Immersion oil and which one to use?
Immersion oil is utilised to increase the optical resolving power of the microscope. It is used by placing a small amount between the coverslip of a specimen and the front lens of an oil immersion objective. There are many different types of immersion oils, all with different properties and it is generally best to use the immersion oil recommended by the manufacturer of the objective. Immersion oil properties are usually matched to the objective lens properties to give optimal image quality. The refractive index of the immersion oil is an important parameter and it is typically 1.515 at 23° C (1), close to that of glass to obtain optimal results. As the refractive index is similar to that of glass, light rays leaving the microscope slide and passing into the oil continue unrefracted, or are refracted less than if they are passed from glass to air. Using immersion oil increases the resolution so that smaller objects can be seen.
As basic light microscopy is routinely used in school science laboratories, the difference in image quality will not be evident, so any manufacturer’s immersion oil will be suitable as long as the refractive index is correct. Immersion oil is available from many scientific suppliers, see the Science ASSIST School science suppliers list.
Other fluids such as glycerine, and also mineral and vegetable oils work, but their refractive indices and dispersive powers vary somewhat from that of glass, and hence cannot be expected to elicit the best imagery from the specimen(2).
Immersion oil should only be used sparingly with an oil immersion objective lens. It should Never be used with any of the other dry objectives, as it will damage them. Immersion oil is best stored at room temperature.
Cleaning microscope objectives
It is best to consult the manufacturer’s guidelines as to the best cleaning method and fluid for your particular microscope.
Generally, the choice of cleaning method and fluid depends on the optical surface that requires cleaning and the substance to be removed. The surface of the optics of a microscope are easily damaged and therefore require extra care in their maintenance. Any cleaning fluid used should not damage any part of the microscope, including the lenses, and it should not leave any residue.
Image quality is dependent on having clean, damage-free optics. Immersion oil should be removed immediately after use and not be allowed to remain on the objective lens. The risk of leaving immersion oil on an objective lens can result in the contamination of other parts of the microscope and the possibility of the oil moving into the objective itself causing irreversible damage(3).
Steps for the removal of immersion oil from the external surface of the oil immersion lens.
- The oil should be removed immediately after use.
- Use clean, lint-free, lens cleaning tissue to gently blot the oil from the lens surface.
- Gently wipe the lens surface with fresh lens cleaning tissue until no oil residue is evident. This will require several changes of lens cleaning tissue.
- Any final traces of immersion oil can be removed using a small amount of lens cleaning fluid. Commercial glass cleaners such as Windex® can be used sparingly but they may have an effect on some coatings used on lens surfaces (4).
- Never use abrasive materials such as dry cotton swabs or facial tissues, as they are likely to scratch the lens.
The choice of immersion oil for many years was Cedar Wood Oil, until the manufacture of synthetic alternatives in the 1940s. Cedar Wood oil is still available, but has many disadvantages: it goes yellow with age, and if not cleaned up properly, will penetrate and damage the cement in the lens. The modern synthetic versions used today are inert, more colour stable and can be obtained in various viscosities(2).
General cleaning advice for microscope optics
- Treat lenses with great care, as they can be easily scratched.
- Solvents are not recommended for cleaning objective lenses as they have the potential to dissolve the cement in the lens assembly and harm other mechanical components, particularly in older microscopes.
- When cleaning lenses, first blow away any dust with a blower brush, then use a lint-free lens tissue and lens cleaning fluid to clean the objectives lenses and eyepieces.
- Do not use paper towel or regular tissues, as they will scratch the lens.
- Never dry wipe a lens, as this may also cause scratching.
- Remove immersion oil immediately after use with lens tissue and lens cleaning fluid.
- Do not remove eyepieces or objective lenses from their location and clean only their external surfaces. Internal surfaces should be cleaned by a professional.
Science ASSIST has also developed a resource on the use of the care of the compound light microscope see: SOP - Use and care of the compound light microscope
(1) Nikon Instruments, 2009. Immersion oil and the microscope FAQs. Melville, NY USA. Micro Video Instruments website, http://www.mviservice.com/Images/Immersion%20Oil%20FAQs%202009.pdf
(2) ‘The oil immersion guide’, Microscopy UK website, http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artmar02/pjoil.html (March 2002)
(3) ’Microscope cleaning and maintenance’, ZEISS Microscopy Online website, http://zeiss-campus.magnet.fsu.edu/articles/basics/care.html (Accessed November 2015)
(4) ‘Immersion oil’ in Murphy, Douglas B.; Davidson, Michael W. 2013. Fundamentals of Light Microscopy and Electronic Imaging, Wiley-Blackwell; New Jersey, USA. https://books.google.com.au/books?id=f8qhtqRjxQMC&pg=PT140&lpg=PT140&dq=...
‘Basic Concepts in Optical Microscopy’, Microscopy Resource Center, OlympusMicro website, http://olympus.magnet.fsu.edu/primer/anatomy/anatomy.html (Accessed January 2016)
Cargille, John J. 1985. Immersion oil and the microscope, New York Microscopical Society Yearbook, Cargille Labs website, http://www.cargille.com/immersionoilmicroscope.shtml
‘Immersion media’, Microscopy Resource Center, OlympusMicro website, http://olympus.magnet.fsu.edu/primer/anatomy/immersion.html (Accessed January 2016)
‘Oil Immersion and Refractive Index’, ZEISS Microscopy Online website, http://zeiss-campus.magnet.fsu.edu/tutorials/basics/oilimmersionrefractiveindex/indexflash.html (Accessed November 2015)