Darkfield microscopy

Bright field and dark field images of commercial slide of Ulothrix Algae

My favourite view from this session – x10 objective, dark field, Zeiss IM microscope, image edited in GIMP2 (other photos below have not been edited, only this one):

My wife looked at the dark field images below and immediately suggested they looked like a star field – to me the cosmic web! The white blobs are I think mountant rather than sample.

These images are part of my ongoing project to get dark field microscopy working well on the Zeiss IM microscope. From my reading, it looks like my previous difficulties obtaining dark field images was due to having objectives that are simply too good – dark field requires a condenser with larger numeral aperture (NA) than the objectives but mine all have enormous NAs!

The following information is taken from “Microscopy Primer by Frithjof A. S. Sterrenburg: 8. SPECIAL MICROSCOPY TECHNIQUES” (http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/primer/index.htm):

Darkfield [microscopy]:

If sunlight enters an otherwise dark room through a slit in the curtains, tiny dust motes that were first invisible are seen against the dark background because they scatter the light. The undeviated light is excluded, the deviated light produces the image. A simple way to obtain such darkfield illumination is to place a central stop in the filter ring of the condenser. This central stop can be a coin, cemented centrally on a plastic disc cut to fit the filter ring (CDROM cassettes are good material). This central stop blocks the direct light, only rays at an angle larger than the “angle of admittance” of the objective in use are allowed to strike the object. The background is dark, the light scattered (deviated) by the object – drawn bright blue in Fig. 43 – is imaged by the objective.

The size of the central stop will obviously depend on the “angle of admittance” (i.e. the NA) of the objective. A central stop of about 15 mm in diameter will generally yield darkfield with objectives up to about NA 0.5, depending on the properties of the microscope condenser. A central stop of 20 mm will generally permit darkfield with a 40x/NA 0.65 objective also. Adjust the focus of the substage condenser for maximum brightness of the central field of view. The field stop of the lamp can be wide open and there’s no major difference between critical or Köhler illumination. For darkfield at higher NA, special “cardioid” darkfield condensers using mirror surfaces are manufactured. These are oiled to the slide (instead of oil, water may be used for convenience) and require exact focusing and centering with their centering screws. Even these high-power darkfield condensers cannot be used with objectives of NA above 1.2 or higher, however. The reason is that darkfield condensers have two NA’s that should be considered: the inner NA (i.e. the portion of the light that is blocked) and the outer NA (which for technical reasons is limited to at most 1.4). For an objective of NA 1.25 one would have to block everything up to at least NA 1.3 and the cone of light that would remain would thus be limited to an NA between 1.3 and 1.4. That’s simply not enough to produce practical darkfield. The inner NA limit attainable with current darkfield condensers appears to lie near 1.0 , corresponding to the medium-power (40 – 60x) oil immersion objectives offered by several manufacturers.

Contrast in darkfield is extreme and resolution is as high as the objective can yield. Darkfield is spectacular for observations of live protozoa or bacteria (cilia or flagella are visible) and for diatoms. I always use darkfield to scan diatom slides at low to medium power because it’s easy on the eyes and even the smallest and faintest diatoms stand out clearly. The limitations of darkfield:

  • thick or extended objects and even dense strews are unsuitable (glare)
  • residual errors in objectives (notably spherical aberration) become maximally visible. This is especially the case with a dry 40x objective and forms the reason why a 40x immersion is highly advantageous here. For examination of live specimens in darkfield a 40x water immersion is ideal.
  • all specks of dirt or traces of grease also become maximally visible. You need a scrupulously clean optical train: top lens of condenser, both surfaces of slide, clean and “dilute” sample, grease-free object glass.

In my experiments with different objectives today, the x10 objective works well in dark field – but then its NA = 0.10.

x40 objective, NA=0.65 – not able to obtain dark field illumination.

Andy

Bright field, x10 objective. The two images differ in colour due to white balance settings in MikroCamLabII software for the Bresser Microcam 5.0 MP camera and show the difference in detail that can be seen with such a simple setting change:

Dark field, x10 objective:

 

Helicon Focus stack, dark field, x10 objective, and 3D model from Helicon Focus from same stack of frames (from AVI video of slide):

x40 objective, bright field:

x40 objective, Helicon Focus stack & 3D model showing difference heights of filamentous algae on slide:

The following picture shows the effect on resolution of closing the illuminator aperture somewhat and switching in the high NA lens on the condenser: