Microbe ISSN 2220-4970 (Online) ISSN 2220-4962 (Print ... · 2 - MicrobeHunter Microscopy Magazine - January 2012 Microbehunter Microscopy Magazine The magazine for the enthusiast

MicrobeHunter Microscopy Magazine - January 2012 - 1

MicrobeHunterMicrobeHunter

ISSN 2220-4962 (Print)ISSN 2220-4970 (Online)

Volume 2, Number 1January 2012

The Magazine for theEnthusiast Microscopist

http://www.microbehunter.comMicroscopy Magazine

Desmid Taxonomy Testing with aMosquito Wing

Victorian “Live Box”Microscope

Victorian “Live Box”Microscope

High DynamicRange (HDR)Imaging

Essay: AmateurMicroscopy - Goodfor the Soul

Desmid Taxonomy

StereoscopicMicrographs

Mosquito Wing asa Lens Test Subject

2 - MicrobeHunter Microscopy Magazine - January 2012

Microbehunter Microscopy MagazineThe magazine for the enthusiast microscopistMicrobeHunter Magazine is a non-commercial project.

Volume 2, Number 1, January 2012 (revision 1)

ISSN 2220-4962 (Print)ISSN 2220-4970 (Online)

Download: Microbehunter Microscopy Magazine can be down-loaded at: http://www.microbehunter.com

Print version: The printed version can be ordered at:http://microbehunter.magcloud.com

Publisher and editor:Oliver Kim, Ziegeleistr. 10-3, A-4490 St.Florian, AustriaEmail: [email protected]: http://www.microbehunter.comTel.: +43 680 2115051

Images and Articles by:

Bianchi, Gino I.Borg, David B.Clinedienst, JeffCrosby, James D.Guwak, MikeKim, OliverKreindler, R. JordanMonzo, LucaThomas, Anthony

Copyright: By submitting articles and pictures, the authorshave confirmed that they are the full copyright owners of the ma-terial. Creative commons and public domain images are indicat-ed with a small text next to the image or in the caption. Thecopyright of all other images is with the author of the article. Youare not allowed to distribute this magazine by email, file sharingsites, web sites or by any other means. If you want to have acopy of this magazine, either order one from Magcloud (see linkabove) or vistit www.microbehunter.com.

Editorial: Article and image submissions are welcome andshould be sent to: [email protected] submission guidelines, consult the website at:http://www.microbehunter.com/submission

Disclaimer: Articles that are published in Microbehunter Micros-copy Magazine and the blog do not necessarily reflect the posi-tion or opinion of the publisher. The publication of these articlesdoes not constitute an endorsement of views they may express.Advice provided in Microbehunter Microscopy Magazine is pro-vided as a service and neither the authors nor the publisher canbe held liable and responsible for any errors, omissions or inac-curacies, or for any consequences (health, hardware, etc.) aris-ing from the use of information of this magazine and the blog (oranything else). Conduct all lab work and (microscopy) hardwaremodifications at your own risk and always follow the instructionsof the manufacturers.

Visit the Forum!

It is now possible to discuss the individual articlesof the magazine. Every issue has a separate sub-forum for discussion.www.microbehunter.com/forum

Facebook

Do you have any microscopy links to share?Do it here on facebook:www.facebook.com/microbehunter

ANNOUNCEMENT

Write for Microbehunter!Please contribute both articles and pictures. Share your expe-riences, problems and microscopic adventures. If you are aresearcher using microscopes, tell the readers what your re-search is about. Please contribute, even if you consider your-self inexperienced. If you are a struggling beginner, tell ussomething about the problems that you encountered. If youare an active enthusiast microscopist then share your proj-ects, experiences and observations. Are you a teacher or lec-turer? Share your microscopic experiences from school oruniversity. This magazine is made by an enthusiast microsco-pist for other enthusiasts. Let‘s work together to make thisproject a successful one.Please send all contributions to:[email protected]

You must own the copyright of the contributions and you re-tain the copyright of all submitted articles and pictures. Whilewe are not able to pay you for your efforts, we will, of course,give you full credit for your contributions.

Guest Bloggers! Yes, guest blogging is also a possibility.Write microscopy-related blog posts, send them to me and Iwill publish them on the web site. Naturally, I’ll put a link toyour blog. Condition: it must be original content and you mustbe the copyright holder of the text (obviously). When submit-ting articles, please indicate if you want to have them pub-lished on the blog or in the magazine (or both).

Before submitting anything, please read the submissionspage on the website: www.microbehunter.com/submissions.

CONTRIBUTE!

Front Cover:Large image: Oliver Kim (synthetic fibers)Left image: Mike GuwakMiddle image: Anthony ThomasRight image: R. Jordan Kreindler

ABOUT

http://www.facebook.com/microbehunter


4 Victorian “Live Box” Microscope Capabilityin 40mmThese small microscopes are not only attractive

collectibles, but also practical excursion companions.R. Jordan Kreindler

10 High Dynamic Range (HDR) Imaging of Micrographs

Sometimes it is impossible to show both bright and dark areas of a micrograph correctly exposed. HDR imaging combines micrographs of different exposures to obtain a single, correctly exposed picture.

Oliver Kim

15 Closterium costatum CORDA ex RALFStarting this issue, Mike Guwak presents a different

Desmid every month.Mike Guwak

17 Making Stereoscopic MicrographsThe free program Picolay can be used to compute

stereoscopic images from a focus stack.Oliver Kim

20 Gallery of MicrographsImages by Jeff Clinedienst, Gino I. Bianchi, James D.

Crosby, Anthony Thomas and Luca Monzo.

28 Amateur Microscopy - Good for the SoulThinking about eye-diving into a microscope? Or are

you already a devotee? A life-long fan ruminates on our fondness for looking at little things.

