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Observing Fluorescent Probes in Living Cells using a Low-Cost LED Flashlight Retrofitted to a Common Vintage Light Microscope

    Authors: G. A. Babbitt1,*, C. A. Hanzlik1,2, K. N. Busse3
    VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: T.H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14623; 2: Confocal Microscopy Lab, College of Science, Rochester Institute of Technology, Rochester, NY 14623; 3: Biomedical Engineering, Kate Gleason College of Engineering, Rochester Institute of Technology, Rochester, NY 14623
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 06 May 2013
    • *Corresponding author. Mailing address: T.H. Gosnell School of Life Sciences, Rochester Institute of Technology, 85 Lomb Memorial Drive, Rochester, NY 14623. Phone: 585-475-7577. E-mail: gabsbi@rit.edu.
    • ©2013 Author(s). Published by the American Society for Microbiology.
    Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 121-124. doi:10.1128/jmbe.v14i1.530
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    Abstract:

    While the application of molecular biological techniques based upon fluorescent probes has rapidly expanded over recent decades, the equipment cost of fluorescent microscopy has largely prevented its adoption in the college and high school classroom. We offer a simple solution to this problem by describing in detail how to build with simple tools, a fluorescent microscope using a common brand of colored LED flashlights and second-hand components of vintage Nikon microscopes. This extremely low cost solution is qualitatively compared to an expensive modern Zeiss system.

Key Concept Ranking

Optical Microscopes
0.6035997
Molecular Techniques
0.45914853
0.6035997

References & Citations

1. Albeanu DF, Soucy E, Sato TF, Meister M, Murthy VN 2008 LED arrays as cost effective and efficient light sources for widefield microscopy PLoS One 3 e2146 10.1371/journal.pone.0002146 18478056 2361193 http://dx.doi.org/10.1371/journal.pone.0002146
2. Hohman B 2007 LED light source: major advance in fluorescence microscopy Biomed Instrum Technol 41 461 464 10.2345/0899-8205(2007)41[461:LLSMAI]2.0.CO;2 18085085 http://dx.doi.org/10.2345/0899-8205(2007)41[461:LLSMAI]2.0.CO;2
3. Martin G, Agostini HT, Hansen LL 2005 Light emitting diode microscope illumination for green fluorescent protein or fluorescein isothiocyanate epifluorescence Biotechniques 38 204 206 10.2144/05382BM06 15727126 http://dx.doi.org/10.2144/05382BM06
4. Robertson JB, Zhang Y, Johnson CH 2009 Light emitting diode flashlights as effective and inexpensive light sources for fluorescent microscopy J Microsc 236 1 4 10.1111/j.1365-2818.2009.03208.x 19772530 2751867 http://dx.doi.org/10.1111/j.1365-2818.2009.03208.x
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/content/journal/jmbe/10.1128/jmbe.v14i1.530
2013-05-06
2017-04-30

Abstract:

While the application of molecular biological techniques based upon fluorescent probes has rapidly expanded over recent decades, the equipment cost of fluorescent microscopy has largely prevented its adoption in the college and high school classroom. We offer a simple solution to this problem by describing in detail how to build with simple tools, a fluorescent microscope using a common brand of colored LED flashlights and second-hand components of vintage Nikon microscopes. This extremely low cost solution is qualitatively compared to an expensive modern Zeiss system.

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Figures

Image of FIGURE 1

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FIGURE 1

A simple retrofit of the Nikon Optiphot brightfield vertical EPI illuminator for fluorescent imaging using a 3W Ultrafire WF-501B tactical LED flashlight as the illumination source. The retrofit includes the modification of the vertical illuminator by (A–B) the removal of internal optics, (C–E) the removal of the internal diaphragm, and (F–I) the attachment of the LED flashlight to the illuminator. More detailed instructions are provided within the body of the manuscript.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 121-124. doi:10.1128/jmbe.v14i1.530
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Image of FIGURE 2

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FIGURE 2

Final assembly and installation of the Nikon Optiphot brightfield vertical EPI illuminator retrofitted for fluorescent imaging. (A) The internal arm supporting the 575DRLP dichroic mirror and 610 nm optical filter suitable for observing live cell mitochondrial or membrane stains. (B) Within the internal housing, the mirror is angled downward at 45 degrees while the filter sits perpendicular to the light path going to the eye. (C) The final assembly showing the adjustment knob for fine-tuning the angle of the mirror.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 121-124. doi:10.1128/jmbe.v14i1.530
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Image of FIGURE 3

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FIGURE 3

Spectral characteristics of an illumination, mirror, and filter system for green fluorescent protein. This graph was produced using Omega Filter’s Curvomatic webtool at http://www.omegafilters.com/Products/Curvomatic. General characteristics one needs in a given system are ( 1 ) an LED light source that excites near the peak of the excitation spectrum, ( 2 ) a dichroic mirror that reflects light in the excitation spectrum and transmits light in the emission spectrum, and ( 3 ) an optical filter that captures light only near the peak of the emission spectrum.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 121-124. doi:10.1128/jmbe.v14i1.530
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Image of FIGURE 4

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FIGURE 4

Images taken using the Nikon Optiphot brightfield vertical EPI illuminator retrofitted to the Nikon model S-Ke for fluorescent imaging. Images are (A) copper-doped zinc sulfide crystals under 400 nm excitation at 100X magnification on our vintage Nikon S, (B) a comparable image from a modern Zeiss Axiovert 200M inverted fluorescent microscope, (C) GFP transformants () under 400 nm excitation at 1000X magnification on our vintage Nikon S. Cells were charged with a solution of arabinose 20 minutes prior to imaging. (D) CHO (Chinese Hamster Ovary) adherent cell line stained with Hoechst nuclear stain – 400 nm excitation and 488 nm emission. (E) CHO adherent cell line stained with Chromeo Live Cell Mitochondrial Staining Kit from ActiveMotif – 520 nm excitation and 610 nm emission, (F) a comparable double overlay image from the Zeiss Axiovert showing CHO adherent cell line double stained as in (D) and (E). Image overlay was produced by merging red and blue channels of independent images using free ImageJ software available at http://rsbweb.nih.gov/ij/index.html.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 121-124. doi:10.1128/jmbe.v14i1.530
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