Reducing Photobleaching

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:

1. Use photostable dyes (Alexa, Dylight, ATTO, Cy3, etc) when possible. "Classical" dyes like FITC & TRITC have poor photostability.
   
2. 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.
   
3. Use photoprotectants. I.E. Oxygen-scavenging medias, mounting media containing photoprotectants. For live-cell work, some vitamins improve photostability.
   
4. Remove/reduce photosensitizers. Some common compounds in cell culture media (e.g. riboflavin and pyridoxal) can increase photobleaching rates.
   
5. 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:

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.

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.

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.

What to add:

Rutin, at 10mg/L, has a significant protective effect, as does trolox, a derivative of Vitamin E, at concentrations of 100-800μM. 1mM glutathione and 100–1000µM ascorbate have been reported to be photoprotective, but only for extracellular labels.

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.