David B. Borg

32 Mosquito wing as a lens test subjectHow sharp is your objective?

Anthony Thomas

Answer to the puzzle (back cover):Cross section conifer needle. Pseudo DIC

15

4

CONTENTS

32

17

10

http://www.facebook.com/microbehunter


Small cylindrical microscopeswere popular at the end of the19th century and the beginning of

the 20th. Figure 1 presents some variet-ies that are still commonly available atantique malls, flea markets, and throughon-line auctions. These are sometimesreferred to as "Pocket Microscopes" or"Pocket Magnifiers". They are descen-dants of the microscope first inventedby Nicolas Hartsocker (Dutch) c. 1689,and popularized by James Wilson (Eng-land) c. 1702. The cylindrical instru-ments shown in Figure 1 come from avariety of makers usually with only mi-nor variations and are generally quitesimilar in size. Many include a remov-able magnifying glass (Fig. 2).

The examples in Figure 1 includetwo somewhat atypical microscopes atthe right. The rightmost microscope dis-plays a somewhat more conical than

cylindrical shape, although of relativelysimilar size. See below and Figure 4 fora brief discussion of the second micro-scope from the right. At the far left amodern Episcope, mounted on a stand,is included for comparison.

Earlier examples of cylindrical mi-croscopes can be found in the RoyalMicroscopical Society collection(RMS)i which also has a related exam-ple ii. Other related examples are in theScience Museum, London iii . The Bill-ings collection provides additional vari-eties iv.

Some of these microscopes comewith a spring-loaded stage which ismoved downward by two short lateralmetal dowels to allow small slides to beviewed. (See three instruments to therear of Fig 1.) Like the centuries earlierWilson screw-barrel microscope, these

instruments' slides are inserted in thesides of the body tube. They were oftenprovided with a Stanhope lens.

Spring-loaded stage pocket micro-scopes were frequently sold in card-board or wood boxes with 3 to 6 smallslides, often with one or more plainslides, a well slide, and some preparedslides with slots to store the slides.Some kits included a stand containingan adjustable mirror. The microscopecould be mounted in the stand and se-cured with a built-in knurled screw.

These microscopes were sold undera variety of names including variouscombinations of the words universal,pocket, and microscope. These includ-ed, e.g., "The Microscope", "UniversalPocket Microscope", "Pocket Micro-scope", "Universal Microscope". Theywere also sold as, "The Thavos Micro-scope", "Achromatisches Universal

These small microscopes are not only attractive collectibles, but also practicalexcursion companions.

R. Jordan Kreindler

Figure 1. Examples of smallcylindrical microscopes

HISTORICALMICROSCOPY “Live Box” Microscope


Figure 3 (left): Storage box for Thavos microscope

Figure 4 (right): Stage focusing pocket microscope

“Live Box” MicroscopeHISTORICAL

MICROSCOPY

Figure 2.: A basic cylindricalmicroscope disassembled to showremovable magnifying glass.


Taschenmikroskop", "The Midgard",etc. Two stands with microscopesmounted are shown at the center rear ofFigure 1, A storage box with micro-scope, stand, and slides is shown in Fig.3. Many of these microscopes weremade in Europe, particularly in Germa-ny and France. A spring-loaded pocketmicroscope was discussed in an inter-esting paper by Martin Mach, in a Mic-scape articlev some years ago.

While many of the cylindrical mi-croscopes shown in Figure 1 focus bymovement of pressure fitting compo-nents, there are cylindrical microscopesthat focus via rotating threads. The mi-croscope shown in Figure 4 has a basesimilar to that of a typical drum micro-scope. It has a threaded cylinder whichcan be screwed up and down the main

body tube to achieve focus by effective-ly moving the stage. Other exampleswere also sold with integral mountedmirrors.

One additional cylindrical micro-scope worthy of mention is the "Insec-toscope", Fig. 5. Its design is sometimesattributed to the French political re-former and scientist François-VincentRaspail (1794-1878). In use, an insectis placed inside the glass cylinder andthe instrument focused by screwing themagnifier, located in the top section ofthe instrument, up or down. This "In-sectoscope" is somewhat similar in pur-pose to the Live Box microscope.However, with its approximately 1-1/2"diameter and 4-1/2" height when open,it is almost twice the size and less porta-ble than the approximately 2-1/3rd" tall(in working position) “Live Box"microscope. Weinervi presents an inter-esting account of Raspail. While thatbiography does not present an "Insecto-

Figure 6. Betrand "Furnace" andPocket 'Live Box' Microscope openand closed.


Figure 5: "Insectoscope", of a designsometimes attributed to Raspail.


scope", it does include a picture of oneof Raspail's earliest box-based instru-ments.

It might be easy for professionalmicroscopists to miss the importance ofthese small and relatively inexpensivemicroscopes which at the turn of thecentury cost about 1s vii. However, formany microscope users these were of-ten the first, and perhaps only, micro-scopes they had. Their low price madethese microscopes quite popular asdemonstrated by the variety of manu-facturers and the number available inthe marketplace, even today.

The microscope discussed here in itsfully open state is similar in height tothe Bertrand "Furnace" pocket micro-scope, c 1840 viii (Fig. 6), but somewhatwider. Closed it is considerably smaller

than the Bertrand "Furnace" or any ofthe brass microscopes shown in Fig 1.Like those microscopes, it can only ac-cept smaller slides, but unlike thoseinstruments it is unique in having itsown convenient built-in live box.

The 'Live Box' microscope can bedisassembled into three parts: (1) thelower section is the live box bottom,i.e., a brass cylinder terminating in aclear glass plate, (2) the middle sectionis the body tube with lateral openingsfor small slides, and also terminating ina glass plate, and (3) the upper sectioncontains the eyepiece with pressure fit-ting collar (Fig. 7).

The microscope focuses with a slid-ing tube inside a friction collar (Fig. 8),allowing it to change focus easily fromslide to live box. The eyepiece is com-

posed of two air separated glass ele-ments. The bottom of the lower elementof the eyepiece extends below its hous-ing and is convex. The eyepiece lens isapproximately 19 mm in diameter, andthe live box about 25.4 mm.

