Practical Digital Photomicrography

  Photography Through the Microscope for the Life Sciences

Brian Matsumoto PhD, 2010


ISBN 978-1-933952-07-9   Published by Rocky Nook Inc., Santa Barbara CA

Recommended retail price: US - $69.95, UK - £53.99.

Typical street price (Amazon): US - $41.70, UK - £29.41.



New and ever-more sophisticated models of digital cameras succeed each other at bewildering speed, and their capabilities continue to expand at such a rate that any book about techniques involving them is likely to be seriously out of date before the ink is dry. Nevertheless, the publishers of this work have apparently found the courage to bite the bullet and present a work which will seem a godsend to the growing army of photomicrographers who are attracted by the peculiar advantages that the digital medium offers them.                  

When the review copy arrived, my first impressions were of a very attractive-looking, rather slim (175pp) volume of large-ish (21cm X 26cm,) format with a striking cover. It appeared that the publishers had given a similar degree of attention to the internal layout though, to my jaundiced eye, the very wide margins and oversized illustrations seemed to constitute a serious waste of space, while a glance at the table of contents was enough to suggest that the organisation of the text might prove a trifle chaotic!

The standard of the illustrations, photographic and otherwise, is generally very high but unfortunately the photomicrographs on pp 36, 48, 78, 154 – which one presumes were chosen as ‘chapter openers’ on account of their aesthetic appeal - are of less impressive quality, with rather low contrast, slightly muddy colours, vignetting to varying degrees and suspect white balance. It is perhaps ironic that these defects could have been removed in minutes by the healing powers of Photoshop as outlined in Chapter 9. For a work aimed at a scientific audience it is odd, to say the least, that the photographs show no indication of scale – neither by bar or statement of the Horizontal Field Width (HFW).

The blurb on the rear cover states that the book is intended for “professionals in the life sciences and photography, educators and students as well as photography enthusiasts”, and makes much of the author’s scholarship and ‘decades of experience’ as a microscopist and photomicrographer who will provide ‘an invaluable guide to learning the skills needed to obtain professional quality images through the microscope.’ The more elevated a publisher’s claims, of course, the greater the potential for disappointment and, as anyone who has had much contact with Reality will tell you: it seldom lives up to expectation. In the case of a ‘reference’ book such as this I tend to measure its shortfall as much by the number of questions that remain unanswered for me when I have finished reading it as by the errors and other sins of commission I may have noted along the way.

In this case, there remained for me quite a battery of questions, many of them fundamental, which might justifiably be aired - and rather too many critical observations. Here are some of the most pressing:

While brightfield illumination, darkground, Zernike phase contrast and Nomarski DIC, by transmitted light, and epi-fluorescence are treated with varying degrees of thoroughness, there is no mention whatsoever of annular illumination (COL), oblique illumination, Hoffman modulation contrast, Rheinberg illumination, epi-illumination techniques other than fluorescence, or of crossed polars techniques other than DIC. Are none of these of value to Biologists? If not, what about the rest of the publisher’s target readership?

‘Specimen preparation’ is specifically listed in the blurb as one of the skills which the author is supposed to ‘demystify’ but I could find little or no trace of it.

The sections on setting up Kohler illumination, (p9 et seq., 28 et seq.)) contain no mention of focusing and centring the illuminant so that it is sharply imaged across the back focal plane (BFP) of the objective. Are these adjustments not available on some recent research instruments?  An important reason for the very existence of Kohler’s technique is that it keeps the image of the lamp filament or arc safely out of the way of the object by locating it there.)

 Users of high power water immersion lenses need to become masters of their correction collars (p27)  but is the rather ‘suck-it-and-see’ technique described on p26 and again on p110 the only means of adjustment known to the author?

If some researchers prefer to use their very expensive NA 1.4 objectives with their condensers ‘dry’ because of the mess caused by oiling them (p31), this practice surely negates the whole point of having such luxuries? In any case, we are told (p31) that “For most transmitted light work, oiling …is unnecessary. With stained slides, the loss in performance is slight to the point where many do not notice it at all” – so why bother with them? Spherical aberration and reflections arising between the underside of the slide and the condenser, both potential sources of glare, are less obvious but still real hazards which result from using immersion condensers dry.

Though probably not the weapons of first choice, it is possible to use some ‘compact’ digicams for the production of high quality photomicrographs (pp54, 55). The lenses of suitable cameras, when placed in near contact with the eyelens of a high eyepoint eyepiece, are capable of projecting an image large enough to fill the camera’s sensor.  Examples of such cameras which I myself have used, or seen used successfully, are the ‘twister’ Coolpixs (990, 995 and 4500), the Coolpix 5000, the Sony DSC W5 and W7. (When an unsuitable camera is placed with its lens just above the eyepiece and ‘zoomed in’, the circular spot which contains the image begins to expand and then contracts before it is large enough to fill the screen.).      

