of the related hazards as many of them are significant.

In both instances, we used the free version of DesignSpark Mechanical (RS Components, Corby, Northhamptonshite, U.K.) to create these files (S1). We further include as Supporting Information detailed instructions on how to build an alternative version of the OPN Scope using parts available at most hardware stores or online (S4). We briefly describe that process here. Specifically, we built a PVC version of the OPN Scope (Figure 2B) using 3/ 4-in. thick PVC board since it is more rigid than most woods and should not warp or rot over time. We made an adjustable microscope stage using 3-in. diameter PVC couplings, and we used 3/4-in. Schedule 40 PVC pipe and related fittings to hold the ocular and objective lenses (as well as the LED flashlight) in place. To help keep costs low, in both designs, we typically employ used fluorescence filter cubes and/or optics, which can be purchased on eBay or other suitable marketplaces for used laboratory equipment, and we generally use a high-intensity (2000 lm) tactical LED flashlight with an adjustable convex lens to focus the beam as our excitation light source. In addition, in the PVC version of the OPN Scope, we use a push-button LED to provide bright-field illumination (Figure 2B). For additional details on the 3D-printed and PVC versions of the OPN Scope, please see the Supporting Information (S1 and S4). We tested our designs using Tetrahymena thermophila cells (a common protozoan), which were stained with two common fluorophores (Rhodamine B or Acridine Orange) or whose cellular cortex was tagged with yellow fluorescent protein (YFP). The latter cells were from the pICY_AA strain and kindly provided by Doug Chalker of Washington University in St. Louis, Missouri. We have further included our protocols for cell preparation (along with the related hazards) as Supporting Information (S5), so that readers can use or adapt them to create their own educational laboratory exercises (as explained in the Discussion section below). We also describe in Table 1 the fluorescence filter set or cube and LED flashlight that we used with each OPN Scope model to view and image the fluorescent cells shown in Figure 4.

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RESULTS We tested both version of the OPN Scope using a variety of different fluorescence filter cubes and LED flashlights (S1 and S4), and we consistently saw sharp and vivid images when looking at fluorescent specimens directly through the eyepiece regardless of the type of cube that we used (CS and JG personal observations; see also Figure 4 for select representative images). For example, similar to other reports that use Rhodamine derivatives to monitor heavy metal concentrations in HeLa cells,20,21 T. thermophila cells stained with Rhodamine B appeared a vibrant red (Figure 4B and 4D), and we were also able to see discrete subcellular structures in some instances depending on the quality of the underlying optics (Figure 4B). Similarly, consistent with other studies involving T. thermophila,22,23 the macro- and micronuclei of cells stained with Acridine Orange appeared bright green while the cytoplasm glowed a lighter green, and acidic vacuoles in these cells (i.e., lysosomes) appeared a pale orange (Figure 4F,H). Finally, T. thermophila cells from the pICY_AA strain, whose cortex was tagged with a yellow fluorescent protein, revealed a pattern of macromolecular structures along their cellular membranes that glowed brightly when excited (Figure 4J,L) and resembled the pattern of basal bodies and related fibers depicted in prior reports.24−27 Thus, both versions of the OPN Scope were able to generate high-quality images similar to the published findings of other investigators. Nevertheless, we did encounter some minor issues with the PVC model. For example, it is sensitive to vibration and movement at high magnification (400×), and readers also need to properly align the holes for the flashlight, lenses, and filter cube when building the PVC shell to fully illuminate the field of view. We describe ways to address these issues in the Supporting Information (S4).