Unlike most similar small micro-scopes the top lens of the eyepiece is notflush with the top of the instrument, butrecessed approximately 7 mm.

The microscope was sold in a singlehinged wood box, with single frontlatch approximately 38.5 mm high x 50mm x 67 mm at its longest dimension.Although protective of the instrumentwhen stored, it was not an ideal com-panion for the pocket. See Fig. 12 for amore convenient carrying case. The mi-croscope's size and design, in particularits integral live box, should make it anice companion for field trips. So, thisraises the question of its optical perfor-mance.

On page 8 are pictures taken thoughthe microscope of two small, approxi-mately 55mm x 17mm, paper coveredVictorian slides of a 'flea' and 'hair'; livebox subject resolution is similar. Thesewere adjusted in Photoshop to approachthe visual view through the microscope.In use, actual views are somewhat crisp-er than the photographs suggest. Fig. 9shows a flea as seen in its entiretythrough the microscope's eyepiece. Fig.11 is a photograph through an Olympusmicroscope of the hair shown in Fig. 10,providing a comparison with a modernbenchtop instrument. Performance issurprisingly good for an instrument on-ly 40mm tall when closed.


MICROSCOPY

Figure 7. 'Live Box' Microscopedissembled.

Figure 8: Top view



The full provenance of the instru-ment is unknown. Before I obtained themicroscope, it was owned by severalgenerations of a Western US family. Ithad been used extensively as demon-strated by the occasional slippage of thefriction focusing tube, which was easilyrepaired.

Although, perhaps, not quite as opti-cally capable as e.g., a later SeibertWetzlar Emoscope, its ability to acceptboth slides and its integral live box, inwhich specimens can be quickly en-closed, makes it an almost ideal, easyto carry companion for field trips. Inow pocket this microscope for excur-

sions stowed in a nicely fitting case,originally made for an Edmund N.J.optical measurement viewer (Fig. 12).

Small cylindrical brass microscopesare still widely available and are usuallyquite inexpensive, in today's, 2011,market from $10 to $50 depending oncondition, completeness, and of coursethe buyers present at the time of sale. Asimilar example to the one presentedhere sold with wood case on eBay inApr 2011 for $50 ix.

The 'Live Box' style of pocket mi-croscope discussed here occasionallycomes to market, and although not up to

professional quality, these instrumentsare still quite capable microscopes thatmake excellent, comfortable to carry,and convenient companions for casualoutings.

The author welcomes any sugges-tions for corrections or improvement.He is always pleased to learn about anyunique small brass cylindrical micro-scopes. He can be contacted at,R. Jordan Kreindler:[email protected]

©2011 Text and photographs by theauthor.

■

Figure 9: Flea seen through 'LiveBox' microscope.

Figure 10 (bottom left): Hair seenthrough 'Live Box' microscope.

Figure 11 (bottom right): Hair seenthrough an Olympus benchtop at100x for comparison.


Figure 12. Carrying case for 'LiveBox' Microscope, adapted from anEdmund, N.J. measurement tool


MICROSCOPY

References

i Turner, G.L'E. The Great Age of the Microscope. The Collection of the Royal Microscopical Society through 150 Years.Adam Hilger: Bristol, England and New York, 1989, Figs. 262, 265, 266, 267, and 268

ii Ibid, Fig. 310.

iii Bracegirdle, Brain. A Catalog of the Microscopy Collection of The Science Museum, London. 2005, Item 1/32 - "Pock-et Microscope", 1/33 "Pocket Microscopes", 4/31 "Pocket Microscope", etc.

iv Purtle, Helen R. (ed.). The Billings Microscope Collection. Second Edition. Armed Forces Institute of Pathology:Washington, D.C., 1974, Figs 294, 296, 314, 393

v Mach, Martin. 30 g of microscope please! Micscape Magazine, August 2000

vi Weiner, Dora B. RASPAIL SCIENTIST and REFORMER. New York: Columbia University Press, 1968

vii Ibid iii, 4/27 "Pocket Microscope"

viii Moe, Harald. The Story of the Microscope. Rhodos: Denmark, 2004, p 189

ix US eBay, April 14, 2011, Item ID 290553686109, 4 bidders, 9 bids, USD


IMAGE PROCESSING Exposure control

Sometimes it is impossible to show both bright and dark areas of a micrograph correctlyexposed. HDR imaging combines micrographs of different exposures to obtain a single,correctly exposed picture. This article shows three ways on how to do this.

Oliver Kim

Some microscopic specimens canhave a large difference in brightand dark areas. Insects and other

arthropods, for example, have a darkchitin exoskeleton which does not allowmuch light to go through. In order to seestructural details of the insect's body, itis necessary to turn the microscope'slamp up and it is also necessary to openthe condenser aperture diaphragm. This,however, may cause other parts of thespecimens, such as the thinner legs, tobecome overexposed. In extreme cases,

the finer structures are not visible at all,as they are completely flooded by light. Often contrast adjustment using dig-ital image processing is not a solution.One may consider to use the “levels”tool of image editing software to makethe dark areas of the image brighter andthe bright areas darker. This only worksto a point, however. Image informationwhich is not there in the first place cannot be recovered this way (Figure 3).

Many microscopic cameras are notable to capture the full range of bright-ness. Some pixels of the digital camera

may go into saturation (in the brightparts), and are thus not capable of cap-turing the image information in theseareas. Other parts of the camera’s sen-sor receive so little light that even in-creasing the brightness in these areaswill not reveal much image detail (Fig-ure 3 illustrates this).

This issue can be overcome to acertain extent by using digital SLR cam-eras that have the capability of record-ing image in the RAW format. Thisformat is capable of storing a far greatercolor depth than JPG images, and it may

Figures 1 and 2: The original images used show the mandibles of a cockroach.Figure 1 correctly shows small hair and irregularities in the chitin exoskeleton (A).At the same time the dark structures (B) reveal very little detail and appear toconglomerate into one large mass. Opening the condenser will reveal moredetails (C) but also results in highly over exposed areas (D). The hair disappearcompletely.

