AAT Bioquest

Cellular Fluorescence Microscopy Troubleshooting & Best Practices

Basic components of the microscope and the light path of fluorescence imaging
Basic components of the microscope and the light path of fluorescence imaging. Instead of a single path, there are two or more filters associated with the excitation and emission light wavelengths, prior to and after the objective lens. Figure made in BioRender.
Fluorescence microscopy is a major tool used in monitoring cell physiology. The most key feature of fluorescence microscopy is the ability to illuminate fluorescent objects, which can be extremely faint and hard to detect otherwise, against a dark background. To optimize fluorescence, image brightness and resolution must be maximized as much as possible. To separate targeted fluorescence and other items in view, filter cubes are used to view the specific excitation light reaching the fluorochrome(s) within the sample.

In understanding fluorescence microscopy, it is important to grasp the underlying process of fluorescence. Fluorescence involves the absorption of light energy (i.e. as a photon) by a fluorescent indicator dye followed by the emission of a portion of this light energy (as another photon) just a few nanoseconds later. Here, energy is lost so the emitted photon has less energy than the absorbed photon. Light emitted from a fluorescent indicator die will usually have a longer wavelength than that of the absorbed excitation light. A fluorescence microscope works, in principle, by separating the emitted from the excited light, thereby separating dim from bright components of the sample.


3 Common Factors that Affect Fluorescence Microscopy: Brightness, Resolution, & Maintenance


The overall brightness of the image will affect how easily fluorescence can be detected and photographed. The sample must be supplied with sufficient light energy to excite the wavelengths needed for each chromophore attached to the specimen. Choice of an appropriate barrier filter will ultimately help to maximize the amount of emitted fluorescence directed to the observation or camera tube, while blocking unwanted wavelengths. For this reason, mercury or xenon burners are ideal lamp sources as they are high-energy and will provide a large amount of excitation energy needed to appropriately illuminate a specimen.

Additionally, the objective lens helps to gather light from the specimen, and directly influences the brightness intensity of the viewed image. It is advised to have an objective lens of high-quality chromatic correction to help produce sharper images. Objective lenses should also be designed so that the lens elements will not autofluoresce when irradiated by light near ultraviolet wavelengths. Oil-immersion objective can be used to help minimize the loss of light caused by the reflection off the glass slide and/or coverslip surfaces. Though fluorescence microscope manufacturers attempt to build microscopes that are completely free from autofluorescent components, this should still be considered.


Appropriate glass coverslips will also help increase image resolution. Coverslips can range in thickness, generally between 0.01 - 0.03 mm, and microscope objectives should be fine-tuned to account for these variations. Sometimes the best route is to purchase highly accurate coverslips to ensure that the objective correction is always performed optimally.

Objective lenses should be cleaned with an appropriate solvent periodically, approximately once a month. Any excess oil has the potential to pick up dust, which will reduce the quality and resolution of the image generated. In choosing oil for an oil-immersion objective lens, make sure the oil is PCB-free, has little to no autofluorescence, and can be applied to the objective-coverslip interface without forming air bubbles.

Effective cleaning method
Effective cleaning method for removing smudges from external microscope lens. Gentle center-outwards circular patterns are best practice, instead of irregular or erratic movements, which can miss areas as well as cause damage. Illustration made in BioRender.


As a rule of thumb, microscopists should keep all optical elements completely free of dust, dirt, oil, solvents, or any other possible contaminants. The microscope should be kept in a room that is smoke free, as clean as possible, experiences little to zero vibration, with no disturbance in the circulated air. The microscope should be covered when not in use, and all accessory components should be kept in air-tight containers where possible.

Note: Standards vary, but in general, minimizing touch is the usual best practice. Gentle treatment is of the highest priority, so as to minimize damage, calibration issues, and other avoidable problems.

