Phase Contrast

The first thing you’ll need is a substage condenser for your BH2 which is compatible with phase contrast. At the very low end, you can use the basic BH2-CD Abbe condenser, as long as you can find the necessary phase contrast inserts for the BH2-CD. The appropriate phase contrast inserts for the specific objective you are using must be pressed up into the bottom bore of the BH2-CD and then the condenser centering must be adjusted to align the phase annulus in the condenser with the phase ring in the objective. Any time you change from one objective to another, or switch back and forth from brightfield to phase contrast, you will be mucking with these inserts and doing this setup. Unless you are using phase contrast with just a single objective this setup makes for a very slow and cumbersome phase contrast experience. Believe me when I say if this is the route you choose, you will soon find this to be very annoying, and you will quickly find yourself avoiding phase contrast. A much better option is either a BH2-PC or BH2-PCD Zernike-style phase contrast condenser. You will get similar performance from these as you would with the BH2-CD solution, since these are all NA 1.25 Abbe condensers. However, compared to the BH2-CD, the BH2-PC and BH2-PCD condensers are a dream to use! Switching from one magnification to another, or back and forth from brightfield to phase contrast, takes almost no effort at all. Just spin the dial to select the proper annulus, and that’s it. Plus, you only have to do a one-time setup to center the phase annuli to the various objectives. So, what’s the difference between the BH2-PC and the BH2-PCD? The BH2-PC contains phase annuli suitable for use with 10x, 20x, 40x, and 100x phase contrast objectives, along with a brightfield setting. The BH2-PCD skips the 20X annulus and includes a simple darkfield stop instead. Take my advice and buy yourself a BH2-PC instead of the BH2-PCD. You won’t miss the darkfield stop if you buy the BH2-PC, since there are other ways to implement darkfield, but you will certainly miss the 20x annulus if you buy the BH2-PCD. It’s easy enough to add a simple darkfield stop to the BH2-CD condenser, or better still, get yourself a real darkfield condenser (BH2-DCD or BH2-DCW) if you’re serious about darkfield. The next step up is a very expensive step. These options rely on either the BH2-UCD universal condenser (with the nearly impossible-to-find phase-contrast inserts) or the BH2-NIC Nomarski condenser. Both of these will give similar phase contrast performance, thanks to their NA 1.40 aplanatic / achromatic design. Unfortunately, both of these will also likely require you to raid your first-born’s college fund for the purchase.

Now here is the controversial part. You should also find yourself a good phase contrast filter. A phase contrast filter passes light over the specific frequency band where the phase rings in the objectives were designed to provide maximum phase retardation, and blocks the out-of-band light where phase retardation is poor. There was a low-cost absorptive filter (45G533) made for the simple BH2-CD solution, and a higher performing interference filter (43-IF550-W45) made for the higher-end solutions. If possible, get the 43-IF550-W45, as its interference design provides better bandpass performance. In practice, most people omit this filter when using phase contrast, since the filter imparts a decided green monochromatic tint to the resulting images. Go look at a few microscopy videos on YouTube, and you will see first hand that none of the phase contrast videos are green. Frits Zernike would of course roll over in his grave if he knew that his phase contrast was misused in such a manner. Zernike of course understood that for the highest contrast results, the appropriate bandpass filter should always be used, with the resulting images photographed in grayscale to remove the objectionable color tint. Phase contrast is a technique that works purely through wave interference rather than absorptive dyes or stains applied to the specimen, and in order to accomplish this, it relies on lighting that falls within a very narrow band of wavelengths. This means that phase contrast, by design, inherently produces monochromatic images. If you omit the bandwidth filter, you will reduce the phase contrast effect, so you should always use the appropriate bandpass filter when using phase contrast. The fact that many people omit the bandpass filter is one of the reasons why phase contrast has a poor a reputation for imaging quality among amateur microscopists. To be sure, halo artifacts are a real thing with phase contrast, but why add to this by omitting the bandpass filter?