Variable-color Phase Contrast ?

AO seems to have dropped the ball with their Polanret Variable Phase Contrast system – too complicated, too expensive, and (for many users) just too hard to understand! Yet AO was not the first maker to venture into the 'variable-phase' waters…

Way back in the late 1960's Nippon Kogaku (Nikon) introduced a "polanret" type system of their own, intended for use on their Model "S" series of stands. However, unlike the later AO system the Nikon system was simple, compact and rather affordable. It also distinguished itself by creating user-controllable interference colors in the phase images!

And, apparently just to ensure that potential users had no idea what it was, they called their marvelous new creation, Nikon "Interference Phase Contrast" (or, more simply, "IPC"). Not even a hint of "Color" anywhere in the official name…!

Now, like the AO system, the Nikon IPC system did not require special objectives but relied on the standard Nikon Achromat types of the day. And, this was probably its Achilles Heel – the excellent Nikon Plan, Fluorite and Apo objectives were simply not supported!

Thus, Nikon offered an expensive accessory that could not be used with Nikon's own highest-quality optics, only with their  lower-end "classroom" type Achromats. What were they thinking…?

But inability to use quality optics was not the only flaw. Since most of their S-series scope had somewhat limited illumination capabilities (the S-Kt and S-Ke models were exceptions), the system relied upon extra-wide openings in its condenser in order to compensate for the light loss within the system. This was coupled with the use of rather generous phase-ring widths in the optical head, a step quite likely taken to simplify system adjustment by allowing a little more "slop" in the optical alignment.

While these measures did allow the system to be used on the lower-cost models, and by relatively unskilled users, it also placed some limitation on the ultimate quality of the resulting phase images.

That said, this is not meant to imply that the Nikon system provides unsatisfactory images – far from it! (See inset detail in the sample image below – click on image for larger versions:)

In fact, with very little effort it is possible to create a wide range of very colorful and highly-detailed phase contrast images using the system. (See end of this post for additional sample images.) In fact, it may be more correct to classify the Nikon IPC system as "artistic," rather than as a primarily "scientific" device.

Now, compared to the rather monsterous AO Polanret system (which weighed in at nearly 15 lbs!) the compact Nikon Interference Phase Contrast system is a marvel of Japanese optical and mechanical technology and innovation. The basic unit, responsible for the phase contrast and interference color functions is less than 10 inches long and sits comfortably (and horizontally) atop nearly any Nikon S-series biological microscope. The matching Interference Contrast condenser is simply a basic Nikon S-series phase contrast condenser with special, extra-wide ring openings, matching those in the optical head.

Control is much simpler than on the AO Polanret unit with a simple horizontal slider-bar holding a set of phase rings and a pair of control knobs to set the optical parameters. Using these the user can set the equivalent of either Bright or Dark contrast, as well as a wide range of contrast amplitudes. (Essentially, one knob selects the background color while the other chooses the level of contrast, although there can be some limited interaction between them at extreme settings.)

One additional, but very important distinction between the AO and Nikon systems is, as one might well expect, that while the AO system is intended for "infinity" type objectives, the Nikon system (naturally) is intended for use with "finite" (or, "160mm") objectives.

Perhaps because of this the Nikon system seems far more amenable to adaptation for use with more modern optics than the simple 1960s-era Achromats for which it was designed. In fact, with little effort the Nikon IPC system functions very well with some of Nikon's best Plan objectives, including many of the CF and CFN Plan Achromats. (And its use is not limited to the old, black S-series either, as it works quite well on a number of more more stands as well, including even some non-Nikon scopes, such as the Olympus BH-series – assuming a proper mounting adapter is used.)

Note that the images below were created simply to demonstrate some typical IPC system image color possibilities and so may not be truly representative of its resolution capabilities…  (Click on image set below for larger versions.) 

Note that focus has been altered very slightly between above images to emphasize different details. 

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Secrets of the AO Polanret System – Part II.

Note: This is an interim release. Because of the total length, Parts I & II are planned to be combined into a single document, with additional photos, as a .pdf file. This will allow continued online viewing as well as easier download and (if desired) printing. 

For Part I, see the post of January 28, 2017, below. 
For sample images, see the post of February 7, 2017. 

Part II

To understand a system like the AO Polanret, or, more specifically, to understand the need for it, you have to recognize that the Phase Contrast technique is primarily affected by just two characteristics of the observed specimen: Refractive Index (RI) and the Thickness of the specimen. (Refractive Index is merely an expression of how much light is slowed by its passage through a material. As an example, for Water, the RI = 1.333 – meaning that light travels only 1/1.333 as fast through water as it does through Air, RI =1.000.)

