UV-Fluorescence+Microscopy

Greg Roth (10/13/14)
UV-Fluorescence microscopy

Basic Description:

Fluorescence microscopy is a technique that is used to examine cells utilizing fluorescence to generate an image. To generate this image, that target specimen needs to be fluorescing so it is typically subjected to genetic manipulation so it has DNA that codes fora fluorescent protein, or it is exposed to a fluorescent protein capable of binding to certain locations within the specimen. The specimen is then illuminated with UV light generated by a mercury or xenon lamp causing the fluorescing material to emit light with a longer wavelength. A spectral emission filter in the microscope is then able to filter the illuminated light from the weaker light of the emitted fluorescence using a dichroic mirror, which allows certain light wavelengths through, and doesn't allow others through. The illumination from the light source in this type of microscopy is first passed through an excitation filter and is then directed toward the specimen by the dichroic mirror. Only the emitted fluorescence is passed back up to the ocular [1, 10].

To explain this another way, the dichroic mirror diverts the light downward, through the objective and onto the slide containing the sample. Electrons gain energy, rising to a higher principal energy level and fall back down to a lower principal energy level by releasing a photon, producing light. Both scattered, short wavelengths and the newly emitted, visible, long wavelengths travel back up through the objective and towards the dichroic mirror. While the desired, visible light can pass through the mirror, harmful short wavelengths are reflected back downward. This process allows for the visualization of a fluorescent specimen [2].

Purpose of Technique:

Fluorescence microscopy has several purposes. It is most often used to identify specific proteins or molecules within cells and tissues. Fluorescent dyes can be coupled with antibodies in an extremely selective binding process which would illuminate very specific macromolecules within cells. Therefore, fluorescence microscopy can be used to tell researchers the relative concentration and location of specific molecules inside living cells [3].

Many cells are transparent in appearance and their dynamic processes cannot be easily studied unless a technique like fluorescence microscopy is applied. Furthermore, fluorescence microscopy allows for the visual separation of different components of a cell based on the stains and marking techniques that are used.

Origin and History:

George Stokes, a British scientist, described fluorescence in 1852, and coined the term when he discovered that fluorspar, a halide mineral, emitted red light when illuminated by UV excitation. He also noted that fluorescence emission light occurred at a longer wavelength than the illumination excitation light used. However, this concept was not used in the biological sciences until the 1930’s when fluorchromes became highly specified for staining organisms and their components [1]. These developments ultimately led to the development of the fluorescence microscope and since then there have been several advancements in fluorescence imaging. The first real advancement was made in 1940 by Albert Coons and Melvin Kaplan, when they introduced the aforementioned antibody-fluorescence coupling. The second major advancement didn’t occur until the 1990’s, when Martin Chalfie, Osamu Shimomura and Roger Tsien discovered and developed the infamous green fluorescent protein (GFP) [4].

Recent Research:

Fluorescence microscopy was processed in three dimensions to measure the surface areas of lipid domains in giant unilamellar vesicles [5]. The data retrieved from the fluorescence microscopy was reconstructed using active surface models. These images and data analysis led to the conclusion that the membrane domains observed do indeed correspond to equilibrium thermodynamic stages meaning that GUV’s are suitable for equilibrium thermodynamic studies of membranes [5]. Additionally, fluorescence microscopy been used for the advancement of nucleic acid treatment for various diseases [6]. Advanced fluorescence microscopy can be used to better understand nanoparticles and their physicochemical and biophysical properties [6]. The advanced fluorescence microscopy techniques include fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single particle tracking [6].

Fluorescence microscopy was used to determine the structure and mechanism of photoactivatable green fluorescent protein (PA-GFP) [7]. Researchers determined that “the activation of PA-GFP is the result of a UV induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form.” The crystal structures, which are brightly fluorescent under 488 nm, would not be as visible if not for fluorescent microscopy [7].

Another study focused on the use of three different fluorescent antibody staining assays for human Cryptosporidium and used them to detect oocytes of Cryptosporidium molnari in fish. Using the fluorescence microscope, it was found that all three kits were capable of detecting C. molnari in fish [8]. Similarly, fluorescent- antibodies were used to visualize the inflammatory receptor CD40 in mice. This non-invasive and specific technique can be used to detect brain inflammation, a common precursor to a stroke [9]. Without fluorescence microscopy, none of the above studies would be able to analyze the results of their experiments.