Corrosive solvents should not be used to clean any part of the instrument, and diluted soapy water is generally the best cleaning agent available. Before cleaning the objective lens, use compressed gas to first remove loose dust and particulate. Dirt on internal lenses should be removed by a professional, so contacting the manufacturer will inform next steps. For external lenses and other easily accessible components, gentle and consistent cleaning and upkeep can extend the useful life of the fluorescence microscope.

Make sure that maintenance on the lens is done gently as even lens paper has the potential to introduce very fine scratches onto the lens surface. Lens cleaning cloths are a common choice. There are a number of appropriate solvents that are purposed for optical surfaces, but absolute ethanol can substitute where necessary. Distilled water is a common choice as well. Importantly, other solvents have the potential to react with coatings that are on the lens. For example, ammonium is known to commonly damage anti-reflective coatings. Acetone can also have damaging effects on plastic components, so be cautious.

Note: When cleaning the lens, first soak a Q-tip in cleaner or ethanol and gently wipe the lens several times, turning the Q-tip each time to prevent reintroducing particulates.

Tips in Troubleshooting Fluorescence Microscopy

  • Photobleaching should be prevented as much as possible, as this will cause damage to the specimen. This can be done by either adding antifading reagents to the sample, using special media, and/or by reducing sample exposure to light in the first place. Reducing this exposure includes methods such as reducing light intensity overall or by blocking the excitation light using the filter slider or shutter in the illuminator of the microscope when not viewing or photographing the specimen.

  • Autofluorescence of the sample can also be limited if the specimen is thoroughly washed after staining to remove excessive fluorochrome buildup before slide mounting.
  • Daylight balance transparency films may help provide the best rendition of specimen colors, resolution, or contrast, though transparency films may be preferred as they can provide enhanced contrast and color saturation.

Recommendations & Preventative Practices

Mounting Media Considerations
The mounting media should:The mounting media should not:
Have an RI as close as possible to that of glass, and be colorless and transparent.Cause the stain to diffuse or fade.
Be able to completely permeate and fill tissue interstices.Become sticky or harden slowly.
Be resistant to contamination, microbial, and fungal growth.Shrink inside the edge of the coverslip and slide upon hardening.
Protect from physical damage and chemical activity such as oxidation or pH changes.Not react with, leach or induce fading in stains and reaction products.
Be completely miscible and combine homogeneously with the dehydrant or clearing agent.Crystallize, crack, shrink or otherwise deform the mounted specimen.
Remain stable with time.Have adverse effects on tissue components.

Anti-fading reagent
U2OS cells were loaded with Calcein AM for 1 hour then fixed with 2% formaldehyde in a 96-well black plate. Anti-fading reagent was added into the samples after removing all the media. The FITC signals were compared at 0 and 30 seconds exposure time using fluorescence microscopy. The same exposure settings were used for all the images.
  • Though ideally all microscopies should be performed in darkened rooms, this is especially important with fluorescence microscopy. This is because the human eye has trouble quickly adjusting to the dark, which can make it particularly difficult to differentiate very dim fluorescence.
  • When choosing the right components for a microscope, it is recommended to use an objective that is equipped with a glass or quartz lens that is transparent near ultraviolet light.
  • Using the lowest magnification projection lens possible will aid in limiting total magnification of the image.
  • Generally, imaging is best performed when using reflected light fluorescence compared to transmitted light fluorescence, though a transmitted light darkfield condenser will help imaging if using the latter.

    Note: The choice of correct film apertures will also help optimize contrast of the fluorochromes within the sample, and it is key that these films match the chromophore excitation and secondary fluorescence properties of the specimen.

  • The best choices for light sources are xenon and mercury vapor lamps or lamps with lasers that can be fine-tuned to specific wavelengths.
  • With all fluorescence microscopy, it is important to use a heat filter between the illuminator and fluorescence filters to limit damage that may be caused by excessive heat.
  • When the lamp starts to flicker or the illumination intensity in portions of the sample appear unevenly illuminated, it is likely time to replace the light source.






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Original created on February 29, 2024, last updated on February 29, 2024
Tagged under: microscopy, maintenance, troubleshooting, fluorescence