December 2013 - MicrobeHunter Microscopy Magazine - 1

MicrobeHunterMicrobeHunter

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

Volume 3, Number 12December 2013

The Magazine for theEnthusiast Microscopist

http://www.microbehunter.comMicroscopy Magazine

Specimen preparation Color temperature Stereoscopicmicrographs

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

Volume 3, Number 12, December 2013

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

Download: Microbehunter Microscopy Maga-zine can be downloaded from:http://www.microbehunter.com

Print version: The printed version can be or-dered at: http://microbehunter.magcloud.com

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

Images and Articles by: Oliver Kim, HansRothauscher, Charisa Wernick

Copyright: By submitting articles and pictures,the authors have confirmed that they are the fullcopyright owners of the material, unless speci-fied otherwise. Authors are responsible for ob-taining permission for copyrighted work that theydo not own. Creative commons and public do-main images are indicated with a small text nextto the image or in the caption. The copyright ofall other images is with the author of the article(unless specified). If you feel that your copyrighthas been violated or that a mistake in attributionhas been made, please contact the editor. Youare not allowed to distribute this magazine byemail, file sharing sites, web sites or by any oth-er means. If you want to have a copy of thismagazine, either order one from Magcloud (seelink above) or vistit www.microbehunter.com.

Editorial: Article and image submissions arewelcome and should be sent to:editor@microbehunter.com.For submission guidelines, consult the websiteat: http://www.microbehunter.com/submission

Disclaimer: Articles that are published in Mi-crobehunter Microscopy Magazine and the blogdo not necessarily reflect the position or opinionof the publisher. The publication of these articlesdoes not constitute an endorsement of viewsthey may express. The authors themselves areresponsible for the contents. Advice provided inMicrobehunter Microscopy Magazine is providedas a service and as a recreational resource andneither the authors nor the publisher can be heldliable and responsible for any errors, omissionsor inaccuracies, or for any consequences(health, hardware, etc.) arising from the use ofinformation of this magazine and the blog (oranything else). Conduct all lab work and (mi-croscopy) hardware modifications at your ownrisk and always follow the instructions of themanufacturers.

Front Cover: Female pine flowerLeft, middle, right image by Oliver Kim

3 Microscope Specimen PreparationDifferent specimens require different preparationtechniques.

Charisa Wernick

4 Web pages to check out!Microscopy does not have to be complicated and

involve expensive equipment. It can, in fact, be very inexpensive and still be fun.

Oliver Kim

5 White and black hairA look at how age-related depigmentation looks under

the microscopeOliver Kim

6 Making stereoscopic imagesOne possibility is to tilt the slide to make stereoscopic

twin images.Oliver Kim

9 GalleryTestate amoeba

Hans Rothauscher

10 The effect of light intensity and bulb age on color temperature

Blue filters can be used to correct the color temperature.

Oliver Kim

CONTENTSBack cover: Pollen grains in topillumination

Hair, p. 5

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Microscope Specimen PreparationDifferent specimens require different preparationtechniques.

Charisa Wernick

HOW TOSample preparation techniques

Microscope specimens must beprepared before they can beviewed under the micro-

scope. There are several methods forpreparation depending on the sample.

Cutting

Animal & Plant Tissues - thesesamples are sectioned by cutting themwith a razor blade or the use of a micro-tome so they can be placed flat betweena slide and cover slip and viewed undera biological microscope. Advanced mi-crotomes can provide samples as thin as1 micrometer. Hand-cut samples aretypically thicker. The thicker the sam-ple, the less light that can pass throughit. Before cutting your sample, it is bestto support it in some manner so thatduring the cutting process it does notbecome distorted. Internal and externalsupport is best. In order to support thetissues paraffin or celloidin can be used.Paraffin is quickest and most common.Because paraffin is soluble in water,alcohol dehydration of the object is aprerequisite to embedding by this meth-od. Freezing before sectioning the em-bedded material can also be successful.