Figure 3: This image shows what happens if one attempts to increase thebrightness of figure 1. The image starts to look posterized, but not much moreimage detail is revealed in the dark areas (E).

1 2

A

B C

D

3 E


Exposure control IMAGE PROCESSING

4: PhotoShop: simple blending 5: PhotoShop: smart blending

6: Enfuse blending 7: PhotoShop: 50% opacity of layers


therefore be possible to recover moredetails from the darker areas of the pic-ture. I have not yet tried this, however.

Combining several images ofdifferent exposures

There is, however, also a differentapproach of solving the HDR problem.If the dynamic range of the specimen istoo large, then it is also possible tocombine several images of differentbrightnesses into one correctly exposedfinal image. The specimen is first photographedseveral times, with different condenserand light intensity settings. The differ-ent images will then have different partsof the specimen correctly exposed, withother parts either being too dark or toobright.

Figures 1 and 2 show the mandiblesof a cockroach. Some parts of figure 1are underexposed (the black circularstructures at the center-bottom). In fig-ure 2 these structures are correctly ex-posed, but the area between themandibles is now white and lacks detail.The hair on the cockroach are also notvisible anymore. Both, the dark and thebright part, has experienced a loss ofimage information. These two imagescan now be combined into an imagewith an overall correct exposure (Fig-

ures 4-7). Figures 8 and 9 show an evenmore extreme example (a tick), withhighly underexposed and overexposedparts.

Enfuse

I have experimented with wo differ-ent programs. The program Enfuse canbe freely downloaded fromhttp://enblend.sourceforge.net/. It doesnot require installation. Enfuse is usedover the command line in Windows. Ihave found out that the standard settingsof the program produces very satisfac-tory results. Blending a large number ofimages together is easy, as one only hasto specify the file names in a separatetext file.

Photomerge

The second program that I used wasthe commercial program PhotoShop El-ements 11.0. The Photomerge Exposuretool offers several modes (simple blend-ing and smart blending), including amanual mode. The simple blendingmode is a single-click option, in whichthe program uses a pre-defined set ofparameters. The smart-blending modeallows the user to control color contrastand saturation over sliders.

This tool can be accessed by overthe menu (File - New - PhotomergeExposure). PhotoShop blending is donecompletely graphically and requires on-ly a few mouse clicks. This may be anadvantage for those people who are notfamiliar with the command line in theDOS box of Windows. You can down-load a 30 day trial version of this pro-gram from the Adobe website, to checkif you are satisfied with the functional-ity of this tool.

Layer Opacity Control

Last, I tried to increase the dynamicrange by blending two layers. This canbe done also with PhotoShop or with thefree program Gimp (or any other pro-gram supporting layers). First, the twooriginal images are copied each into oneseparate layer. The opacity control isthen used to make the top layer trans-parent. Choose a value of 50% to weighthe two images equally. PhotoShop’sSimple Blending tool and the 50%blending using layers produced verysimilar images. I suppose that the algo-rithm used to compute these images isthe same.

On page 14, I have included a briefexplanation on how to blend imagesusing Enfuse and layers.

■

Figures 8 and 9: The original images to be blended (a tick). This was a difficult specimen. The condenser had to be openedwidely to allow light to pass through the specimen. This resulted in very over exposed parts, of especially the legs.

Figures 4-7 (previous page) and 10-13: Comparison of different blending tools. PhotoShop’s “simple blending” as well as theblending done by Enfuse resulted in the best images. Smart blending did produce considerable artefacts, especially aroundthe edges of the specimen. Adjusting the opacity of the layer (image 13) also produced acceptable results.

IMAGE PROCESSING Exposure control


10: PhotoShop:simple blending

11: PhotoShop:smart blending

12: Enfuse blending

Exposure control IMAGE PROCESSING

13: PhotoShop:50% opacity of layers


Using LayersHere you need a graphics programwhich is capable of supporting lay-ers. The screenshots show Photo-Shop.

Step 1: Open the first original imageand click on the Create a new layerbutton

Step 2: A new empty layer is createdon top of the image.

Step 3: Open the second image,press CTRL-A to select the whole im-age and CTRL-C to copy the image tothe clipboard.

Step 4: Switch back to the first imageand press CTRL-V to paste the sec-ond image into the empty layer 1, ontop of the first image.

Step 5: Use the horizontal slider(green circle) to adjust the opacity ofthe top layer. The two images will beaveraged this way.

Step 6: Merge all layers or flatten theimage (menu Layers). Then save thenew image.

Using Enfuse

Step 1: Download enfuse fromenblend.sourceforge.net and copythe file enfuse.exe into the directo-ry containing your original images.(no installation required).

Step 2: Use a text editor to make alist (called list.txt) of original filesthat are to be processed. Press En-ter at the end of each line. The lastline must be empty.

Step 3: Open the command line win-dow by running the program cmdfrom the start menu. Change intothe directory containing the pro-gram enfuse and the original imag-es. Type:enfuse @list.txtto process the files.

Step 4: The program has now gen-erated a file called a.tif. This is theoutput file containing the HDR im-age.

IMAGE PROCESSING Exposure Control


Name: Closterium costatumCORDA ex RALFS var. costatum

Original publication: Ralfs, J.(1848) p. 170 and p. 312.

Synonym: Closterium striolatumEHR. var. costatum (CORDA)KLEBS

Origin of name: The name costatumrefers to the characteristic costae, thestriations in the cell wall (Fig. 2).

Size: 200-400mm by 30-45mm

Shape: The cells are 6-10 times lon-ger than wide and are arched. Thecell wall is striated. In contrast toother Closterium species, the striaeare conspicuous, far apart and rib-

like (costate). Slight dots are visiblebetween the striae. This costate appear-ance is a distinguishing characteristicfor this species and allows for a differ-entiation from the similar looking C.striolatum, whose striae are muchdenser. The apices (Figs. 3, 4) arewidely rounded and possess a thick-ened cell wall.