The ‘Nyquist criteria’ - which apparently limit the range of useful magnifications achievable with a digital camera – must be of overwhelming interest to photomicrographers  (who are possibly coming across them for the first time here) but they appear (p42), without preamble, in a section devoted to the ‘1000x NA Rule’ (the theoretical basis of which is itself not really explained). If there is any aspect of his subject that the author needs to explain in detail - and immediately - this is it, but all we get is “The principal (sic) of the 1000x NA rule is the same with the camera. You can increase the magnification to a level at which no finer detail is captured by the camera. If your camera has photodiode size of seven microns, you can apply the Nyquist criteria to come up with a camera magnification rule: in this case, the maximum useful magnification is 70 times the NA.” What is the ‘rule’ then? What if the diameter (?) of your camera’s photosites is not 7 micrometres? The anxious reader will search in vain for an explanation of what these ‘criteria’ are and how precisely they will affect his or her future practice because what follows is a new section entitled “Magnification Changer and Eyepiece”! This is not to say that there is no further mention of the ‘70X Rule’ because the above statement actually reappears, almost word for word’ on the next page, followed by worked examples relating to modern (Olympus?) microscopes fitted with bespoke ‘professional’ digital cameras. However, it is not until page 85 that an actual explanation appears, this time under the heading ‘Pixel (Photosite) Size and Resolution’.

Unlike the Nikon version mentioned, most ‘simple tube adapters’ for SLRs (pp63, 64) merely consist of empty tubes, and an eyepiece is used to project the image directly onto the film or sensor. Unfortunately, eyepieces for visual use are designed to project an image at infinity and, if the objective is refocused in an attempt to produce a real image a few centimetres away, spherical aberration is introduced and what is more, the objective will no longer be parfocal with the rest of the set. The seriousness of these problems increases directly with the NA of the objective in use and inversely with the projection distance, to the point where the quality of the image is likely to be compromised.

The book shows considerable bias toward the products of some manufacturers. For instance, the text includes detailed instructions for use of the old Nikon PFM photomicrographic attachment, the Nikon D300 and D700 DSLRs, while a couple of pages are devoted to comments about the Olympus E3 and the Panasonic G1 and Gh1, but references to Canon cameras, for instance, are almost non-existent. The reader is entitled to know if this is merely because the author’s experience is limited to these brands or if there are more serious implications.

Readers other than well-heeled professional researchers may be irritated by the emphasis on the use of very expensive instruments equipped with infinity-corrected objective systems.

In photomicrography, cleanliness is surely of paramount importance and the whole optical system needs to be inspected frequently and kept scrupulously clean. Merely relying on a camera’s ‘flat-field correction’ software (p57, repeated on p81)) – even if available - is a grave mistake because it does not detect the loss of contrast caused by fine films of dust or grease.

The use of oil immersion objectives on uncovered smears is described as ‘acceptable’ (p107) on the grounds that oil and coverglass (if present) have identical optical characteristics (p108) In fact, however, (most?) modern oil immersion systems are no longer truly ‘homogeneous’ (hence the vital importance of using the oil specified by the manufacturer) and for critical work on uncovered smears, Zeiss recommended the use of a special oil with RI of 1.5 – see ‘Optical Systems for The Microscope’ 1971 p22.)

If researchers cannot afford dry darkfield illuminators and are consequently forced to fit patch stops to their brightfield condensers, it would have been useful to include a note on how to calculate the diameter of the stops they will need for various objective/condenser combinations? Should merely taping a ‘quarter’ over the bottom of the condenser, (pp111 and 112) really be a serious option at a ‘professional’ level?

Also noticed:

‘Depth of Field’ confused with ‘Depth of Focus’(p25)

‘microphotography’ for ‘photomicrography” (p45)

‘X1000’ substituted for ‘X100’ in the calculation on p86

The 2 whole-page illustrations (pp124 and 125) purporting to show the difference between positive and negative phase contrast images have had their captions transposed!

I must admit to having looked forward to the arrival of this book with particular pleasure. Though I have been engaged in ‘film-based’ photomicrography for the best part of a lifetime, I switched entirely to digital work only about five or six years ago and so still have much to learn - even about the basics – and consequently relished the thought of having a new (and apparently experienced) brain to pick.

According to the blurb, the publishers hope that with the help of this volume readers will learn “the skills needed to obtain professional quality images through the microscope” but, sad to say, the treatment of the subject is insufficiently thorough in some key areas for this to be fully realisable. This is not to say that many in the Life Sciences will not find it a useful introduction to the subject – and it contains information that they may not easily find elsewhere – but those who aspire to rank among the real masters of photomicrography will need to wait for the second edition!

Spike Walker. 2010


Micscape Acknowledgment: The Micscape Editor thanks the publishers Rocky Nook for kindly sending a review copy on request.



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