Table 1. Fluorescence Filter Set or Cube and LED Flashlight Used To View T. thermophila Cells with the 3D-Printed and PVC Versions of the OPN Scope Fluorophore

3D Printed

Rhodamine B

Zeiss DAPI/FITC/Texas-Red filter set in an OPN Drawer; Outlite WT03

Acridine Orange YFP

Nikon B2A; Outlite WT03 Nikon B2A; Outlite WT03

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DISCUSSION Given the simplicity of these designs and their ease of use, we hope that both versions of the OPN Scope and the OPN Drawer will be helpful tools for educators and researchers alike. For example, the parts fit together intuitively, and once assembled, these designs can be kept intact indefinitely or disassembled for ease of storage. Moreover, if incorporated into an introductory chemistry, biochemistry, biology, or physics course, either version of the OPN Scope could help demonstrate many of the chemical principles and properties giving rise to fluorescence. Similarly, if used in upper level classes on biochemistry, histochemistry, photochemistry, or molecular biology, the OPN Scope could be used to teach more advanced concepts and techniques. For example, using Rhodamine B (or a different fluorophore), instructors could demonstrate key concepts of photochemistry, which are central to fluorescence microscopy, such as the excitation of electrons at a shorter, higher energy wavelength (green in the case of Rhodamine B) followed by the emission of photonic energy at a longer, lower energy

PVC Leitz/Leica N2.1; Coast G20 Leitz/Leica I3; Coast G20 Leitz/Leica I3; Outlite A100

As shown in the Abstract Graphic, we photographed all of the images at 400× total magnification using a Lenovo ThinkPad T430s and a 14-MegaPixel OMAX A3514OU digital camera along with its corresponding ToupView 3.7 software. However, other computers or digital cameras will suffice. We further adjusted the exposure time and gain for the OMAX camera in the ToupView program (as well as the brightness and contrast of the resulting digital photographs) to more faithfully represent the images as they naturally appeared when viewed through the eyepiece of the microscope. C

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Figure 4. Bright-field and fluorescent images of T. thermophila cells (400×) taken using the 3D-printed (left) and PVC (right) versions of the OPN Scope. (A−D) Cells stained with 50 μL of Rhodamine B (100 mg/L) added to a 450-μL culture sample. (E−H) Cells stained with 50 μL of Acridine Orange (500 mg/L) added to a 450-μL culture sample. (I−L) pICY_AA cells whose cortex was tagged with a yellow fluorescent protein. The scale bar in the lower right corner applies to all images in the figure.

wavelength (here, red).2,18,28 In addition, for more advanced laboratories, other Rhodamine derivatives could be used to explore various biochemical phenomena inside living cells, such as changes in pH levels,29,30 the effects of increased concentrations of heavy metal ions (e.g., Cu 2+, Hg2+, Cr3+),21,31−33 or the identification of key structures involved in cellular respiration (mitochondria) or cell signaling (dopamine receptors).34,35 Moreover, because Acridine Orange differentially stains nucleic acids orange or green depending upon whether they are single- or double-stranded (respectively),23 this fluorophore could be used to demonstrate the dual nature of many fluorescent probes. For example, students could use the stain to identify the location of important intracellular structures (such as the macro-nucleus and any micronuclei, which will appear bright green given the double-stranded DNA that comprises them). At the same time, students could use the fluorophore as an in vivo biosensor to identify differences in pH levels inside a cell since lysosomes will appear light orange as Acridine Orange molecules accumulate within these acidic vacuoles. Instructors could further take this opportunity to explain how the chemical composition of Acridine Orange gives rise to these distinct photochemical properties. Specifically, as a cellpermeable cationic dye, Acridine Orange interacts with DNA by intercalation to fluoresce in the green spectrum and with RNA by various electrostatic attractions to fluoresce in the red spectrum.36,37 Moreover, because Acridine Orange is an amphipathic weak base, inside the cell, this organic dye enters acidic compartments due to the pH gradient across these membranes and then becomes protonated to emit an orange glow when excited by blue light.23,38 Since the different spectral characteristics of Acridine Orange further depend on whether the compound exists as a monomer or forms di-, tri-, tetra-, or octamers in solution,37,38 in a single lab exercise, teachers could tie together (and, thus, simplify for their students) rather complex notions of how the composition, structure, and function of a molecule are inter-related while also demonstrat-