Fluorescence microscopy was used to allow the researchers to gain a clear and direct view of different structures within the stigma of hermaphroditic plants. Using fluorescence microscopy the authors could see the individual structures and were able to differentiate why certain pollen was incompatible with other members of the same species of plant, or with the same plant the pollen originated from [11].

Batard et al used fluorescence microscopy in order to better understand how plant pathogens have become resistant to certain antibiotics and what exactly they do with the antibiotics that have been administered. They found that by tagging the antibiotic with a fluorescent marker they saw that it simply accumulated <span style="font-family: Times New Roman,Times,serif;">around the colonies of bacteria without actually entering their cells, rendering it useless [12]. <span style="font-family: Times New Roman,Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;">Kaščáková et al used fluorescence microscopy similarly to that of Batard et al, but instead they looked at individual cells that up taken in an antibiotic but are still resistant to it. They wanted to see where the antibiotic ends up within the cell in order to better understand the development of resistance and how it could possibly be avoided down the future [13].

<span style="font-family: 'Times New Roman',Times,serif;">References:
====<span style="font-family: 'Times New Roman',Times,serif;">[1] Spring, K. R., & Davidson, M. W. (n.d.). Fluorescence Microscopy. In Microscopy-U. Retrieved March 7, 2012, from []. ====

<span style="font-family: 'Times New Roman',Times,serif;">[2] Staley et al. Microbial Life. 2nd. Sunderland, MD: Sinauer Associates, Inc., 2007. 81-82. Print.

<span style="font-family: 'Times New Roman',Times,serif;">[3] Alberts et al. Molecular Biology of the Cell. 4th. New York, NY: Garland Science, 2002. 302-430. Print.

<span style="font-family: 'Times New Roman',Times,serif;">[4] Vonesch et al. “The Colored Revolution of Bioimaging.” IEEE Signal Processing Magazine. 23.3 (2006): 20-31.

<span style="font-family: 'Times New Roman',Times,serif;">[5] Fidorra, M., Garcia, A., Ipsen, J. H., Hartel, S., & Bagatolli, L. A. (2009, August 21). Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach. Biochimica et Biophysica Acta, 1788, 2142-2149. Retrieved March 7, 2012, from ScienceDirect (10.1016/j.bbamem.2009.08.006).

<span style="font-family: 'Times New Roman',Times,serif;">[6] Braeckmans, K., Buyens, K., Naeye, B., Vercauteren, D., Deschout, H., Raemdonck, K., & Remaunt, K. (2010, September 9). Advanced fluorescence microscopy methods illuminate the transfection pathway of. Journal of Controlled Release, 148, 69-74. Retrieved March 7, 2012, from ScienceDirect (10.1016/j.jconrel.2010.08.029). <span style="font-family: 'Times New Roman',Times,serif;">[7] Henderson et al. "Structure and Mechanism of the Photoactivatable Green Fluorescent Protein." Journal of the American Chemistry Society. 131.12 (2009): 4176-4177.

<span style="font-family: 'Times New Roman',Times,serif;">[8] Barugahare et al. “Fluorescent-antibody staining assays for human Cryptosporidium detect oocysts of Cryptosporidium molnari from fish.” Applied and Environmental Microbiology. (2011).

<span style="font-family: 'Times New Roman',Times,serif;">[9] Klohs et al. “In Vivo Imaging of the Inflammatory Receptor CD40 After Cerebral Ischemia Using a Fluorescent Antibody.” American Heart Association Journals. (2008)

<span style="font-family: 'Times New Roman',Times,serif;">[10] Freudenrich, C. 2012. How Light Microscopes Work. <span style="font-family: 'Times New Roman',Times,serif;">http://science.howstuffworks.com/light-microscope4.htm

<span style="font-family: 'Times New Roman',Times,serif;">[11] Saumitou-Laprade, P., et al. 2010. A Self-Incompatibility System Explains High Male Frequencies in an Androdioecious Plant.

<span style="font-family: 'Times New Roman',Times,serif;">[12] Batard, E., et al. 2011. Diffusion of Ofloxacin in the Endocarditis Vegetation Assessed with Synchrotron Radiation UV Fluorescence Microspectroscopy.

<span style="font-family: 'Times New Roman',Times,serif;">[13] Kaščáková, S., et al. 2012. Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution. <span style="font-family: Times New Roman,Times,serif;"> =<span style="font-family: 'Times New Roman',Times,serif;"> =