Occurrence: This is a very adaptableand widely distributed species. It hasbeen found in acidic moorland pools aswell as in neutral and even weaklyalkaline lake shore zones.

Note: The images show dead, emptycells. Live cells are green due to theirchloroplasts. The striae can be seenbetter in dead cells.

References

"Closterium costatum Corda ex Ralfs" Algae-base: Listing the World's Algae. N.p., n.d.Web. 15 Jan. 2012.<http://www.algaebase.org/search/species/detail/?species_id=28163>.

Lenzenweger, Rupert (1996). Desmidiaceen-flora von Österreich: Teil 1. Berlin, Stuttgart:J. Cramer

Ralfs, J. (1848). The British Desmidieae. Lon-don: Reeve, Benham & Reeve.

Image Credit

The images are copyrighted byMike Guwak (2012).http://www.mikroskopie-main-taunus.demike.guwak@mikroskopie-main-taunus.de

Reference Plate DESMIDS

Closterium costatum CORDA ex RALFMike Guwak

1


DESMIDS Reference Plate

2

3 4

http://www.starosta.com/3dshowcase/ihelp.html


Yes, the pictures on these pages are real micrographs and not drawings! The free programPicolay can be used to compute stereoscopic images from a focus stack.

Oliver Kim

On these pages, I want to showyou some stereoscopic imagedof radiolarians. The images on

page 16 and 17 can be viewed by cross-eyed viewing. The two images on page18 are anaglyphs, which require the useof red-cyan 3D glasses.

Cross-eyed viewing does not needglasses. Look at the images and crossyour eyes until the two images start toappear as four (out of focus) images.Then slowly uncross your vision so thatthe middle two of the four images over-lap. You will then see the radiolarian in3D.

The images on page 18 requireanaglyph glasses. The right eye looksthrough a blue/cyan foul, while the lefteye looks through a red foil. Theseglasses can also be made at home, byusing an ink-jet printer and printing thetwo colors on an overhead transparencyfoil.

How the images were made

The free program Picolay was usedto make these images. The program is astacking software, which is able to com-bine different images that have differentparts in focus into one single image, onewhich is in focus throughout. The pro-gram is also able to compute stereo-scopic images from the stack. Thecondenser diaphragm should be openedto lower the depth of field. This waymany pictures have to be taken and it isthe task of the program to assemble themany pictures into a final image. Theintensity of the stereoscopic effect canbe specified as well.

The images were originally in color,but the anaglyphs can be can be betterviewed in grayscale. The program al-lows for a conversion to grayscale.The procedure was relatively simple. Aseries of about 20 images were taken,each picture at a different focus. The

program assumes that the first image isthe top most image and that all furtherimages are deeper into the sample. Theimages were loaded into Picolay andstacking was started by pressing the F1key. After completion, a new menu (3Dedit) starts to appear. It is then possibleto convert the stacked image into eithera red-cyan anaglyph or into images forcross-eyed viewing. The newly generat-ed images are all automatically saved.

The program is relatively elaborateand allows for much fine-tuning. Theprogram can be downloaded from:www.picolay.de. It is advisable to studythe documentation to fully understandall of the features. I have found out thatthe standard settings provide very goodresults for radiolarians, however.

Help for cross-eyed viewing:http://www.starosta.com/3dshowcase/ihelp.html

Radiolarians in 3D STACKING

http://www.starosta.com/3dshowcase/ihelp.html


STACKING Radiolarians in 3D


Radiolarians in 3D STACKING

These images erquire red-cyan 3Dglasses (red over the left eye, cyanover the right).


GALLERY Crystals

A common pain relief medication with acetaminophen, aspirin, and caffeine as the main constituents. Stack of 5 images usingCombineZP. Partially polarized and oblique lighting. Amscope T490, 10x objective, Canon 7D. By Jeff Clinedinst (www.jclinedinst.com)

Crystalized B/W film developer containing phenidone, hydroquinone, sodium sulfite and sodium bisulfite. Polarized lighting using AmscopeT490, 4x objective, Canon 7D. By Jeff Clinedinst

20 - MicrobeHunter Microscopy Magazine - January 2012 - Send images to [email protected]


Top: A 7 micron histologic slide of human breast with lactational change , colored with hematoxilin and eosin. Camera used: Coolpix 5000with adapter. Labophot microscope with Nikon e-plan objective. By Gino I. Bianchi

GALLERYLarva and Histology

Mosquito larva in darkfield.Amscope T490, 10x objective,Canon 7D. By Jeff Clinedinst

Send images to [email protected] - MicrobeHunter Microscopy Magazine - January 2012 - 21


Top and right: Lichen from King’sCanyon, CA

Page 23, top: Easter moss

Page 24, bottom: “The lonelysporophyte” (moss).

All images on page 23 and 24 byJames D. Crosby

GALLERY Lichens



GALLERYMoss



Butterfly EggsGALLERY

The three images show the eggs of Mourning Cloak butterfly (Nymphalis antiopa)

Right: yellow freshly laid egg mass.

Bottom: Images of single eggs darker than when freshly laid due to developingcaterpillar; top view and side view.

By Anthony Thomas



GALLERYAquarium Life

The top two images were obtained witha Bresser trinocular microscope (TrinoResearcher II model). They show anamoeba with cyanobacteria inside. Thesample was taken from the algalmaterial of my aquarium. I used a patchof black paper to obtain an obliqueillumination. The photos were processedwith Acdsee software, increasing thecontrast and detail with the tool“unsharp mask”.

The image on the left was obtained withthe same technique of the previousimages. For all the pictures I used aSLR Canon Eos 350D at 1600 ISO andautomatic exposure with the software"DsrlRemote" to control remotely thecamera from my laptop PC.

By Luca Monzo



Water LifeGALLERY

The images on this double page show water fleas of my littlewater tank. The original source is a fountain located in the publicgarden of Udine, Italy (see photo on the left). I used a piece ofblack paper to obtain oblique illumination on some images. Iprocessed them with Helicon Focus or ACDSee software.

