How to Improve PhotographyThrough the Microscope

•••••••+.lA.. _3.

How to get the most out of this bookletThis booklet is divided into four main sections. Items marked with a • list the minimum informa-tion that newcomers to the art of photomicrography need to know, while items left unmarkedare addressed to users who have already taken photos with the microscope but are not satisfiedwith the results. By referring to sections 1 to 3 for picture taking and section 4 for checking,you will find this brochure a reliable help the next time you want to take a photomicrograph.

From preparation to observationMicroscopes suitable for photomicrography 8Equipment suitable for photomicrography 9Various types of photomicrographic equipment

and pertormance 10• Equipment needed for photomicrography 11

Suitable locations for setting up the microscope 12.Objectives and photo eyepieces suitable for

photomicrography 13• Differences in the peripheral images depending on

objective design 14• Differences in resolution depending on the type

of objective 15.Some hints about the objective 16-18

Selection of a condenser 19.Observation procedures 20- 21.Handling of specimens 22

Use of the field iris diaphragm 23Use of the aperture iris diaphragm and its effects .. 24- 25

• Basic focusing methods 26- 27How to spot dirt and specks of dust in the optical

systems of the microscope and the photomicro-graphic attachment 28

• How to clean the microscope frame 29• How to clean the optical system 30- 31• How to clean an oil-immersion objective 32• How to clean specimens 33

How to obtain good printsHow to obtain good color prints 60-61

.Preparing black-and-white prints 62Marks on the photo 63How to avoid marks during development 64- 65

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Section!From preparation toobservation

Section 2When taking picturesin photomicrography

Section 3How to obtaingood prints

In the case of poor photos

Section 4Trouble-shooting

I Ireduction of equipment suitable forrvation and photomicrography, basicledge, observation procedures,Ing methods, etc. are explained inBy-to-understand manner.

10 knowledge and explaliilaHorl ofpi procedures needed for photo-hy, diffe~erncesin films and purchase1mB,etc.

rrect processing can ruin the best! This section deals with film de-ent, how to order color prints, etc.

rranging finished photomicrographsrdlng to their characteristics you canthe causes for failure and take cor-IV! action. In many instances, the

I -shooting section can also serve, a check-list.

When taking pictures in photomicrographyBasic information in photomicrography

Operating instructions for models PM-10ADandPM-1OADS(with 35mm camera back) _.. 36-37

Magnification of photographic equipment 38Differences in resolution according to the combinations

of objectives and eyepieces 38Setting of photographic magnification (effective

magnification) 39Framing of the specimen 39Checking of the finished photomicrographs 40-41

Photomicrography techniquesExposure adjustment techniques 42-43Use of the AE lock 44-45Compensating for a film's reciprocity failure 46

Color photomicrography• Color film 47• Types of filters 48• Differences in color rendition depending on the

combination of film and light source 49• Differences in color reproduction depending

on differences in color temperature 50• Differences in color rendition depending on the type

of film 51• Purchase of color film 52

Use of COlor-compensating (CC) filters when takingcolor photos 53

• Test photography 54Black-and-white photography

• Black-and-white film 55• Comparison of different film brands 56• Filters ' 57

Photography with Polaroid film 58Trouble-shooting

Problems In finished photos and their correctionPoor color reproduction 68- 71Blurred image 72-73The image is in focus but not sharp 74-79Objects other than the specimen image appeared

on the film 80-81Uneven brightness 82-83

o

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From preparation to observationSection!

Microscopes suitable for photomicrography 8Equipment suitable for photomicrography 9Various types of photomicrographic equipment

and performance 10• Equipment needed for photomicrography 11

Suitable locations for setting up the microscope 12• Objectives and photo eyepieces suitable for

photomicrography 13• Differences in the peripheral images depending on

objective design 14• Differences in resolution depending on the type

of objective 15.Some hints about the objective 16-18

Selection of a condenser 19.Observation procedures 20-21• Handling of specimens 22

Use of the field iris diaphragm 23Use of the aperture iris diaphragm and its effects .. 24 - 25

• Basic focusing methods 26 - 27How to spot dirt and specks of dust in the optical

systems of the microscope and the photomicro-graphic attachment 28

• How to clean the microscope frame 29• How to clean the optical system 30 - 31• How to clean an oil-immersion objective 32• How to clean specimens 33

Microscopes suitable for photomicrography1. The phototube accepts the photomicro-

graphic camera attachment. ---------------,

3. The desired light intensity matchingthe specimen conditions can be setby the optical path selector. ----------''''

2. The image can be focusedthrough the binocular tube. -----,

9. The microscope stand is resistantagainst external vibrations. -------- <The photo shows model BHS.

------.-4. Objectives should have high resolution

and good flatness (flat image all the wayto the periphery of the visual field). -----~