Wood - very thin slices or sectionsof wood are commonly required formicroscopy examination. When fresh,most wood can easily be cut with ablade. If the wood is not fresh, soakingit in hot water or in an alcohol-glycerinemixture (equal volumes) for severaldays is a common procedure to allowcutting thin sections. Fibers - because fibers are oftenfairly flexible, they require some specialsupport during sectioning. Drawing to-gether bundles of fibers through a verysmall hole in a thin metal plate allowsyou to make transverse sections bydrawing a cutting edge with the metalsurface across the bundle. The hole willusually be about 0.5mm diameter andcutting is done on both sides of the platewith a razor. Fibers can be embedded incelloidon or gelatine, then frozen andcut in a conventional microtome. Thismethod is most frequently performed inmaking longitudinal sections of fibers.Fibers are most commonly viewed un-der either a stereo microscope or if thefibers are extremely small, a metallurgi-cal microscope is used. Bone - cutting any materials thatcontain lyme such as bones or animals

with calcareous shells can be made pos-sible by decalcification. The material isfirst fixed and then treated by specialacid mixtures. A good decalcyfyingagent is trichloracetic acid, which decal-cifies rapidly and hardly changes thestructure of the tissue. Bone is typicallyviewed under a biological laboratorymicroscope in very thin sections. Largerpieces of bone are viewed under a stereozoom microscope.

Grinding

Rocks and minerals - creating thinsections of very hard materials is usual-ly done by grinding. Small chips ofrocks, minerals, or other such materialsare cemented to microslides and theupper surface ground smooth on a flatmetal or glass plate. When the uppersurface is flat, the chip is removed bysolvent action on the cement and re-mounted, flat side down. Then finalgrinding in a similar manner, but to amuch finer degree, is continued until thematerial is of the proper thickness.Rocks and minerals are typicallyviewed under a polarizing microscope.

Microfossils - similar grinding pro-cedures as those listed above are com-monly used to expose chambers inmicroscopic shells or tests of microfos-sils. The grinding is continued until thesection or specific attributes of the ob-ject are revealed.  ■

Minerals captured under a polarizingmicroscope.

Author: Charisa Wernick with source: RobertB. McLaughlin, Special Methods in Light Mi-croscopy (Microscope Publications Ltd. 1977),130.Image: Microscope WorldLink: www.MicroscopeWorld.com

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MICROSCOPYNEWS Microscopy on the Web

Combining two microscopictechniques

Researchers at the Fraunhofer Insti-tute for Production Technology IPThave combined two techniques, digitalholographic microscopy and opticaltweezers, in order to reduce the numberof measurements needed to determinehow individual cells react to new drugingredients. This reduces the time re-quired to perform the measurements by50%-80%. A fluorescence microscopeis used to produce a three-dimensionalimage of a cell, which can be viewed ona computer. Optical tweezers are usedto move and place individual cells intomicroscopic wells with the help of alaser. The response of the cells to differ-ent chemical can then be observed inthese wells.Reference: http://www.fraunhofer.de/en/press/research-news/2014/january/faster-testing-of-new-pharmaceuticals.html

Correlative Light ElectronMicroscopy

In order to study the movement ofcertain proteins inside a cell, both lightand electron microscopy are needed toobserve the same specimen. Light mi-croscopy allows for the rapid observa-tion of live and fixed cells, whileelectron microscopy provides a higherresolution. In Correlative Light Elec-tron Microscopy the cells are first ob-served using a fluorescence microscopeand are then rapidly fixed and preparedfor subsequent electron microscopic ob-servation. The cells are first prepared usingmarkers. These bind specifically againstcertain intracellular structures (usingantibodies) to make them visible. Theproblem is, that the fluorescent markerswhich are needed for light microscopy

cannot be seen under the electron mi-croscope. Electron microscopy, in turn,uses gold particles to label certain intra-cellular structures. These cannot be seenwith the light microscope. The chal-lenge was to find a labeling method tomake the structures visible for both mi-croscopic techniques. Ideally one probecontains both a fluorescent marker anda gold particle, but the article also ad-dresses other methods.Reference: http://link.springer.com/article/10.1007/s00709-013-0597-5

Facebook page: History ofMicroscopy

A new Facebook page dedicated tothe history of microscopy, has beenfounded. The page is dedicated to “pre-1900 microscopes, slides and their mak-ers”. Please support the Facebook pageby “liking” it! The Facebook page be-longs to the website microscopist.net,which documents a large collection ofhistoric microscope slide makers.Reference: https://www.facebook.com/microscopist.net

Nikon Small World Competition

Vim van Egmont won this year’sNikon Small World photomicrographycompetition with a picture of the colo-nial marine diatom, Chaetoceros debi-lis.Reference: http://www.nikonsmallworld.com/galleries/photo/2013-photomicrography-competition

Olympus BioScapes Competition

Igor Siwanowicz won the 2013 mi-crography competition with a pictureshowing Utricularia gibba, with single-cell organisms inside.Reference: http://www.olympusbioscapes.com/gallery/2013/