By Luca Monzo



GALLERYWater Life



Oliver Kim, the exemplaryfounder of Microbe Hunter,states the website and maga-

zine's main mission is to encourage andmotivate. He wants this project to in-duce enthusiasts to broaden their inter-ests in the miniature world and share theresults. He also hopes to inspire new-comers to enter into the magical realmof microscopes for the first time. Insupport of these laudable aspirations Ioffer a few thoughts – some 'philoso-phizing' – for everyone's consideration,but especially for novices, on whatmakes our “great hobby and pastime”so satisfying and worthwhile.

On microscopy itself

“The development of microscopyrevolutionized biology and remains anessential technique in the life and bio-logical sciences.” (From an article inWikipedia)

The compelling word in that sen-tence is: “remains”. Consider that lightmicroscopy, invented centuries ago, hasnot yet been made obsolete and replacedby engineering advancements based ontotally different chemistry and physics.How many other vital technologies arethere in our lives today about which thesame may be said? Very few.

Breakthroughs such as electricity,internal combustion engines, electro-magnetic wave transmission, electron-ics and digital technologies haveconsigned scores of antiquated technol-ogies to the dustbin over the last centuryor so. Nothing strange about that – infact it is expected. That's why it isnotable (even thrilling, if you are thecontemplative sort) to find a venerable

way of doing something important –very important – still in the forefront.

Optical magnifying lenses have notonly survived, they continue to triumph.Thus far, no one has come up with abetter, more economical way of resolv-ing small objects, particularly tiny liv-ing things. And note that people havebeen crafting glass for this purpose forfour hundred years. (The invention ofthe compound microscope is often cred-ited to Dutch spectacle-makers HansJansen and his son Zacharias in 1590.)

Engineering continues to improvelens-making and microscope design,but the basic physics has not been su-perseded in any practical way. That letsyou, the amateur microscopist, standside by side with the ghosts of Antonievan Leeuwenhoek and Robert Hooke.You can feel the same astonishment andexhilaration they did in seeing cells and“animalcules” for the first time in yourlife, in exactly the same way, which isstill the best way.

Empathy bridging centuries. Mi-croscopy is an venture whose livinghistory is continuous and progressive,not a resuscitated tradition whose timeof relevance and innovation came andwent. There are no “re-enactors” in ourendeavor.

On the noble traditionof the amateur

“Amateur” is a word whose reputa-tion has been degraded by changingtimes. Currently, the adjective shares inmost people's minds a dual definition.One is neutral (engaging in a pursuitwithout payment; nonprofessional).But the other is disparaging (contempt-ibly inept or unskillful). How much

worse could one's sincere efforts bedenigrated than by being described as“amateurish”?

It is a shame to have come this.There was a time when amateur scien-tists were so highly regarded that presti-gious institutions were commissionedby courts and governments to encour-age their efforts and allow the benefitsof their discoveries to accrue to thegreater good of all citizens and subjects.(The Royal Society of London for Im-proving Natural Knowledge, wasfounded in November 1660 by KingCharles II's grant of Royal Charter. It ispossibly the oldest such society in exis-tence.)

Back then, an Amateur was some-one with time on his hands, whichmeant he (or alas, rarely, she) was edu-cated and had money – someone ofaristocratic birth, inherited wealth ordeserving of patronage by way of anevident and prodigious brilliance.

These scientists were often free ofthe stifling constraints of commercialconsiderations; they pursued their inter-ests with abandon and without dead-lines, and most shared the results oftheir efforts with the world withoutavarice. In our avocation, we 21st cen-tury amateur microscopists can be justas free.

We can rejuvenate the older defini-tion's sense of altruistic virtue with ourown attitudes and behavior. And whatbetter attitude to have than that which isat the etymological heart of the worditself:

ORIGIN late 18th cent.:from French amateur, 'lover of',

from Italian amatore,from Latin amator ‘lover,’

from amare ‘to love.’

ESSAY

Thinking about eye-diving into a microscope? Or are you already a devotee?A life-long fan ruminates on our fondness for looking at little things.

David B. Borg


And reflect upon whose footstepswe can humbly follow, in earnestness ifnot necessarily with similarly toweringintellects: Charles Darwin and GregorMendel were “amateurs”.

On left brain/right brain

Thinking and feeling. Analyzingand creating. Science and Art. Dothese divergent human faculties everintersect? Constantly – often in mo-ments of supreme insight.

Take the music of Mozart for exam-ple. Are there mathematics in its beautyor beauty in its mathematics? Or Ein-stein's formulations. Did he think thesublime or did the sublime condescendto “think” him?

As a musician (formerly profession-al and now happily “amateur” again) Isometimes feel a tangible expression ofmusic, already existing in perfection far

above me, show me the way by allow-ing a tiny shadow of itself to alight for amoment in the lower world of soundwaves in air. It turns me into its tempo-rary vessel, a self-less screen on whichto project the slightest hint of its grace.

Afterwards I ponder, with all theself-love ego can muster, “Wow, howdid I do that?” Of course, “I” was notthere at the time and had nothing to dowith it. But my rapture in such experi-ences is transcendent. Hardly my pri-vate domain – anybody doing anythingcan find themselves in the “Zone” ifthey work at it. Many do.

What work? My job is to strive withmy best effort and be fully present withno expectations. My job is to study andpractice never-endingly. My job, as thevocabulary of technical proficiency andmental understanding grows, is to openthe door to emotion, let the vocabularyflow in to find expression and quietlyinvite beauty to join me.

The job description is exactly thesame with microscopy. Follow your

bliss, as Joseph Campbell said, and joywill be yours if you apply yourself.There is an infinity of science to learnwith an organized mind, and no less towork with creatively. One side of yourbrain stimulates the other.

