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 Unfortunately, time has a way of gradually making printed material all too quickly outdated -- especially these days -- and so, this blog was created partly as an attempt to address that issue.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 In addition, much previously unpublished material is now planned to be released, including high-resolution color images.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...

Note -- The new e-mail for issues concerning this Blog is:

Thanks for visiting!

Tuesday, June 27, 2017

Summer Hiatus...

Due to increased obligations in other areas, it will not be possible to update this site until after Labor Day, Sept. 4, 2017

For now, you may expect normal updates to resume shortly thereafter... 

Also – note that the contact e-mail listed above will not be monitored during this period.

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Friday, June 9, 2017

3D Image Enhancement made Easy – Part IV.

Combining contrast methods

In prior posts we described a simple method, "Radial-3D Masking," for microscope image enhancement with could produce both contrast enhancement and a perception of "3D" (relief effect).

However, while useful on its own, it is also possible to combine this method with at least one other popular contrast method, namely, "Circular Oblique Lighting", ("COL"). This new method has the potential to not only further improve contrast levels in the COL image, but also to add a 3D effect.

Unfortunately, the results of this combination, in some instances, can become a bit excessive. For this reason we will introduce a more moderate version of the Radial-3d Method, termed here the "Diffuse Half-Mask" ("DHM") method.

This is. basically, little more than half of the Radial-3d Method, using only a single diffusion strip, rather than two. It produces results that are a bit less dramatic than the Radial-3d Method, but which may be better-suited for combining with other optical contrast methods. Details of this method, as well as the original Radial-3d Method are shown below:

Examples of just how simple and effective these "combo" contrast methods can be are depicted in the photo set immediately below.

The image set on the left depicts increasing levels of "3D" effect, beginning with 'Brightfield' (no 3d enhancement), then 'DHM' (moderate 3D enhancement) and, finally, 'Radial-3D' (full 3D enhancement).

The image set on the right depicts the same series, but now in combination with COL.

Click anywhere on the above image for larger versions. 

In general, these methods appear to be most effective when applied to subjects which exhibit fine structural detail. Still, they may also be useful with less demanding specimens, as shown in the next photo set:  

Click anywhere on the above image for larger versions. 

Note that when the image contrast is already high (e.g: Phase Contrast) additional contrast enhancement using these methods is likely to be rather limited, if at all. 

In any case, thoughtful experimentation is highly recommended! 

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Tuesday, May 9, 2017

3D Image Enhancement made EASY – Part III.

Comparing Simple Contrast Methods – cont'd.

Part I (April 25th.) of this series introduced a new method of image enhancement (simple '3D') and gave examples of its use.

Part II (May 3rd.) offered further examples of results using this method, particularly as compared to Brightfield and COL.

Part III continues this effort with examples at higher magnification and higher resolution.

Now, in casual microscopy, considerable time is often spent using the 40x ("high dry") objective. This lens offers the combined advantages of reasonably high magnification and resolution, decent working distance, and ease-of use. Also, almost every standard microscope has one. Further, more than 90% of what maybe seen (at high magnification) with Transmitted Light Microscopy may be seen using this type of lens. So, it seems only proper that we should determine just how well the 3D Mask method works with this lens…

In the first set, below, we compare results obtained using Brightfield with those using the Radial 3D mask method. This  set shows the same diatom photographed using each of these methods, with the results adjusted to approximate the actual visual appearance of the object. (If anything, these images understate the advantages of the 3D method, in part due to loss of image quality due to the use of the JPEG image format during image processing.)

Click anywhere on the above image for larger versions. 

Note that the measured width of diatom used in these photo is approximately 21 microns, which results in a calculated 'dot spacing' of about 1.0 microns. As this is reasonably within the resolution capability of the NA 0.70 objective, the performance of the objective itself should not be a limiting factor in these tests.

Not apparent in the photo set is the slight loss in overall image brightness associated with the 3D mask. However, this loss is a small penalty to pay for increased image contrast and resolution.

Radial 3D versus 'COL' – more surprises? 

In the second set, below, we compare the Radial 3D mask method with COL, a currently popular alternative method of contrast enhancement. Once again, the relative levels of image brightness are not preserved in these images, but here, COL typically suffers a much greater loss of overall brightness (typ. 3 to 4 times greater) than does the 3D mask method.

