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Photobleaching is a common issue with both fixed and live samples. Proper sample preparation, and the addition of photoprotectants, can dramatically reduce the degree of photobleaching during an experiment.

 

=== What is Photobleaching? ===

Photobleaching is a chemical reaction. Typically, an excited fluorophore reacts with oxygen, but reactions with other compouds can, and do, occur. The oxidation of the fluorophore destroys its fluorescence. When using a microscope, photobleaching is observed as a continued decrease in the fluorescence intensity of a sample. If too serious, this can prevent the capture of images as the fluorophore depletes before an image can be captured. 

=== ===

=== General Advice: ===

#Use photostable dyes (Alexa, Dylight, ATTO, Cy3, etc) when possible. "Classical" dyes like FITC & TRITC have poor photostability. 
#Avoid excessive excitation and/or reduce excitation intensity. This lowers the frequency of fluorophore excitation, thereby reducing the likelihood of a photobleaching reaction occurring. This is not always an ideal solution - lower excitation intensities often require longer exposures, thus providing the same degree of photobleaching; lowering overall exposures do reduce photobleaching, but at the cost of image quality. 
#Use photoprotectants. I.E. Oxygen-scavenging medias, mounting media containing photoprotectants. For live-cell work, some vitamins improve photostability. 
#Remove/reduce photosensitizers. Some common compounds in cell culture media (e.g. riboflavin and pyridoxal) can increase photobleaching rates. 
#DO NOT use gluteraldehyde for fixation. Paraformaldehyde or methanol are preferred. 

 

=== Fixed Samples: ===

===== Wet-Mounted: =====

Wet-mounted fixed samples are best protected with oxygen-scavaging medias. These include: 
<pre>Beta-mercaptoethanalomine (cystamine) Media:
-50 to 100mM cystamine or cystamine HCl dissolved in PBS.

Note: Media will have to be re-pH'd after cystamine addition. Media should be prepared fresh, or frozen as aliquots for no more than 1 month.

To use: Mount sample in media, using a depression slide. Seal edges with a sealant. Image within 3 hours of mounting.
</pre> <pre>Glucose-Oxidase Media:

Solution 1: PBS containing 11% (w/v) glucose
Solution 2: PBS + 20% glycerol + 5 mg/mL glucose oxidase and 400 μg/mL catalase.

Note: Solutions can be prepared in advance, and frozen at -20C until needed. Solutions are stable for 2-3 months at -20C.

To use: that aliquots, dilute solution 2 1:10 in solution 1. Immediately mount sample, using a depression slide. Seal edges with a sealant and image within 3 hours of mounting.

Note: Cystamine can be added to solution 1, 50-100mM, although it is unclear if this adds additional sample protection.

</pre>
===== Hard-Mounted: =====

Polymerizing (hard-mounted) mounting medias containing photoprotectants are available from a number of companies (e.g. Prolong gold [Invitrogen]). 100mM to 200mM of N propyl galate can be added to any hard mount media that lacks photoprotectants. 

 

=== Live Samples: ===

Protecting live samples from photobleaching is more difficult, as many photoprotectants are cytotoxic and/or scavenge oxygen. As a rule, anti-oxidants tend to protect cells, while photoreactive compounds (e.g. flavinoids) tend to act as photo-sensitizors. Common medias contain photo-sensitizors, thereby necessitating specialized media for long-term imaging. 

===== What to avoid: =====

Riboflavin and pyridoxal are found in most tissue culture medias, and are potent photo-sensitozers<ref>Bogdanov AM, Kudryavtseva EI, Lukyanov KA. Anti-fading media for live cell GFPfckLRimaging. PLoS One. 2012;7(12):e53004. doi: 10.1371/journal.pone.0053004. EpubfckLR2012 Dec 21. PubMed PMID: 23285248
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053004</ref>. 

===== What to add: =====

Rutin, at 10mg/L, has a significant protective effect<ref>Bogdanov AM, Kudryavtseva EI, Lukyanov KA. Anti-fading media for live cell GFPfckLRimaging. PLoS One. 2012;7(12):e53004. doi: 10.1371/journal.pone.0053004. EpubfckLR2012 Dec 21. PubMed PMID: 23285248
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053004</ref>, as does trolox, a derivative of Vitamin E, at concentrations of 100-800μM<ref>Sun X, Wong CCF, Keefe J, Chan GK. The effects of antioxidants on photo-toxicity in fluorescence live-cell imaging. http://www.focusonmicroscopy.org/2010/PDF/285_Sun.pdf</ref>. 1mM glutathione and 100–1000µM ascorbate have been reported to be photoprotective, but only for extracellular labels. 
<pre>Base Imaging Media:
-150 mM NaCl
-5 mM KCl
-1 mM MgCl2
-100 µM EGTA
-2 mM CaCl2
-20 mM Hepes (air) or 2g/L sodium bicarbonate (5% CO2 chamber)

Dilute everything in water, then pH to 7.4. EGTA can be left out, but is reuqired if preparing media Ca-free. If EGTA is left in, media will have 1.9mM free Ca2+. This formulation is suitable for short-term imaging (<1 hour)

Medium-Term Imaging Media:
For imaging sessions lasting 1-4 hours, add to the base imaging media:
-2g/L d-glucose
-upto 5% FBS

Long-Term Imaging Media:
RPMI or DMEM (appropriately buffered, with FBS) should be used when imaging cells for >4 hours. Rutin &/or trolox shoudl be added to counter the effect of the riboflavin & pyridoxal found in the media.
</pre>
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Reducing Photobleaching - Revision history

Admin: Created page with "Photobleaching is a common issue with both fixed and live samples. Proper sample preparation, and the addition of photoprotectants, can dramatically reduce the degree of photo..."