The microscope magnifies and re-solves. Your mind and heart perceiveand synthesize. The more you under-stand about what you are looking at, themore you see, and the more beauty isdisclosed and admired. This all leads tocritical insight and new understanding.Art and science merge and advancetogether.

I must admit, biology might neverhave been my favorite subject, norwould microscopes have held so muchinterest for me had it not been for thepictures! Images feed my soul, as doesmusic. And, just as with other fine art,the interpretation and meaning associat-ed with elegant microscopic images ex-pands appreciation far beyond the mereperception of abstract designs.

Amateur Microscopy – Good for the Soul ESSAY

Citric acid in polarized light.

(Image: Oliver Kim)


Today, with the digital technologyof photo-microscopy so available anddiverse, an arena of boundless creativityis open to us that was mostly closed tothe frugal amateur just a decade or twoago. And there is a booming communi-ty out there (of which Microbe Hunteris a very friendly subset) with which toshare splendid images. I cite, as thelofty tip of the iceberg, the yearly festi-vals put on by Nikon (Small World) andOlympus (BioScapes). Google, gawk,emulate and enter. Join the party; posta picture.

On serendipity

You never know what, exactly, youare going to see, and what you do seethrough a microscope is always uniqueto you. Even if your subject is com-monplace, your awareness, if sharpenough, and your mind, if open enough,will discover something new, possiblyeven “important”, but certainly fasci-nating to you, if no one else. Develop-ing these qualities in yourself –awareness and openness – rewards youwith the sustained elation of continuousadventure!

Microscopy is also a perfect domainin which to test the truth of the old

maxim “Luck is where opportunitymeets preparation”. There is opportuni-ty every time you examine a specimen.Diligence and persistence define prepa-ration.

When you reflexively snap the shut-ter on that bewitching image that sud-denly – unexpectedly – appears in yourfield of view, or when that technical“accident” produces results ten timesbetter than anticipated, some may thinkit is good fortune. But we know better.You get to enjoy the private pleasure ofappearing to be smiled upon by “luck”while knowing that you yourself were awilling and essential participant, andthat your next piece of luck is rightaround the corner.

On the directness of perception

Imagine accessing information –truth – that has not been debased by spindoctors.

Imagine this truth emanating fromthe moment to moment reality of anunfolding universe of being.

Imagine yourself perceiving thistruth directly from its source in the formof visual images produced by your eyesand brain.

Imagine your perception being as-sisted only by the applied understandingof natural laws, developed over severalmillennia by synergistic scientific dis-covery, in the form of instruments andtechniques that make the formerly un-seeable, seeable.

Intrigued? This is the great gift ofmicroscopy. It is pure. It is unlike somany readily available “entertain-ments” and distractions, digital and oth-erwise. There is no interposed opinion,political agenda, religious belief, cultur-al bias, market research or profit motiveshaping your perception or influencingyour interpretation of it.

You are sitting there in your chair.Something real is sitting there on aslide. A tiny slice of the electromagnet-ic spectrum of radiant energy is mediat-ing the space between you and thatsomething real through a handful ofchunks of carefully crafted clear glass.And suddenly you are in direct commu-nication with a formerly invisible, un-imaginable world of incalculablevastness and gracefully organized com-plexity.

You are touching the infinite withyour own eyes, unfiltered – undefiled –by narrow human interests. Realizethat, be receptive enough, and awe willsweep through you. This kind of awe isgood for what ails all of us. It is whatkeeps me coming back to the micro-scope for another peek.

On humility through theexperience of scale

Would you like to generate within your-self the healthiest of humblenesses?Here's a recipe:

1. Calculate the linear measure-ments of various specimen features un-der magnification every time you focusyour microscope. In other words, al-ways know the actual sizes of the ob-jects you are looking at.

2. Consciously relate them againand again to your own body's measure-ments and those of planets, molecules,galaxies, subatomic particles and the

ESSAY Amateur Microscopy – Good for the Soul

Stem of a sunflower, inverted colors.

(Imag

e: Ol

iver K

im)


expanding, curving vastness ofspace/time.

Make a personal study of scale as aconcept. Consider nanometers and lightyears and everything in between andbeyond in both directions; feel them inyour bones as units and multiples ofarbitrary increments on one impossiblylong tape measure, the ends of whichnever end.

3. Reflect on the limitless eleganceof this continuum of scale, inside ofwhich you are apparently sandwichedexactly at the theoretical center-point.Yes, think about that.

Look at your hand. Know deeplythat there is a bacterium, 3 micrometerslong, one million five hundred thousandtimes smaller than yourself, with thesame survival imperative as yourself,alive on the surface of that hand eventhough you cannot see it.

Look at the horizon. Know deeplythat as big as the earth seems, the sun islarge enough to contain over one mil-lion earths. And it is so far away, ittakes over eight minutes for its light,traveling at 671 million miles per hour(1.08 x 109 kilometers per hour), toreach us. Perform these exercises regularlyand each time your self-importance willsuffer a fresh diminishment. And whata relief that will continue to be for you!Awareness of one's insignificance gen-tly banishes anxieties of all kinds, andvigorously tempers the ego by remov-ing its burden of false pride.

On the cultivation of gratitudeand trust

Microscopy, as a vehicle of pro-found pleasure and insight, invites youto be grateful for your life and the giftsof sight and reason. You can accept theinvitation and live better, longer.Someone said, “You can't be angry ifyou are grateful.”

The perfection of what you see alsoengenders a sense of deep trust, a stress-dissolving antidote to the media's re-lentless panic-pandering. How can youremain fearful while contemplating theconsummate form and function in suchstunningly beguiling, ancient creaturesas diatoms and protozoa? Thus far, they

have survived evolution's toughest tests,and when it is time, they will depart thestage, as will all species, to be replacedby new ones. Life trusts itself. Itdoesn't watch the news.

On cheap thrills

And you can have all this for a song.Aspiring to afford an adequate micro-scope is not limited to the wealthiestamong us. To get started, costs forequipment and supplies can be quitemodest. (As a financial investment, therisk-to-reward ratio is extremely favor-able!)