ing the valuable role that this particular metachromatic fluorophore plays in the fields of cyto- and biochemistry. Additionally, instructors could use cells tagged with fluorescent proteins to demonstrate similar biochemical phenomena inside a cell, such as measuring the concentrations of various ions (e.g., H+, Ca2+, Cl−) or organic compounds (e.g., ATP, cAMP),5 detecting redox reactions or other enzymatic activity,3,8 or observing interactions between different proteins. Instructors could also design lab activities where students mark certain proteins with a given fluorophore and then conduct time course studies as these tagged macromolecules are taken up by and distributed within the cell. By further varying the chemical environment surrounding these cells, students could explore how differences in pH level or the creation of other electrochemical gradients impact the movement of these proteins across the cellular membrane. Moreover, the ability to have several working epifluorescence microscopes on the bench opens up a world of possibilities for a teaching or research lab. In particular, using the OPN Scope, students or others could perform many sophisticated analyses without the need for the expensive or often cumbersome equipment that is typically required for fluorescence microscopy. For example, as explained in the Supporting Information, we estimate that printing the basic parts for the OPN Scope (with just one drawer) through the 3D printing Web sites MakeXYZ39 or Shapeways40 would cost between roughly $85 and $136 (S1) at March 2016 prices, and buying the raw materials to make the PVC version of the OPN Scope would generally cost between $53 and $134 depending on the exact supplies used (S4). Thus, with a relatively inexpensive used filter cube ($250) or filter set ($175) and moderately priced light sources ($20), a fully assembled 3D-printed or PVC version of the OPN Scope should cost less than $500 (not including the price of the underlying 3D printer, microscope, or other tools). Plus, even with a moderately priced new filter set ($450) in the OPN Drawer, the 3D-printed version of the D

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OPN Scope should cost between roughly $570 and $615 if printed on the MakeXYZ or Shapeways Web sites,39,40 which is still less than many new (and even some used) filter cubes. Indeed, absent a steady stream of used cubes in a variety of different spectra, buying a new filter set may be best option for many educators and researchers. Thus, we hope that these designs can help to expand the use of fluorescence microscopy and the valuable insights that it can provide in the classroom, teaching lab, and beyond.

CONCLUSION Recently, our lab has started to focus on ways to make science and scientific learning more accessible to others, especially for those who may lack the funding for what can often be an expensive pursuit. Given the simplicity, affordability, and versatility of these designs, we hope that both versions of the OPN Scope and the OPN Drawer will help to foster learning and promote scientific inquiry and investigation among students, educators, and others. In this respect, we hope that these designs can help complement the exceptional work that others have done in this field and provide further meaningful experiences for those in the sciences. We also invite others to use or modify our designs to fit their educational or research needs as we continue to expand and improve upon our ideas as well. ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.5b00984. (S1) Our protocols for 3D printing the parts for the OPN Scope and OPN Drawer (PDF) (S2) The underlying CAD files for the components of the 3D-printed version of the OPN Scope (ZIP) (S3) The underlying STL files for the components of the 3D-printed version of the OPN Scope (ZIP) (S4) Instructions for building the PVC version of the OPN Scope (PDF) (S5) Our protocols for preparing T. thermophila cells for fluorescent viewing and imaging (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS From Washington University, we would like to thank Doug Chalker for providing us with several strains of T. thermophila cells that were tagged with a fluorescent protein, including the pICY_AA cell pictured in Figure 4I−L. From the St. Olaf College Biology Department, we would like to thank Eric Cole for helping us keep and care for these and other strains of T. thermophila cells. Finally, the digital camera used to capture images of the fluorescent cells set forth in Figure 4 was paid for in part by an award from the Dr. E. Gordon and Alice Behrents Endowment for Biology Research at St. Olaf College. E

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