5. The stage is rotatable. ------------.,,-

6. The condenser is equipped with anaperture iris diaphragm and centeringdevice for aligning to the optical axis. ----'''''----''''l

7. The microscope is also equipped witha field iris diaphragm. --------------11

8. Sufficient light intensity is assured.Light intensity is adjustable to match theobservation and photographing condi-tions. Uniform illumination from lowto high magnification. --------,---------

10. Provision for insertion of filters. -----------------------------'

Equipment suitable for photomicrography

1~-:----- 1. The camera attachment accepts 35mm.~=~=c:::=========-~ or large-format films. The 35mm camera~•• ~.F=="':=9"•• ~ back is equipped with automatic film, advance,

,f -_. -

~ OLYMPUS C 35M 2 _ WINnIN(,

1----- 2. The optical path can be changed tomatch photographic conditions.

3. The measuring area can be changedto match the specimen. (Integratedmetering 30%-spot metering 1%)

----- 4. The camera attachment is firmlyclamped on the microscope.

5. Device for measuring color temperature.

7. Compensation for reciprocity law failureis carried out automatically for longexposure.

----+-- 8. Exposure adjustment, matchingspecimen conditions, is possible.

g. Manual exposure is possible.

-The photo shows model PM·1OADS.

6. Wide range for setting ISO/ASAsensitivity.

10. Special applications, such as 16mmcine and 35mm time lapse photography,can be performed.(A special control unit is required inthese cases.)

11. An exposure lock mechanism (AE lock)is built-in.

Various typesof photomicrographicequipment and performance

PM·10ADSI PM·10M PM·6 OM Series SLR cameraPM·10AD and t-adapter

Ease of0 0 0

The focusing screen must be re-focusing placed for photomicrography*'

Photographic0 0 0 Shutter blur may result when 40X

quality and 100X objectives are used*2

-Manual winding of -Manual winding of filmEase of -Use of cable releaseoperation 0 35mm film -Use of Varimagni Finder for-Use of cable release focusing

Exposure mea-0

Use of exposure meter -Use of built-in exposure metersurements (EMM-7)

Use of largefonnat film

O· 0 X(4" x 5" sheet, X

Polarol~) .

*, Concerning the focusing screen forOlympus OM System cameras:The screen can be changed to suit differentuses (OM-1, OM-2, OM-3, OM-4). For photo-micrography use screen No. 1-12.

*2 Shutter blur may occur if magnification isincreased. When using 40X and 100Xobjectives, adjust the light intensity so iha jthe shutter speed is 1 - 2 sec. In this case,some types of color film may require colorcompensation.(For color compensation refer to page 53)

,

Equipment needed for photomicrography.st check the chart t~ see if the equipment

required for photomicrography has been com-pletely assembled. (The types listed below arethe most basic attachments needed for takingphotosof stained specimens in transmitted light.

Equipments required in photographingCD Focusing magnifier (F1)

Focusing Telescope35mm camera backAutomatic exposure body

® Photo eyepiece®Connecting cord

Large-format camera backAdapter for large-format camera

®Automatic exposure control unitPower cordSpecimenInstruction manualsFiltersFilmForm for recording dataCleaning utensils: blower, cleaning liquid,lens cleaning tissue

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Suitable locations forsetting up the microscope

Reasons for unsuitable locations and corrective measures tUnsuitable room Consequences Treatment

-Located too close to mechanical appliances or -Remove the microscope from the sourcemachinery that can cause external vibrations

Blurred image as a result of vibrationsof vibrations

-Places in which the vibration of persons -Use a sturdy table as supportwalking past can be transmitted -Use a vibration-proof table

-Use of the microscope near a window Bright light from the window prevents correct -Set up the microscope near a wallfocusing -Position the microscope in such a way

that the overhead light falls in Slightly infront of the microscope.

-Place where room light enters the eyepiece Room light or flares are reproduced on the -Cover the eyepieces with caps

photo -Shut out stray light getting into the eyepieceor the focusing telescope by changing theoptical path selector

-A dusty and dirty room Black spots are reproduced on the specimen -Set the microscope up in another room-Place near a window where dust can enter image -Cover the whole microscope with a dust-

from the outside proof covering

,

Example of a suitable room for photomicrography

Objectives and photo eyepieces suitablefor photomicrography

~ojectivesFor photomicrography high-resolution objec-tives with flatness all the way to the peripheryof the visual field are required. Of the LB (Iong-barrel) objectives, series S Plan Apochromat,S Plan Achromat, and D Plan Achromat,and of the short-barrel objectives, PlanApochromat and Plan Achromat types arerecommended.Photo eyepiecesThe photo eyepiece is optically compensatedto permit the objective to deliver its fullperformance on the film plane. It must becorrectly matched with the objectives.