And compared with so many otherflamboyant and extravagant leisure pur-suits, amateur microscopy leaves a tinyenvironmental footprint. Microscopydoes not, in its inherent organizing prin-ciples, abet our subconscious existentialdesire to consume ever more resourcesjust to feel alive for a moment. Neitherdoes it promote compulsive acquisition.(Some collectors of antique instrumentsmay disagree!)

Microscopy could be the prototypi-cal avocation for the adage “Less IsMore”. All tools are limited in one wayor another, so even the most elaborateset-up can't give you everything youcould hope for. The trick is making themost of what you have in front of younow. No matter what that may be, youcan imagine innovative and enthrallingways of using it.

And there are many micro-tech-nique “do-it-yourself” articles and vid-eo tutorials available enabling you toexpand your capabilities for little or nomoney. Microscopists are a generousclan when it comes to offering informa-tion.

Satisfaction through striving to havewhat you want is fleeting, because, onceyou have it, you will soon want more,and that is not a happy or tranquil state.Conversely, happiness through wantingwhat you do have is permanent andcalm, even as what you have changestoward more or toward less.

On solitude

Microscopy creates superb space inyour life to be silent and alone. We all

need time away from the inundatingcacophony of the 'infotainment' streamand the demands of all our social net-works. It's good to let go of the i-devic-es for a while.

In the stillness of your solitary re-treat, the quiet white light streams upthrough the column of crystals, freeingthe micro-genies to reveal their myster-ies – for your eyes only.

Let it be a meditative practice. Youcan watch extraneous thoughts comeand go while you purposefully bringyour attention back to observing all thatis going on inside the microscope andyourself. (Nine out of ten enlightenedbeings recommend it.....)

In conclusion

Clara Schumann, the wife of nine-teenth century romantic composer Rob-ert Schumann, herself a noted concertpianist and composer, left us a poignantquotation musing on the quality of herinner life while she composed music. Itapplies succinctly to all creative activi-ties, and I will paraphrase it, with a fewitalicized words, into my own personalexperience:

Microscopy gives me great plea-sure… there is nothing that surpassesthe joy of creative observation, if onlybecause through it one wins hours ofself-forgetfulness, when one lives inan infinitesimal world of visual splen-dor.

This essay is dedicated with deep affec-tion and respect to Richard A. Cloney,Ph.D., Professor Emeritus of Zoology,University of Washington in Seattle, mymicroscopy mentor and friend, whosefamously rigorous undergraduatecourse in comparative vertebrate histol-ogy I had the greatest delight and honorof attending both as a student and sub-sequently as a teaching assistant andlaboratory instructor.

For those readers who wish to respondto this essay: please post comments tothe forum About MicrobeHunterMagazine on microbehunter.com. ■

Amateur Microscopy – Good for the Soul ESSAY


Compound microscopesvary enormously in price.The newest infinity-type

are likely not within reach of any-one not associated with a re-search laboratory. The older usedfinite-type microscopes, espe-cially the Olympus BH2 seriesand the Nikon Labophot, are pop-ular with keen amateur microsco-pists but command a high price,anywhere between $1,000.00 to$3,000.00. In contrast, some newmodels from certain manufac-tures can be obtained for around$500.00.

I have a trinocular OlympusBH2 with a set of S Plan Apoobjectives. I thought it may beworthwhile to compare imagequality from my setup with imag-es obtained from a new lower-priced microscope. Hopefullythese new lower-priced modelswill give images as good as theolder used (expensive) scopes.However, I have only this onescope and will leave it to othersto post images for comparison.

For comparison purpose sub-ject matter is critical. I am usingthe wing of a northern mosquito,possibly an Aedes or Culex spe-cies, mosquitoes of these generaare to be found in many parts ofthe world. The wing was de-tached from the fly, dehydratedin Isopropyl Alcohol, rinsed inToluene and mounted in a pineresin.

An Olympus 2.5x NFK relaylens fits in the photo tube of theBH2 and projects an image,4,288 x 2,848 pixels, onto the23.6 x15.8 mm sensor of a Nikon

D90 camera. Obviously one can-not display the images at full sizeso I have reduced them to 1,000pixels wide (but they may be fur-ther reduced to fit on the page)but have also included images ofactual pixels. i.e., images of1,000 x 1,000 pixels as recordedby the camera's sensor. Becauseof the very limited depth of fieldwith the higher power objectivesthe following images are stacksof several frames using ZereneStacker software.

The first image is an overallimage of the detached wing takenwith a 2x Olympus S Plan FL2and shows the area along thetrailing edge that is used in theother images centering on theisolated wing scale which isabout 13 microns (0.013 mm)wide.

The second image shows theentire image of 4,288 x 2,848pixels recorded by the camerawith the 10x objective but re-duced to 1,000 pixels wide to fiton the page. Below this image,image 3 shows an area of 1,000 x1,000 actual pixels as recordedby the camera with the 10x ob-jective.

The fourth image is with the20x objective, and image #5 isthe 1,000 x 1,000 pixels crop.The 6th image is with the 40xobjective, and image #7 is the1,000 x 1,000 pixel crop.

You can contact the author at:[email protected] ■

How sharp is your objective?Anthony Thomas

Figure 1: Mosquito wing

TESTING Sharpness of Objectives


Figure 2: Portion of wing, 10x objective +2.5x relay lens

Figure 3: 1,000 x 1,000 pixels crop of 10ximage

Sharpness of Objectives TESTING


Figure 4: Portion of wing, 20x objective +2.5x relay lens


TESTING Sharpness of Objectives


Figure 6: Portion of wing, 40x objective+ 2.5x relay lens


Sharpness of Objectives TESTING


What’s this? Answer on page 3.

Documents

Microbe ISSN 2220-4970 (Online) ISSN 2220-4962 (Print ... · 2 - MicrobeHunter Microscopy Magazine - January 2012 Microbehunter Microscopy Magazine The magazine for the enthusiast