I was working on a client project recently which called for me to do some optical microscopy to image the droplets present in a skin cream. How you do microscopy has a huge impact on the nature of the images you get, and things like changing the wavelength of the light and direction the light comes from can have quite big effects on the final image and how it looks. I’ve seen various ‘qualities’ of images papers and scientific reports, and it has been my experience that many companies are losing technical experts from the their departments which means the experience of using things like microscopes is often disappearing. I thought I’d share some images of a topical oil in water emulsion using my modified Olympus BHB microscope and show the effect of changing the light source spectral distribution and lighting direction on the image, as well as going to high resolution to see what size features can be expected to be seen.

I’ll keep the product anonymized for now, but this is a topical skin cream with oil droplets in an aqueous matrix. A dot of cream was placed on a standard glass slide and a 0.17mm coverslip placed on top. This was gently pressed to flatten it out. The microscope is an Olympus BHB which has been modified to allow me to do UV work (although I am only using visible light here). Two objectives have been used, first a 20x Nikon Plan Apo NA 0.65 one, then a 63x Leitz Pl Apo NA 1.4 oil immersion one. Condenser was an Olympus Aplanat Achromat, used in brightfield and oblique configurations (and oiled to the underside of the slide when used with the 63x objective). 2.5x Nikon CF PL photoeyepiece. Monochrome converted Nikon d850 camera. Light source was a white LED light which was left unfiltered, or filtered using a 450nm, 40nm FWHM bandpass filter from Thorlabs. The microscope was focused just below the underside of the coverslip. Single images were taken and processed in Photoshop (denoised, sharpened – unsharp mask, and auto contrast). They were then cropped to 1600×1200 and kept at original resolution.

Enough waffle. First two images – brightfield lighting, white light and 450nm light, and 20x Nikon Plan Apo NA 0.65 objective.

Both brightfield images look pretty good. The droplets can be seen. If you go in close the 450nm light image is slightly higher resolution than the white light image. This is simple physics – resolution of a microscope setup is highly dependent on wavelength. The shorter the wavelength the better the achievable resolution for a given setup. White light contains a mixture of light from 400nm to about 700nm. This spread will have an ‘average’ wavelength which is longer than the 450nm filtered light. Also, having a range of wavelengths puts greater stress on the optics being able to focus all those wavelengths to the same point. The objective is a Plan Apo (Plan – flat field, Apochromatic) and is a good one, but it will always be harder to correct for a wide range of wavelengths vs a small range.

Next oblique images. Oblique lighting, white light and 450nm light, and 20x Nikon Plan Apo NA 0.65 objective.

Changing the lighting to oblique (basically pulling the condenser slightly to the side) has the effect of creating a pseudo-3D image compared with brightfield, as it produces directional shadows. The effect is subtle here, and is easier to see when oblique and brightfield are put next to each other. Again, both the white light and 450nm light images look good, and the 450nm light one has slightly better resolution as before. I am a big fan of oblique lighting and use it a lot for my diatom photos. Brightfield images can be quite flat, but oblique lighting can make the structures in an image more tangible. Oblique lighting tends to be overlooked these days in favour of other approaches to improving contrast in images, which I think is a bit sad. It is such a simple technique and so much cheaper than something like Differential Interference Contrast (DIC). In fact oblique lighting has been called “poor man’s DIC” in the past.

While a 20x objective image is often good enough to see what is going on, sometimes higher magnifications and resolutions are needed. I also imaged this slide using a 63x Leitz Pl Apo NA 1.4 oil immersion objective, using 450nm light, and with brightfield and oblique lighting. Some people are scared of oil immersion, but it’s not that bad and enables the highest NA objectives to be used (and therefore the highest resolutions to be reached). These images are shown below.

Yet again, both images look good. However as expected the brightfield image is quite flat, while the oblique image has that pseudo-3D look about it, which can help with visualizing smaller features. For instance I picked on one of the small droplets in the image and used ImageJ to measure its diameter (which turned out to be 498nm).

Keep in mind with these 63x objective images that the depth of field is tiny – probably a few hundred nm, so very minor shifts in the stage will bring things in and out of focus, especially the smaller droplets. Sub micron resolution is not too hard with these high NA objectives, and it should be possible to see things down to about 4-500nm without too much difficulty. Below that things get a bit tricky without getting more complex in your approach.

Optical microscopy is a very powerful technique for the skin care formulator as it allows emulsion structure to be visualized which can impact everything from product stability to appearance and aesthetics on application. As with all imaging techniques the more you know the more you get out of it, and simple tweaks like choosing the right wavelength of light to use, and even how you illuminate your sample can have a marked effect on the image produced. While this can help with just seeing what is going on, if the images are to be used in marketing the product, getting the right ones can make the difference between a successful product and one which doesn’t live up to its potential.

As always, thanks for reading, and if you’d like to know more about my work, I can be reached here.