Introduction . . .

This is a brand new blog, by a brand new blogger. However, some readers may recognize this blog's title, taken from a series of books of the same name. Unfortunately, time has a way of gradually making printed material all too quickly outdated -- especially these days -- and so, this blog was created partly as an attempt to address that issue.

As we move forward from here on-going efforts will be made to transfer selected content from the Better Microscopy books series into this new format, not only to provide to provide more effective distribution, but also as a means for making timely additions and overdue updates to that material. In addition, much previously unpublished material is now planned to be released, including high-resolution color images.

The current plan is to aim for a content mix that is both interesting and educational -- perhaps even inspiring -- and which will address the needs and interests of a wide range of user levels, from beginner to semi-professional. With more decades of Microscopy experience than I care to admit, I hope I will be able to contribute something to others in terms of both knowledge and enjoyment.

I hope you find something of interest in new undertaking as it takes shape and gain much from its content, now and well into the future!

Just beware of the occasional attempts at humor...

Thanks for visiting!

Sunday, February 19, 2017

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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