Correct combinations of objective and eyepiece(1) Combination with NFK t e 2 Combination with FKlP types

S Plan Apochromat series

S Plan Achromat series

D Plan Achromat and D Achromat series

\'~81 It· '~IIt-- .. ~'. . . I, -> -~ .••..•... .=-\,._,~ r > f;""~~

... -•••••

Short-barrel Plan Apochromat series

Short-barrel Plan Achromat series

Short-barrel Achromat series

Differences in the peripheral imagesdepending on objective design

According to the type of objective, the peri-phery of both the observed image and thephotographed image may appear out of focus.This effect is caused by the performancecharacteristics of Achromat type objectives,Using Plan Achromat objectives, however,will result in a sharp and flat image extendingall the way to the periphery of the fieldof view.

The field flatness of a D Plan Achromat objective extending all the way to the periphery is superior tothe one provided by a D Achromat objective.

D Achromat 10X, NFK 3.3XLD

Difference in resolution dependingon the type of objective

vsolution in the cen er of the field of boththe observed and the photographed imagesdiffers according to the type of objective.The top class objective series, S Plan Apo-chromats, as well the S Plan Achromatseries provide superior resolution.

o Plan Achromat 40X, NFK 3.3XLD

Gives sharp resolution of the whole image, allowing observation of minute details.

Some hints about the objectiveIt is important to choose an objectivesuitable for your specific purposeFor all objectives, proper use depends on thespecific purpose, some types requiring someadjustments. In order to fully utilize theobjective, you should know the meaning ofthe various numbers and letters engravedon the objective barrel.

1. Objective with correction collarIn objectives with a numerical aperture (N.A.)above O.6-excluding oil-immersion types-the thickness of the cover glass stronglyaffects the image quality. Cover glass thick-ness is theoretically designated at O.17mm,although in actual practice this may vary by± O.3mm. By optically compensating for thisthickness deviation, the correction collarassures the best image.

Objective type and magnificationMechanical tube length ---t--- Numerical aperture (N.A.)of the microscope -------j;;;:;;;;;;;.!:~!Z.==r-- Thickness of the cover

glass to be used

Method of adjustment(1)Set the scale to the O.17mm position,

then focus.(2)Rotate the correction collar 2 or 3 gradua-

tion marks (O.02-0.03mm) in the pirectionof 2, and refocus. If the image is sharperthan under (1), rotate the collar another2 or 3 graduation marks in the samedirection and focus again.

(3) If you are not satisfied with the imagequality, try rotating the collar in the oppositedirection for 1-2 graduation marks, re-focusing and comparing the image.

(4)Gradually reduce the amount of rotation ofthe correction collar, and try to find the op-timum condition by repeating steps (2H3) .

• The above adjustment procedures mustbe repeated every time the specimenis changed.

0.17 0.23

1017mm

2. Objective with iris diaphragmSome objectives above 40X are equipped withan iris diaphragm. This diaphragm preventsdirect light from entering the objective indarkfield or transmitted light fluorescenceobservation. By watching both contrast andresolution of the image, the aperture canbe adjusted to the optimum position.

D Plan 50X, S Plan Apo 100X, NC S Plan Apo 100X

,

This mark Indicates that no coverglass is needed -------,---+N

3. No-cover (NC) objectiveFor specimens without cover glass such assmears, objectives bearing the mark NCof the cover glass affects the image clarity,a fact which is particularly obvious in the caseof objectives with a large numerical aperture.As a result, when observing specimens notprotected by a cover glass at high magnifi-cation, a special NC objective is used.When viewing uncovered specimens throughnormal objectives, only poor images withmany flares and insufficient resolution canbe obtained.

The Olyrnpus LB series no-cover objectives:NC S Plan 40X, NC D Plan FL 6OX, NC S PlanApo 100X, and NC S Plan 100X dry

4. No-cover 100X dry objectiveHigh-magnification 100X objectives are usuallyof the oil immersion type, but for the no-cover100X objective a dry type is available. Usingthis objective, together with other non-immersion objectives, e.g. no-cover 40Xand 60X, obviates the need to put immersionoil on the specimen slide. The 100X dry-typelens can also be used for photomicrography,but for achieving optimum picture quality,the use of an oil-immersion objective isrecommended.