Click anywhere on the above image for larger versions. 

Here the 3D method appears to be at a disadvantage, at least initially, but only because the Condenser Iris was fully open (e.g: 100%).

However, as shown in the third photo set (below), this limitation is easily overcome by simply reducing the Iris opening.  This results in a sort of "variable contrast" mode, where the overall image contrast is readily controllable by means of the Condenser Iris. For Iris openings down to about 60%, there is very little loss in resolution, while even smaller openings (down to about 40-50%) may be used to achieve further increases in image contrast.

Click anywhere on the above image set for larger versions. 

Note that the COL method has no equivalent mode!

With COL results are fixed, as determined by the annulus diameter and opening width. To adjust contrast with COL either the entire Iris must be exchanged or a variable condenser, such as the scarce (and costly) Leitz 'Heine' Condenser, must be used. (Note that the Heine condenser will typically fit only a few older microscopes, whereas the Radial 3D mask method may be used on nearly any microscope equipped with a standard Condenser!)

Finally, remember that, with COL, usually the entire condenser annulus (or the Heine condenser setup) must be changed with every change of objective, while, with the Radial 3D mask method, only a simple Iris "tweak" is all that is typically be needed, if at all.

Next, in Part IV of this series, we will explore some of the unique possibilities for even greater image enhancement using the Radial 3D mask method…

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Wednesday, May 3, 2017

3D Image Enhancement made EASY – Part II.

Comparing Simple Contrast Methods

A recent post (April 25, below) has revealed a very simple method of optical contrast enhancement, "Radial 3D Effect", which also adds a measure of 3D-like effect to an overall contrast improvement.. The method is very easy to implement and to use, functioning with a wide range of common objectives and yet requiring no special adjustments with objective changes.

Yet, in spite of these these and other operational advantages (described below), the basic question remains, "Just how well does this new method work?"

In this post we begin to address that question, starting with a direct comparison of images from this new method with those from two common alternative methods: (a) Simple Brightfield, and (b) Circular Oblique Lighting (or, COL). (These other methods also share the traits of simplicity, ease-of-use, and of not requiring any special objectives.)

So, how do these methods actually compare? 

To see, we can begin by examining the image set below… 

Click anywhere on the above image set for larger versions. 

The top image is in Brightfield mode, with illumination by the Kohler Method, with the Condenser Iris set to about 100% of the full objective aperture. This is to serve as the "Reference" image. It reveals a reasonable amount of detail but one could hope for greater contrast. Also, the image appears "flat," revealing little of the object surface texture.

The middle image is basically identical, but with the addition of the Radial "3D" Effect mask discussed earlier. Note that this image shows not only increased object contrast and detail, but also reveals object surface contours, especially evident in the specimen on the left.

The final image was made by replacing the "3D" mask with a standard Condenser phase ring (annulus) to produce COL contrast. Note that here there is also increased contrast (relative to Brightfield), but the "3D" effect seen in the middle image is absent.  However, much unlike the Radial 3D Mask method, image results with COL can be quite dependent upon the exact choice of annulus, as well as 'annulus-specimen' matching. Thus, with COL, each objective potentially requires using a different annulus. (The 'Radial 3D Mask' technique has no such sensitivities.)

One important factor, not shown by the above images, is the difference in overall image brightness which occurs with each of these methods.

Brightfield, of course, offers the highest image brightness level, with the Radial '3D' Mask method running second with somewhat less than half that level (depending on the exact diffusion material used). The COL method, however, is far behind this with overall brightness at 15% or lower, depending upon the size and width of the Condenser annulus selected. (Note that some specially-made COL rings may support slightly higher overall image brightness levels.)

Condenser iris Effects

Both Brightfield and Radial 3D methods also support a basic level of user control over "depth-of-focus" in the specimen plane, simply by adjusting the Condenser Iris opening. Reducing the opening, even by a small amount (e.g: from 100% to 80%) can result in a significant increase in not only depth-of-focus, but also in overall image contrast. These effects are depicted in the image set  below:

Click anywhere on the above image set for larger versions. 