Obtaining depth information

Researchers from the Institute ofMolecular Pathology (IMP) Vienna,Austria, developed a method to quicklyobtain three-dimensional information ofa specimen sample. The researchersused fluorescent markers to label thecells and were able to convert the dis-tance of the marker to the slide to acolor change of the emitted light. It istherefore not necessary anymore to scanthe different depths of the sample andonly one measurement is necessary toobtain full three-dimensional informa-tion of the fluorescent markers in thespecimen. The depth of the marker inthe cell could be represented by thecolor given off by the fluorescing mark-er. The researchers achieved this byplacing the labeled specimen on a spe-cially prepared quartz microscope slide,which was covered with a thin silverfilm and a dielectric layer.Reference: http://www.imp.ac.at/news/press-releases/press-release/press-release-pushing-the-limits-of-light-microscopy/

Observing batteries in action

Researchers have developed a meth-od to microscopically observe the elec-trodes of a battery during operation. Theresearchers could see that the electrodeschange their thickness as they give offand accept positively charged ions fromthe surrounding electrolyte. Up to thispoint, the observations were based ontransmission electron microscopy in dryconditions. The researchers now devel-oped a method to observe the electrodessubmerged in a liquid electrolyte, in wetconditions.Reference: http://www.newswise.com/articles/ batter-ies-as-they-are-meant-to-be-seen

Web pages to check out!What’s going on on the web? Here is a summary ofmicroscopy related publications for January 2014.

Oliver Kim

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MICROSCOPYNEWSMicroscopy on the Web

Increasing the resolution

Researchers from Switzerland couldsignificantly increase the resolution ofconventional light microscopes usingoil or water immersion objectives. Theyachieved this by simply placing micro-spheres made of barium titanate glasson top of the sample. This way theycould observe structures which weresignificantly below the resolution limitof the microscope.Reference: http://www.nanowerk.com/spotlight/spotid=33865.php

DNA-PAINT

Scientists of the Wyss Institute(Harvard Univeristy) developed a meth-od to surpass the 0.2 micrometer resolu-tion limit of conventional microscopes.They designed DNA probes with a fluo-rescent marker which are able to selec-tively bind against cell-internalstructures. Once bound, they create a

blinking effect, which can be picked upby the microscope. This method is ableto overcome the diffraction limit of mi-croscopes and is therefore able to visu-alize extremely small structures.Reference:http://www.molecularimaging.net/topics/Molecular-imaging/biomarkers/%E2%80%98blinking%E2%80%99-dna-probes-revolutionize-optical-microscopy

Low-cost webcam microscope

Researchers of the University ofHelsinki and Karolinska Institutet, inSweden constructed low-cost micro-scopes from inexpensive webcams. Theoptics of these devices were removedand the specimen was placed directly onthe image sensor. They were able toidentify several pathogenic parasitesthis way.Reference: http://yottafire.com/2014/01/stripped-mobile-phone-camera-turned-mini-microscope-low-cost-diagnostics/

Even more low-cost DIY webcammicroscope

As a weekend project, why notmake your own microscope? This postgives a step-by step instruction on howto modify a webcam to be able to viewmicroscopic objects.Reference: http://www.instructables.com/id/Save-money-on-your-DIY-laboratory-robust-easy-to-f/

Microscope in space

Already in 2011, NASA carried alight microscope to the InternationalSpace Station (ISS). Now the spinning-disk iMIC light microscopy platformwas introduced. This system is able toperform 3D imaging in high resolutionin microgravity. It is able to fit into a 44cm diameter rocket and will allow thestudy of the space environment on cells.Reference: http://www.bioopticsworld.com/articles/2014/01/an-optical-spin-on-rocket-science.html

White hair and black hair

The picture above shows blackand white hair of the same person.Two separate pictures were stackedin order to increase the depth of field.

Achromotrichia is the loss of haircolor due to aging. The hair folliclescontain stem cells, which form mel-anocytes. These produce the pigmentmelanin which is responsible for thehair color. As the person becomes old-er, more and more of these stem cells

die off and insufficient pigments canbe formed. As a result the hair turnsgray or white. It is still not understoodwhy certain hair follicles lose the mel-anocytes, earlier than others.

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Making stereoscopic imagesOne possibility is to tilt the slide to make stereoscopictwin images.