Objective type and magnificationN I I t (NA) SPlanApo 100 This mark indicates an

:~~~~ic~~~~~elen~t;,===t~'~oo"1~o~~;~~~~===l- oil-immersionobjectiveof the microscope Thicknessof the cover glass

to be used

5. Oll-lmrnerslon objectiveUse of the oi/-immersion objectiveOil-immersion objectives have a numericalaperture above 1.0 and use manufacturer-specified oil between the front lens of theobjective and the specimen. (Oil-immersionobjectives carry the mark "oil".) In order tomake full use of the resolving power of theobjective, it is preferable to use oil alsobetween the condenser front lens and thespecimen slide.

How to apply the oil(1)Focus on the specimen, with the 10X,

or 40X objective and bring the desiredspecimen detail in the field of view.

(2)Rotate the revolving nosepiece so that theoil-immersion objective is pointing towardsyou, and apply oil to the front lens ofthe objective.

(3)Apply oil to the specimen surface androtate the nosepiece until the oil-immersionobjective, its tip likewise covered with oil,

* Use only oil specified by themanufacturerIf you use old Cargill oil or cedar oil, theobjective cannot display its full potential,since their diffraction coefficients differ fromthe nominal value. If the oil is tinted, it affectsthe quality of color rendition in color photos.Therefore, it is advisable to use only manufac-turer-specified oil. In particular for fluorescenceexamination, use only the fluorescence-freeoil provided with the fluorescence microscopes.

clicks into the light path. Make sure thatthe objective front lens is fully immersedinto the oil on the specimen slide.

(4)If the image is not in view, slowly rotate thefine focusing knob till it comes into focus.

• Make sure that the objective front lens doesnot get too close to the specimen, sincethe image will deteriorate if air bubbles getinto the oil. If a haze seems to cover partsof the image, even though it is in focus,swing the nosepiece 1 or 2 times from theclick stop in order to remove the air bubbles.

If the image still does not improve, removethe eyepiece as illustrated above, checkfor air bubbles by viewing the back lensof the objective, wipe off the oil andreapply oil.(For the method on how to clean an oit-immersion objective, refer to page 32)

Selection of a condenser~ purpose of the contlenser is to efficiently

iocus the light, emanating from the lightource, on the specimen, to create lightingonditions matching the objective, and thus

to provide a better image. Depending on theIntended use, several types of condensersIre available. Particularly with ultra-low mag-nification objectives such as 1X, 2X, and 4X,problems like uneven illumination and lnsut-

ficient amount of light at the periphery arelikely to occur. Therefore make sure to usethese objectives in combination with theultra-low magnification condenser.*In order to obtain better photos with theS Plan FL 2X objective, use of the ultra-lowmagnification condenser BH2-ULC isrecommended.

Combination of BH2 series condensers and LB objectives

BH2-AACAchromaticAplanaticcondenser

BH2-SCAchromaticSwing-outcondenser

BH2-CDAchromaticcondenser

BH2-ULCUltra-lowcondenser

S Plan FL 1X,S Plan FL 2X,differentdesign 4Xobjectives

Observation proceduresNow we are finally getting to observation,but first make sure that no dust or dirt ison the objective, eyepiece, and specimen.Make it a habit to check for dirt before youuse the microscope, since dirt preventsfocusing and results in poor image quality.(For dust and dirt detection, refer topage 28, for cleaning methods refer topages 29-33)

1 Placement of filters 2 A~justment of interpupilla.____ distance

Turn on the main switch and adjust thevoltage to position Photo (ca. 9V).

(1)Place the light balancing filter (LBD-2N)on the light exit window.

(2)Place an ND filter in the slot close to thelamp housing. Depending on the objectivemagnification and the density of thespecimen, use this filter so that it providesenough brightness for easy examination.

(3)Place the specimen on the stage and focuswith a 10X objective.

(For the method of focusing refer topages 26-27 )

(4)Adjust the interpupillary distance until bothleft and right view fields merge into one.

Di'c;)pter adjastim.ent

Adjust the diopter to suit the observer'syesight. The method differs according to

the eyepiece used.Unless the diopter is adjusted, parfocalitywill not be maintained when the objectiveis changed.

(5)When using WHK 10X eyepieces, thefocus is adjusted with the focusing knobswhile observing through the right eyepiece.The diopter adjustment ring ® on the leftside is then adjusted for maximum imageclarity for the left eye.

•(6)When using a finder eyepiece for photomicro-

graphy, adjustment is made by rotatingsection @ of the eyepiece at the right eye.