As expected, reducing the Condenser Iris opening in either Brightfield or 3D mode provides a slight increase in image contrast, but in the 3D mode this more significantly enhances the apparent depth-of-focus for the specimen. In general, Iris openings from 100 percent open, down to 50 or 60 percent open, can prove useful. At less than about 50% open, image resolution can begin to suffer noticeably.  

Unfortunately, the COL method (described above) does not directly support such control – with COL the only recourse is to switch to using a smaller diameter annulus, which often can have unpredictable and undesirable effects on the overall appearance of the specimen. 

Be aware that the image set presented above is not intended as definitive basis for comparing these methods, but merely as a basic indication of the results produced by each method. More precise imaging techniques for these three methods, as well as for some additional methods, will be needed to permit extending this evaluation to a more definitive level

Note that the above images are limited by minor variations in focus between modes, as well as a slight residual camera motion which may obscure finer detail. Both these issues will be addressed in the test setup before any additional comparison photos are produced.

This comparison will continue is a subsequent post, or two…

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Tuesday, April 25, 2017

3D Image Enhancement made EASY – Part I [Updated April 27]

Click on image above for larger version.

Recent posts covering the Goerz '3D' Condenser have sparked interest in creating an alternative approach suitable for use with ordinary (Abbe-type) condensers.

The goals of this new effort were: (1) Low cost and Ease of construction, (2) Improvement in image contrast and apparent object depth, and (3) Ease of construction and use..

It is important to remember that the goals here are not maximum image contrast nor maximum resolution, but rather to simply achieve meaningful improvements in both (relative to normal Brightfield imaging) by use of the simplest and most generally applicable method possible. 

The method presented here appears to satisfy these goals and results in a simple addition which may be easily applied to an ordinary microscope condenser and, unlike other methods, (e.g: oblique illumination or phase contrast) may be used without any additional/special optics or concern for adjustments during use.

Also, since the light loss is minimal, the device is suitable for use on instruments having limited illumination and/or at higher magnifications than these common alternatives.

The device is constructed as a simple disk having an array of three diffusion segments, which modify the illumination in a predetermined manner and is essentially the same for all objectives, from 10x to 40x, or more. The "radial" approach both eliminates the need for adjustments when changing objectives and also allows the Condenser Iris to remain fully usable as an additional means of image control.

The construction of this Condenser "Radial Diffusion mask" is depicted in the following diagrams:

Note: Click on any above image for a larger view. 

Installation of the mask, its preferred use and example specimen photos will be presented ASAP… 

* * * * *  
Basic Construction Notes:  

Most of the construction details for this mask are non-critical. If the tape intersection point is reasonably close to the optical center of the Condenser when installed, then the device should function as intended. The precise angles are also not critical – anything approaching 90-degrees should work just fine. 

The specific type of "matte tape" to be used is also not critical. Ordinary, generic "dollar store" types seem to work about as well as anything else. As long as the tape width is about 3/4" then disks up to about 37.5mm diameter would seem feasible. Scotch (brand) Matte finish tape, however, apears to be superior to their "Satin finish" variety, which seems to exhibit less desirable diffusion characteristics, at least for this use. 

The acetate disk material is also non-critical. The basic requirements are that it be basically clear, self-supporting and thin enough to be trimmed with scissors. However, if your Condenser accepts a clear Daylight filter, then this might  serve as an alternative substrate. 

The positioning of the Opaque segment was chosen so as to not create uneven illumination with objectives of less than about 40x. However, it may be positioned closer to the disk center if contrast enhancement is desired for objectives in the 16x to 25x range also. In this case, about a third, instead of halfway, from the center to the disk edge may be tried. However, if detailed examinations with 40x and greater objectives are not the primary use, then the Opaque segment may simply be omitted with minimal loss in overall performance. (It can always be added later, if desired.) 

The Opaque segment may be created with a single strip of black PVC tape (plastic electrical tape), located on the reverse side of the disk, so it does not interfere with the matte segments. This allows the opaque segment to be re-positioned, if desired, without interfering with the other segments. 

* * * * * 
An External Mask

One common problem with many modern microscopes is that they often use Condensers which lack any sort of proper filter holder. Naturally, this sort of shortcoming might seem to be an issue when attempting to implement even something as simple as a Radial '3D' Mask… 

Fortunately, the design of this Mask is remarkably forgiving when to comes to placement in the Condenser, even to the extent that it can be perfectly acceptable to place the mask outside the Condenser! This can be especially true for use with objectives of >10x, where the loss in Mask performance is basically trivial. (For objectives of ~10x, the loss is most typically limited to a slightly uneven background light level.) 