The basic appearance of Phase Contrast image depends upon the difference in RI between the specimen and the surrounding material. Since nearly all biological material has an RI greater than that of Water, the various components of a specimen in Water will typically be displayed as darker than the background.

However, this is only true for phase objectives of the Dark (or Positive) contrast-type – the most common type. For objectives of the opposite type, Bright (or Negative) contrast. the specimen and its details will typically be brighter than the background.

Now, while all that may seem quite simple, in practice things are not always so straight forward…

If, for example, cells are in a culture medium which has an RI that is nearly the same as the cell bodies, then the level of contrast provided by normal phase objectives (typically, "Medium" contrast) may be inadequate. This is because, if the RI difference between the specimen and is surrounding medium is low, then the contrast level will also be low, and so an objective of higher-than-normal contrast may be required to show the specimen properly.

However, under the same circumstances, consider what happens when the internal contents of the cell are examined. Here, the minute bodies of interest (e.g: cell nucleus, or its contents) may well have an RI that is quite different from the surrounding cell material. In fact, due to the normal variations in RI within the cell, the potential image contrast levels can easily vary quite widely from location to location.

This means that the ideal contrast characteristic for the phase objective can easily vary as well.

If the objective contrast is too high, then details may be obscured, and, if the objective contrast is too low, then some details may not be visible at all. Thus, for the most effective observation, the objective's contrast characteristic might best be made variable, such that the user could adjust it as necessary for each specific observational situation. This is the premise for the AO Polanret system (and similar "Polanret" type systems).

To examine this point further, let's consider a case perhaps more familiar to many users… 

Consider, now, the use of Phase Contrast on "mounted diatoms."

Historically, these specimens (RI=1.46) have been mounted in media of high RI (RI=1.72, or even higher). This was done to improve the visibility of the delicate markings on the specimens, a technique dating from long before Phase Contrast was invented.

However, when viewing such mounts with Phase Contrast we have two undesired effects: (1) the RI relationship between the specimen and the media is "reversed" (e.g: RImedia >> RIspecimen), and, (2) inherently high contrast within the mounted object.

Now, the first issue results in an "reversed" phase image (e.g: when "Dark" type objectives yield a "bright" image), and the second issue often results in a "phase halo" that may easily mask the very details being sought! These issues have led some makers to seek technical solutions (such as "B minus contrast," or other specialized-contrast phase objectives) in an effort to achieve a more acceptable image. (Another, frequently-used alternative is to simply abandon the use of Phase Contrast altogether, and opt instead for a different method, such as COL or Oblique Illumination.)

But, with a Polanret system, the user only needs to adjust the controls so as to "dial-in" the most appropriate levels of Amplitude and Contrast for the particular specimen at hand!

All this now brings us to the point of discussing just how these changes are produced by the Polanret system…

Actually, there are two main sections involved – an "image transfer" (or, "relay") optical system, which shifts the objective's rear focal plane into the optics of the Polanret system, and a Phase processing system which permits optical manipulation of the images from the objective, based on phase differences.  

Also, understand that there are actually two images formed by the (any) objective: 

  

(1)  the Intermediate Image, which is located near the eyepiece and forms the actual image of the object, and, 

(2)  the Rear Focal Plane image which, in this case, holds an image of the Phase Condenser's annular ring. 

In an ordinary Phase Contrast objective the Rear Focal Plane is also the location of the Phase Plate which is responsible for determining the characteristics of the final Phase Contrast image. But, in the Polanret system which uses non-phase objectives, this plate is not there. Instead, it is positioned inside the Polanret unit where the system's optics "relay" the necessary Rear Focal Plane image to it. 

Now, the Phase Plate in the unit (typically, one for each objective magnification) differs from those found in ordinary Phase objectives in that it is comprised of a pair of concentric rings made of polarizing material. These are arranged such that, when placed between a Polarizer and Analyzer, rotating the Polarizer will darken one ring or the other, selectively. This feature allows adjustment of the Amplitude characteristic of the system. 

This plate is followed by a quarter-wave plate and adjustable Analyzer, which allow varying the Phase Shift ("Phase change" sensitivity) of the system. Thus, both of the important characteristics of the system are made user-adjustable  and the user may select and degree of Bright or Dark contrast, or any degree of phase change sensitivity within the system's limits.  