(7)At that time, adjust the image so that thecross lines in the center of the frame maskare clearly distinguished as two separatelines. Then adjust the focus by rotatingthe fine-focusing knob so that the crosslines and the specimen image are in focussimultaneously. After completing right-eyeadjustment, also adjust the diopter for theleft eye by rotating section ® as in (5).

• Since the type of the finder eyepiece differsaccording to the size of the film used,choose the type suitable for your particularpurpose.

[][jWHK10X 3Smm film

~WHK1OX 4xS" sheet film

0WHK10X 4 1/4 x 3 1/4" Polaroid"

IMH I WHK10X 16mm cine

(8)After focusing on the specimen with the10X objective, rotate the field stop ® inthe direction of the arrow and reduce thefield iris diaphragm diameter to a minimum.Then slowly move the condenser from topto bottom by using the condenser heightadjustment knob and stop at a positionwhere the field iris diaphragm imageis sharply defined.

• Move the field iris diaphragm image to thecenter of the visual field with the con-denser centering knobs © .

(9)Open the field iris diaphragm image untilit almost touches the periphery of the visualfield, and make some final centeringadjustments. For normal observationconditions, make the diaphragm slightlylarger than the visual field.

• If the field iris diaphragm cannot be sharplyfocused, check the thickness of the speci-men slide. Use slides with a thicknessbetween 0.9 and 1.2mm.

Handling of specimensMake it a habit to clean the specimen regularlyboth before and after observation. Just aswith lenses, it is most important to work witha clean specimen. Make sure that no dustparticles stick to the specimen when youstore it and do not touch the glass surfaceswhen you handle it.

Notes on specimen placement on the stageWhen you place a specimen on the stage,the specimen holder is opened wide, and ifit is released rapidly, it will hit the edge of theslide and damage the glass. After confirmingthat the specimen has been firmly put in place,the spring-loaded specimen holder must beretracted slowly so that it gently touches theedge of the slide.

In case the slide is damaged, carefullyremove the tiny glass fragments. If fragmentsare left on the stage, they may cause injuries,or the specimen slide may be placed in atilted position on the stage, causing one sideof the visual field to be out of focus.

Hold the glass slide at the corner only, or hold the glass slide between your fingers.

Fingerprints on the cover glass.

Put the specimen firmly in place and slowly retractthe specimen holder.

The image Is blurred along the traces of yourfingerprints.

t:,~,

One side of the visual field is blurred because of atilted slide.

Use of the field iris diaphragm•. le field iris diaphragm serves to adjust the Using the PM-10AD turret mask focusing telescope (with 35mm film)

Illuminated area on the specimen dependingon the objective power. This diaphragm playsIcrucial role during photomicrography, and if

opened wider than necessary, illuminating lightI reflected and scattered irregularly on thepecimen, resulting in a loss of image contrast.topping down the field diaphragm to just

beyond the frame reticle area will result inphotographic images with improved contrast.

If the field iris diaphragm is stopped downtoo close to the frame reticle, the photo-graphed image may be cut at the corners.The diaphragm should therefore be openedslightly more than the reticle shows.

The corners are cut because thefield Iris diaphragm has beenstopped down too much.

Use of the aperture iris diaphragmand its effects

The diaphragm mounted on the condenseris called aperture iris diaphragm. The functionof this diaphragm is to maintain optimumconditions of image resolution, contrast andfocal depth by adjusting the numerical apertureof the illumination system depending on thenumerical aperture of the objective in use.For most specimens optimum image qualityis achieved if the aperture diaphragm isadjusted to between 60% and 80% of theobjective numerical aperture.

How to adjust the aperture iris diaphragmThere are two methods of adjustment:Pull out the eyepiece with the specimen infocus and then adjust the diaphragm whilewatching the iris at the rear focal plane ofthe objective, as in photo (1); use the gradua-tion marks on the condenser, as in (2).

Rear focal planeof the objective

Aperture Irisdiaphragm

(1)Direct viewing, with the eyepiece Iremoved.

Reducing the aperture iris dia-phragm to 60-80% of the numericalaperture of the objective.

•

(2)Using the graduation marks on thecondenserExample: Using a 10Xobjective with

a numerical aperture of0.25 and reducing it to80%, the graduation markon the condenser should

• be set at 0_2(= 0.25x 0.8).

.fects of the aperture iris diaphragm Example: When using S Plan Apo 20X, NFK 2.5XImage resolution deteriorates if the apertureiris diaphragm is stopped down too much.With the exception of specially stained thin•pecimens the diaphragm should not belopped down lower than to 60% of the

numerical aperture of the objective.An effect similar to stopping down theaperture iris diaphragm can be achievedby moving the condenser downward, butthis tends to interfere with the basic illumina-tion function of the condenser and resultsin uneven illumination. Thus always use thediaphragm and do not move the condenser.