For Condensers which have a significant bottom flange (e.g: most base-mount types), external mounting of the mask should be possible, as long as the width of the flange's bottom surface is sufficient to allow retention of the tape strips. 

The tape strips are simply applied across the width of the flange, such that the "inside corner" formed where the strips overlap is located approximately in the center of the objective aperture, as viewed from the eyepiece position. (For the most accurate alignment, with the least difficulty, use of the 10x objective is suggested for this process.)  The two diagrams below depict both proper tape placement and proper overall mask alignment (centering).  

(Note: Click on either image above for a larger view.)

Be aware that the tape placement should be performed only after the Condenser has been properly centered in its mount, as for Brightfield use. Once the Condenser itself is centered, any centering mechanism present should not be used to correct any misalignment of the tape strips. If the overall positioning is unacceptable, then the strips should simply be removed and re-positioned properly. Just remember that exact alignment is not essential for successful use!  

The photo below shows typical results when using the External '3D' Mask method:
(Image cropped and downsized from 16Mpix JPEG original. No sharpening used.) 

(Note: Click on above image for a larger view.

Additional photos using the Radial '3D Mask' will appear in the next post – so, stay tuned…

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Thursday, April 13, 2017

Coming soon: '3D' Imaging, for Free?

The recent examination of the Goerz '3D' Condenser (below), and its performance, seems to have raised the dual issues of, "Can we do any better?," and, "Can we get '3D' effects from an ordinary Condenser?"

Further testing demonstrates that there may be qualified "Yes!" answers to both of these questions.

For details, '3D' images of typical subjects, and complete instructions, just stay tuned to this Blog… 

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Wednesday, April 5, 2017

The Goerz 3D Condenser – many Surprises?

The primary mystery surrounding the Goerz 3D condenser is, "What is this thing supposed to do, and, how is it supposed to do it?"

Fortunately, careful testing of this 'Mysterious' condenser has led to a better understanding of its intended workings. However, not only that but this testing has also uncovered a number of surprises surrounding its design and use.

All of this hinges on understanding the functions of strange plate located in built into the bottom of the condenser, a few mm above the Condenser Iris. This plate has a clear central strip (or, 'slit') about 3.5mm wide separating two partially-silvered areas, which exhibit about 25% light transmission, and together fill the remaining aperture area.

The key to understanding this Condenser seemingly lies in understanding the intended function of this central slit.

To better appreciate this, consider a normal condenser where the Iris is closed down such that the objective aperture (as seen in the rear of the objective) is only about 1/4 to 1/3 open.

Under these conditions the effective NA of the objective is reduced considerably (to just a bit more than 1/2 of its rated value, actually), the image contrast is increased, and also the 'depth-of-focus' in the Object plane is increased somewhat. However, all this is accomplished at the cost of object resolution, which is reduced to about 1/2 of its maximum value. Even worse, this form of masking results in an image that is objectionably harsh and prone to various undesirable artifacts.

Now, consider what happens if the normal, circular Iris opening is replaced with a slit opening of the same width, but whose length spans the full aperture of the Condenser…

In this case we might expect full resolution in the direction of the length of the slit but also an increased depth-of-focus in the direction of the width of the slit. In this way we might hope to achieve improved image contrast and 'depth-of-focus' while sustaining only minimal loss of resolution. However, although image brightness is somewhat better than with the minimally-open  iris method (above) the image is still a bit darker than normally desired and some image artifacts may still be observed.

Now, making the "sides" of this slit semi-transparent, instead of fully opaque, would seem to offer the prospect of moderating the least-desired effects – increasing image brightness and minimizing possible image artifacts, while also allowing the device to be used "normally", if desired, with little loss of imaging performance. This seems to be the Goerz approach, based on examination of the device and initial testing.

So, this is the current thinking on how the Goerz might have been intended to work – no magic, no Mystery; just some basic physical optics!

In other words, the "3D" effect seems to be mostly marketing hype for a slight, directionally-biased increase in 'depth-of-focus' resulting from the use of a somewhat unique, built-in slit aperture.