Oliver Kim

Greenough-style stereo micro-scopes use two different objec-tives and produce two separate

images of the specimen, one for eacheye. The objectives are not parallel toeach other, but oriented at an angle ofaround 11 degrees. Each objective,therefore, observes the specimen from adifferent side – one more from the left,the other one more from the right. Thesetwin images are merged by the brain toproduce a stereoscopic image. Parts ofthe images which are the same (such asthe background) will be perceived asbeing further away. Parts of the imageswhich are shifted will appear to be clos-er to the observer. Compound microscopes, in compar-ison to stereo microscopes, produce aflat, non-stereoscopic image. In binocu-lar compound microscopes the lightfrom the objective is split into two iden-tical beams and both eyes receive thesame picture. For this reason no three-dimensional image is formed. Already in the January 2012 issue, Ihave described how to use the focusstacking software Picolay to make ste-reoscopic images. This program is quite

versatile. It is able to merge severalimages of limited depth of field (i.e.with parts of the image being not infocus) into one final image in which allparts are in focus. The program is alsoable to use this depth information toproduce stereoscopic images which canthen be viewed either cross-eyed or us-ing red-cyan glasses. Instead of using a computer algo-rithm to make stereoscopic pictures, it isalso possible to horizontally shift thespecimen to the left and the right side ofthe field of view and take a picture ineach case. The specimen is then seeneither slightly from the left or from theright. These two images can then becombined to make a stereoscopic im-age. This method, I found out, is notquite as effective because the stereo-scopic effect is not very strong.

Tilting the slide

The third method for obtaining ste-reoscopic images is by tilting the speci-men slide. In this case too, the specimenis either viewed from the left or fromthe right side and the two images can

then be combined. It is this method thatI want to talk about in this article. Con-sidering the fact, that software stackingprograms already give you so muchcontrol over making a stereoscopic im-age, I consider the tilting method moreof a proof of concept, but certainly oneworth being explored more. For obtain-ing the best results, images have to befocus stacked anyway. One possible advantage with thetilting method is that the specimen canbe tilted to much larger angles thanwhat can be achieved by software alone.For making stereoscopic images, theselarge angle are not needed, but can be an

Figures 1 and 2: One side of the slidewas raised, then a picture was taken.For the twin image the other side wasraised the same way.

Figures 3 and 4 (opposite page): Ste-reoscopic images of radiolaria ob-tained by tilting the slide. You canobtain a stereoscopic impression byusing cross-eyed viewing of theseimages.

HOW TO Stereoscopy

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HOW TOStereoscopy

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advantage when making so-called rock-ing GIFs. These are animated images,which alternatively show the specimenfrom two different angles and therebygive a perception of depth. Figures 1 and 2 show an improvised,but functional method of tilting theslide. I placed blank slides beneath thespecimen slide in order to lift them afew mm. I took a series of pictures andstacked them and then repeated the pro-cedure with the other side of the slide.

Choosing the right angle

Lifting the slide too much will resultin twin images which are too differentto each other for the brain to combinethem. If the two images are too similar,on the other hand, then the stereoscopicimpression is not very strong.

Choosing the right specimens

Thin microtome cuts of tissue areprobably less suitable, as they (inherent-ly) do not have much depth variation.Whole mounts, such as the flea in Fig-

ure 5, give a much stronger impression.These specimens, however, must be fo-cus stacked in order to get the best re-sult.

Disadvantages

Tilting the slide does have severaldisadvantages as well. First, the bestresolution can not be obtained anymoreas the light passes diagonally throughthe cover glass. This way the effectivecover glass thickness is increased andespecially for high numerical apertureobjectives the consequence can be a lossof resolution. I found, that the loss ofquality for a 4x objective is minimal,however. A second disadvantage is thatat high tilting angles the objective mighttouch the slide. For making stereoscopicimages, however, this angle is not verylarge anyway, but may become a prob-lem if you want to make a rotating ani-mated GIF image. The thirddisadvantage is that the tilted slide willcause some parts of the specimen befurther away from the objective than

others. As a consequence parts of theimage will be out of focus.

Cross-eyed stereoscopic viewing

It is possible to view the twin imag-es stereoscopically without and addi-tional tools. It is necessary to crossone’s vision. Four blurry pictures be-come visible. It is then necessary torelax the vision until the center twoimages start to form a clear stereoscopicimage. This only works if the two picturesare of the same size and orientation. Thepicture for the right eye must be locatedon the left, and vice versa. If you do notwhich side is the correct one, then thefastest way is simply trying it out.

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Figure 5: The dog flea. Use cross-eyed viewing to get a stereoscopicimpression.