•

Fully opened position

Overall contrast is low.

80%Contrast is enhanced. Details are alsoclearly visible, and focal depth isincreased, resulting in an optimumimage.

30%

Resolution deteriorates as a resultof diffraction.

Basic focusing methodsFocusing for observationA 10X objective is used as the standard forfocusing, then the objective is changed from10X to 4X and from 10X to 40X and furtherto 100X. Do not change abruptly from low mag-nification (2X and 4X) to high (40X and 100X).As a result of the limited eye of the observerand the large focal depth of low magnificationobjectives (2X and 4X), the front mount ofthe high-magnification objective could touchthe specimen surface when the revolvingnosepiece is rotated.

Focusing for photomicrographyFocusing during photography is done either 1through the focusing telescope of the photo-graphic attachment or through the eyepiecesof the binocular tube. When focusing throughthe eyepieces, a finder eyepiece must beused. Prior to photomicrography the findereyepiece has to be focused, by means of itsfocusing front lens, to make clearly visibledouble cross lines as two parallel lines inthe center of the framing reticle.

~II-- --

IIII

II---II

Blurred Clearly visible astwo distinct lines

1

After focusing on the specimen with 10Xobjective, set the upper limit of the coarseadjustment excursion with the prefocusinglever.

Move the specimen detail to be examined tothe center of the visual field and increasemagnification by rotating the nosepiece.

•

2

Focusing telescope of the photographicattachment

For photography the specimen focus mustbe adjusted with the same eye with whichthe cross lines were focused.

Finder eyepiece of the binocular tubeFocusing through the binocular tube ispossible with microscopes Vanox, BHS, BHT,and BHTU.

If the image is slightly out of focus afterchange of magnification, use only the fine-focusing knob for refocusing. In particular,do not use the coarse-focusing knob forfocusing a high-magnification objective, sincethere is a danger that the objective will runInto the specimen and get damaged.

The finder eyepiece is provided with a locatingpin. The pin must be firmly inserted into thegroove of the eyepiece sleeve. If the pin isimproperly inserted, correct focusing becomesimpossible, and the image will be out of focus.

1_ Focusing when using 1X, 2X, and 4Xobjectives

Focusing errors occur quite frequently withlow-magnification objectives. Therefore usea focusing magnifier and adjust the focusby following the procedures listed below.But before that the photographer shouldadjust diopter at the cross lines of thefocusing telescope or the finder eyepiece.(refer to page 21)

Clamp the focusing magnifier on thefocusing telescope, and slide the top sec-tion in or out, thereby readjusting the focusat the cross lines. Focus is correct ifboth the cross lines and the specimenimage are clearly visible simultaneously.

2. Focusing when using 10X and 20Xobjectives

Focusing is achieved by adjusting thecross lines so that they are clearly visible,and then rotating the fine-focusing knobuntil both the cross lines and the imageof the specimen are clearly visible simul-taneously. By slightly moving your eyesto all sides, see if the positions of thecross lines and the specimen image do notshift. If this is the case the picture isin focus. If they do shift, the focus is not

properly aligned and must be readjustedwith the fine-focusing knob.

3. Focusing when using 40X and 100Xobjectives

Adjust the cross lines so that they areclearly visible, then slowly adjust the focusof the specimen image with the fine-focusing knob until cross lines and imageof the specimen are clearly visiblesimultaneously.

How to spot dirt and specks of dust in theoptical systems of the microscope andthe photomicrographic attachment

Dirt and dust particles are sometimes notedduring observation or photogrpahy, but pin-pointing their exact location may be difficult.When photographing important specimenswhich cannot be photographed again it canbe really frustrating if dust particles are visibleon the picture. An effective method is there-fore required that will help you detectcontamination.

~'----'---.,;==:!!.lTfiZi~@

~-----=~"'--~------'

• Since dirt at locations no.CDand@maycomefrom partictes of the film itself (loading, filmtransport), regular checks are necessary.

• If the aperture diaphragm is reduced below30%, dirt and dust specks become clearlyvisible.

.Dust specks on the prism inside the obs »1vation tube, on the inside of the objective,and on the inside of the microscope basecannot be wiped off easily. If cleaningbecomes necessary, contact your Olympusdealer for service.

Loca- Description Obser- Photo- Method of verificationtion vat ion graphy

Relay lens for Remove the adapter for large-format cameras

CD large-format 0 and check for dirt by peering in through the top.If you spot dirt, unscrew and remove the relay lenscamera and clean it.