Since it was not practical to remove the critical 'slit plate' from the Goerz condenser, testing of this concept was performed on a similar (but totally normal) common NA 1.25 Abbe-type condenser.

Different Slit-type masks were compared with normal Bright field and with common COL-type masks in a preliminary effort to determine whether Slit-type masking provides any observable benefits, and, indeed, any detectable "3D" effect.

The initial results of these tests are summarized in the photos below…

(Click anywhere on the above image panel for a larger version.)

The there are two big surprises revealed in these photos: (1) despite its novel design and construction the Goerz design actually performs rather poorly as a "3D" device, barely equaling the depth-of-focus performance of a number of more common alternatives (as shown), and, (2) the real surprise lies in the number of alternative methods which easily equal, if not surpass, the performance of the Goerz device, both in terms of resolution and image contrast!

In other words, despite its novel design, elaborate construction, assortment of optics (optional tops) and unique optics-offset adjustment, the device seems to do nothing which cannot be accomplished by simple alternatives.

It only genuine attractions are its low-power (NA ~0.5) Brightfield mode (using no top), and its Darkfield modes, all of which are mildly compromised by the presence of the non-removable "3D" slit gizmo embedded in the basic design.

As far as possible "alternatives" are concerned, it may be noted in the photos that these all seem to offer results which are basically similar, differing mainly in the presentation of image details, as well as ease-of-implementation and ease-of use. All of this, of course, being potential food for a future post or two…

See the post of March 28, 2017 (below) for more on the Goerz '3D' Condenser. 

* * * * * 

Tuesday, March 28, 2017

The mysterious Goerz '3D' Condenser! [Updated 30 Mar 17]

A little-known child of the "Flower Power" era, this unique device was served-up with a thick layer of marketing hype, such as "plastic field," and similar nonsense. 

Sold by the C. P. Goerz American Optical Company, the device had nothing to do with the "real" American Optical Co., and the cryptic labeling was a cause for much confusion (and perhaps even some litigation). 

Still, it seemed to offer at least some promise as a potential, low-cost alternative to more the established methods, like Phase Contrast and DIC. 

But, what Secrets does it really hold? And, will it really measure up to all the wild claims made for it? 

Stay tuned to this Blog to find out!  

 * * * * *  

The sample unit (shown above) was contained in a fitted wooden box, together with an aperture viewing telescope, a set of three slip-on tops and an adapter ring for the mounting sleeve. An iris diaphragm and swing-out filter holder were attached to the base of the main unit with three small screws, which would allow the iris unit to be rotated, if needed, and then locked into position. 
The basic condenser was provided in a European-standard 37.0mm sleeve mounting. Unfortunately, instruments using this size mount are not common in the US and this diameter is a bit to large to fit the JIS-size sleeve mount (36.75mm) which is far more common (e.g: Nikon-S, Olympus BH and CH, etc).  
However, included in the set was an expansion sleeve (adapter) which enlarges the Condenser mount size to 39.5mm (the old Leitz/Wild size). Reportedly, there is also an adapter sleeve for 38.75mm mountings (RMS, B&L) but this was not present with the sample tested. 
The condenser unit itself appeared to be a conventional 3-lens design, with the third lens provided as a slip-on option. A brightfield top of NA 1.2 ("A 1,2") and a pair of darkfield tops ("A 0,65" and "A 0,8-1-1,2" – as shown above) were included. (The later top presumably provided NA 0.8-1.0 "dry", and 0.8-1.2 when used with oil.) Oddly, there were only two positions in the fitted case for such tops, although a total of three were actually provided. (There were also three slots for filters, but none were included with the sample unit. These were reportedly Blue, Green and Ground glass types.)  
By itself (with no top) the condenser appeared to have a NA of approximately 0.45, or so, since it filled the rear aperture of a 20x/0.40 objective but only about 2/3 to 3/4 of the aperture of a 40x/0.65 objective. When the A1,2 top was added, the rear aperture of the 40x/0.65 objective was easily filled, with the condenser iris a bit more than 1/2 open. (Details of the internal optics of the device will be presented in the next update.) 

Still, the main question to be answered remains, "What, if anything, does this thing actually do?" 

And that can only be answered by actual testing…  

* * * * * 

Saturday, February 25, 2017

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. 

* * * * *  

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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