HOW TO Stereoscopy

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MicrobeHunter Microscopy Magazine - March 2012 - 9Send images to editor@microbehunter.com - December 2013 - MicrobeHunter Microscopy Magazine - 9

The picture shows a 117 µm high Nebela, probably of the tincta/collaris group. It was taken under obliqueillumination and shows that the plasma body does not fill the test, but is fixed to the inner wall by tiny strandsof plasm, called epipodia (Reference: www.arcella.nl).

Image by Hans Rothauscher

Testate amoebae GALLERY

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The effect of light intensity andbulb age on color temperatureBlue filters can be used to correct the color temperature.

Oliver Kim

Halogen and other tungsten lightbulbs have a few characteris-tics, which can not be found in

LED lamps. First, they have a compara-tively broad color spectrum, with astronger emphasis on the red end of thespectrum. Conventional incandescentlamps also have the tendency to changetheir color temperature as they age, withthe color becoming increasingly redderover time. This is due to tungsten de-posits forming on the inside glass sur-face of the bulb. Halogen lamps show a lower ten-dency to do this compared to otherbulbs with a tungsten filament. Halogenbulbs are filled with a halogen gas,which is responsible for removing the

deposited tungsten from the glass andre-depositing it on the filament. Last, the color temperature changeswith the light intensity. The brighter thelight, the hotter the filament and thecooler the color temperature. At highbrightness levels the color containsmore of the blue spectrum. At low lightintensity levels the red proportion of thespectrum is clearly visible. The back-ground of the specimen appears yellow-ish and not in a pure white. All of these effects can make it diffi-cult to make reproducible pictures andit might be necessary to apply a digitalwhite balance filter. The red compo-nents of the color spectrum can also bereduced by placing a blue filter into the

light path. Some microscope condens-ers already come with a built-in bluefilter, and also digital cameras and web-cams frequently have a filter installedright over the sensor in order to reducethe red components of the light. From practical experience I can saythat these filters are not able to com-pletely remove the color shift. I there-fore always use my microscope with anadditional blue filter placed over thelamp. This blue filter is a so-called day-light filter from the time of analog pho-tography. During the time of analogfilm photography, it was possible to buydifferent film types for different lightsituations. Daylight films produced thebest results in the sun, while films made

1. Low intensity, no filter

4. Low intensity, with filter

2. Medium intensity, no filter 3. High intensity, no filter

5. Medium intensity, with filter 6. High intensity, with filter

OBSERVATIONS Testing color temperature

December 2013 - MicrobeHunter Microscopy Magazine - 11

for artificial light were best used in-doors. The blue daylight filter nowmade it possible to use a daylight filmin artificial lighting conditions. The fil-ter essentially made the warm artificiallight cooler to adapt it to the daylightfilm. I have not exchanged the halogenbulb of my microscope for about 16years now - it is still the first bulb whichwas installed and the bulb still worksfine. I now wanted to test if an exchangeof the bulb has a significant effect onthe color temperature.

The method

I took pictures at three different lightintensity settings, with and without adaylight filter. I then repeated ex-changed the bulb with a new one andtook the same images. I used the auto-matic exposure settings of the camera.In order to standardize the brightnessstill further, I manually adjusted thehistogram in order to stretch the con-trast to a maximum (Figure 7) to makethe images more comparable. The ad-justment of the brightness did notchange the white balance.

The results

The results for the old light bulb canbe seen in figure 1-6. First, the age ofthe lamp did not make any notable dif-ference. The pictures had the same col-ors as those of the old bulb. For thisreason I did not include them. At low intensities the blue filter de-creased the brightness significantly.This is due to the red components of thelight, which are absorbed by the filter.At medium intensities the blue filterworks best (Figures 2 and 5), giving thepictures a neutral color. At high lampintensities the blue filter causes an un-

naturally blue hue (Figure 6). The bluefilter is therefore best used at mediumintensities.  ■

Figures 1 - 6: Color temperature de-pends on light intensity. At mediumintensities the blue filter significantlyenhances the image. The backgroundappears less yellow. The color of thespecimen (individually the bluestained cells) is also more visible. Athigh intensities, the blue filter is alsonot suitable, making the image tooblue.

Figure 7: The levels dialog box in aphoto editing program can be usedto both correct contrast and whitebalance. In order to make the imagesmore comparable, I manually adjust-ed each image to maximum contrast,but did not do a white balance.

Figures 8 - 9: The blue daylight filter.

OBSERVATIONSTesting color temperature

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What’s this? Answer on page 2.