<V Focusing 0 Rotate the top lens element as you observe thetelescope image.

@ Camera prism 0 Set to Time mode, open the shutter, and peer inthrough the top.

Photo Either remove photo eyepiece and check for dust® eyepiece 0 particles or leave photo eyepiece in place, rotate

it and check for moving dust particles.

® Eyepiece 0 Rotate the eyepiece as you observe the image.

Optical path Change the optical path while alternately observing® selector prism 0 0 through the focusing telescope or the finder

eyepiece.Tube length Remove the observation tube from the microscope

(J) correction 0 0 body and check the prism surface for fingerprintsprism or contamination.

® Objective 0 0 Remove the objective from the nosepiece andcheck it for dirt or contamination. 1\

® Observe the specimen and move it in the field. ~ l.ISpecimen 0 0 If dust is on the specimen, it will also move.

@ Condenser 0 0 Remove the condenser from the microscope andlook for dirt and oil deposits.

(jj) Filter 0 0 Check the filter after removing it from the micro-scope base.

@ Light exit 0 0 Switch on the illumination and examine the lenswindow at an angle.

@ Frosted glass 0 0Remove the lamp housing from the base, andcheck the frosted glass.

@ Collector lens 0 0 Remove the lamp housing and check the lensesin the collector assembly.

@ Bulb 0 0 Remove the bulb from its socket and check forsigns of blackening, fingerprints, dirt, etc.

How to clean the microscope frameins on the microscope frame are first Materials used for the microscope

wiped with a piece of cloth wetted with a r-------------=-----------------------,mall amount of neutral detergent, and then

wiped clean with a piece of cloth that hasbeen immersed in luke-warm water. But makesure not to touch the lens section whilecleaning the microscope frame.Avoid using organic solvents which maydamage plastic parts.

For dust contamination that adheres topainted parts and is difficult to remove, wipewith a piece of cloth or soft tissue paper thathad been soaked in a mixture of 7 parts etherand 3 parts alcohol. Keep the mixture awayfrom plastic parts to prevent damage.

D Plasticparts

DLens

D Paintedsurfaces

o

o

How to clean the optical systemKeeping the optical system clean at all timesis most essential. But if dust gets onto a lenssurface, it can normally be removed with ablower. You should, however, make a habitof covering the microscope with a dustproofcover, after each use.

Cleaning methodIf dust spots on optical glasses such as lenses,prisms, and filters are left unattended, thedust becomes difficult to remove and maycause mold. By always keeping optical glasssurfaces clean, you avoid maintenance prob-lems and prolong the life of your microscope.Cleaning of the lens surfaces applies only toexposed areas of objectives, eyepieces, filtersand condensers. If internal or major cleaningbecomes necessary, please contact yourOlympus Microscope dealer.

Required tools

-.....- -- \I \I~( \ 1"1'1';\'.'1 \

,"~~."..,,,

CD Air gun or blower brush® Cleaning mixture of 7 parts ether and

3 parts alcohol, or lens cleaning fluid.® Q-tips, wood stick® Soft gauze, lens tissue® Magnifying glass. An eyepiece can also be

used in place of the magnifier.

~--------------------------------~J!I~1 2 ----~--~~------------

To prevent scratches on coatings and opticalglass, remove dirt and dust that sticks totheir surfaces with an air gun or blower brush.

When cleaning large glass surfaces on bothsides of an accessory such as a filter, foldtwo or three layers of lens tissue soaked inthe cleaning mixture, hold the accessory atits edges, and wipe from the center towardsthe periphery as you slowly rotate it.

ebb@!s ® ~ ® ~

Wrap the lens tissue around a wooden orbamboo stick as illustrated.

6 ~ __

@w ®n;> @flY/~LS~@t~®~

When cleaning the surfaces of the condenserand of the light exit glass hold a piece oflens tissue between your middle and indexfingers, fold it and wrap it around your indexfinger. Then hold the tissue down with yourthumb while wiping the lens surfaces clean.

Clean the lens by putting a small amount ofI ns cleaning fluid or cleaning mixture on thetip of a lens tissue. Discard each lens tissuefter a single use.

After cleaning, examine the lens surface witha magnifying glass. If color reflected fromthe lens surface looks uneven, it is an indi-cation that there are still dust specks anddirt on the lens.

By viewing through the bottom of an eye-piece, you can use it as a magnifying glass.

When cleaning a large lens surface, wipefrom the center towards the periphery ina circular motion.