Note that the unit's optics are arranged such that the Intermediate Image is unaffected by these operations, except for the addition of Phase Contrast information to the final image. 

So far, so good – but now it's time to consider Reality!  

In order for all of this to function properly, certain operating conditions must be met:  

   

  (a) The objective to be used must be precisely positioned relative to the matching Phase Plate in the unit. This means that the objective must be almost exactly concentric with the Plate, and the image of the Condenser Phase ring in the objective's Rear Focal Plane must be almost exactly the correct distance from that same Phase Plate in the unit.  

   

  (b) Now, the objective's concentricity is controlled by mounting of the Polanret unit to the microscope  body (over which the user has little control – this is mostly a matter of manufacturing tolerances), AND the centering adjustment of the microscope's nosepiece (which the user can control, somewhat). However, the nosepiece centering needs to be accurate within a fraction of a millimeter, if the centering function for the Condenser Phase Rings is to have the proper effect.  

   

  (c) The distance of the Phase Ring image (in the Rear Focal plane) is controlled by several factors, the design of the Phase Condenser and the Phase Rings being largely beyond user control. (The exact focus of the Condenser is under user control, but has only minimal effect.) Phase ring centering is, of course, under user control, as above. 

   

  (d) Surprisingly, what is critically important is the adjustment of the microscope's stage height! This is because with AO's nosepiece focusing, the stage height is what determines where the objective will be positioned, relative to the Polanret unit, when the object is in focus – and that is the only point that matters when considering the location of the objective's Rear Focal Plane! If this height is off more than a millimerer or two, then the image of the Condenser Phase Ring will not be in the correct position within the Polanret unit. 

   

  (e) You need a whopping amount of light to run this thing! The use of Polarized light technology within the unit exacts a rather severe penalty in terms of light throughput. The Series H10 scope boasts a 20Watt Halogen illuminator, which puts out roughly 400 lumens. That is barely adequate for the system in its most transparent modes. But, if you expect to run the unit in more "normal" modes you simply need more light!

The H20 (and 120) microscope models feature illuminators based on 100 Watt lamps – good for about 3200 lumens (if you pick the right lamp). This is just about sufficient to run the unit properly, but can be a bit marginal if there's a beamsplitter in the image path (as for photography) and/or a moderately dense color filter in the illuminating path (to reduce color artifacts from the Planachromat objectives, for example). 

And as entertaining as all this might seem, it gets even more so when the job includes aligning the basic microscope from scratch and working without access to the proper Condenser… 

So, taken all together, it is now easy to see why AO recommended against using anything but a factory-aligned system. 

There is no known documented procedure from AO to allow users (or even AO dealers) to make all the necessary adjustments to permit the Polanret system to operate as intended – although a practical, user-level system alignment procedure is currently being developed by this author.

Despite its complexity, and minor issues, the AO Polanret stands as a potentially useful analytical instrument. However, it does not stand alone as there is another Variable Phase Contrast system out there – as you will soon discover – and one which promises much more colorful results! 

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Looking Forward -- beyond Polanret?

As we prepare to wrap up the discussion of the AO POLANRET system some of you may begin to wonder whether that device is just one-of-a-kind, "oddball" invention, or whether there may be other, similar instruments lurking out there, perhaps by other makers?

Perhaps the image panel below will give you a clue…

Stay tuned to this Blog for answers!  

(Click on the image panel above for larger versions, then use the Return or Back button in your browser to return to this page.) 

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Images from Polanret!

As efforts continue to shift this Blog into more visual presentations, here two sets of images made from a single diatom at random setting for the instrument.

The in the first set, immediately below, colors from the camera images have been left largely intact. However, note that the colors are, to some extent, due to the interactions between the residual phase information from the instrument and the internals of the digital camera, and are not present to the same extent in the visual images.  (Such effects are common in phase contrast imaging and are not peculiar to the Polanret. They may. however, vary somewhat with the specific camera used, in this case a Canon 2400is.)

The next set contains exactly the same images as above, but with the extraneous color information suppressed. This provides a better representation of the actual visual appearance of the specimen.

This set more clearly demonstrates both the Polanret's ability to vary the image contrast level continuously and the ability to switch seamlessly between Bright and Dark contrast modes. (Click on an image set for larger versions, then use the Back button in your browser to return to this page.)

A more technical discussion of the Polanret system will follow in a day or two… 

Also, see the Blog Post of January 28, 2017 for an earlier discussion of this system. 

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