8After cleaning, breathe lightly on the lens,surface until the whole surface has turnedwhite, then check whether the haze dis-appears uniformly. Spots where the hazedisappears only slowly are not yet wipedclean.

When cleaning a large lens with lens tissuewrapped around your finger, you should wipefrom the center towards the periphery in acircular motion. Always, use a clean portionof the lens tissue as you rotate your indexfinger.

How to clean an oil-immersion objectiveClean the oil-immersion objective duringexaminationAfter finishing observation with an oil-immersion objective, wet a pad of cotton-woolor a piece of lens tissue with a small amountof cleaning mixture containing 7 parts etherand 3 parts alcohol, to remove oil adheringto the objective. Since an oil film will oftenadhere to the objective front lens, it shouldbe wiped clean twice after each use.

Use only factory recommended immersionoil and remove it after examinationIf the oil remains on the objective for a longperiod of time, it will harden (e.g. cedar oil),making it difficult to remove even if youwipe the lens repeatedly. The lens may bedamaged in the process. Use only specifiedoil, and after use, wipe the oil from the immer-sion surface of the objective, keeping itclean at all times.

If you frequently use an oil-immersion objec-tive, oil may contaminate the surface of adry-type objective when you change objectives.To prevent the oil from adhering to theobjective, carefully rotate the nosepiece afterlowering the stage, so that the oil does nottouch any Objective.* If the image of a dry high-magnification

objective appears fuzzy, check for oil thatmight have adhered to the tip of theobjective.

Wipe the front lens of the objectiv )1 ~

,

Lower the stage slowly so that no oiltouch the front lens of the dry objectiv

After lowering the stage, carefully ro Inosepiece and click the dry objective Inoptical path.

How to clean specimens

There are two types of specimens: those withcover glass, and those without a cover glass,

such as blood smears. Cleaning specimens with cover glass1. Cleaning specimens with cover glassesJust as when cleaning lenses, wipe off theoil and dirt with a piece of lens tissue lightlymoistened with cleaning mixture. Becausethe oil cannot be completely removed withone wipe, repeat wiping until the oil filmIs removed.

Cleaning specimens without coverglasses

Oil adhering to uncovered specimens cannotbe wiped off. You can, however, remove theoil by immersing the specimen for 5 to 10minutes in a xylene bath. There are containersfor both horizontal and vertical immersion,the proper selection of which depends onyour particular need.

e it a habit to clean each specimen bothI) fore and after observation. Otherwise, dirtInd dust that you failed to notice during

observation might appear on the photo.I or cleaning the specimen a soft cloth, gauze,or piece of lens tissue may be used withoutGleaning liquid. But if the contamination isdifficult to clean, breathe on the specimenI) fore wiping it. When cleaning the specimen,wipe both surfaces.Points to note during cleaning

(I) Removal of oil as well as routine cleaningcan be done more easily if the specimenis removed from the stage.

(2)When using cleaning mixture or lenscleaning fluid, use a moistened cloth orcleaning tissue. Be certain not to applyexcessive fluid, as it may seep underneaththe cover glass and damage the specimen.

Cleaning specimens without cover glass

•

•

When taking picturesin photomicrography

Section 2Basic information in photomlcroqraphy

Operating instructions for models PM-10AD andPM-1OADS (with 35mm camera back) 36 - 37

Magnification of photographic equipment 38Differences in resolution according to the combinations

of objectives and eyepieces 38Setting of photographic magnification (effective

magnification) 39Framing of the specimen 39Checking of the finished photomicrographs 40-41

Photomicrography techniquesExposure adjustment techniques 42-43Use of the AE lock 44 - 45Compensating for a film's reciprocity failure 46

Color photomicrography• Color film 47• Types of filters 48• Differences in color rendition depending on the

combination of film and light source 49• Differences in color reproduction depending

on differences in color temperature 50• Differences in color rendition depending on the type

of film 51• Purchase of color film ' 52

Use of color-compensating (CC) filters when takingcolor photos 53

• Test photography 54Black-and-white photography

• Black-and-white film 55• Comparison of different film brands 56• Filters 57

Photography with Polaroid film 58

Basic information in photomicrography

Operating Instructions for models PM·10ADand PM·10ADS (with 35mm camera back)

1 Setting the film format11---

2 Load the film properly--~-...;;;~

Push the button for 35mm camera.

7Center the specimen to be'III,""_~ photographed in the visual field

Move the area of the specimen you want tophotograph into the center of the visual fieldwith the stage controls. When you use modelPM-10ADS, center the area to be photo-graphed in the spot metering section of theviewfinder.

o xLoad the film into the camera and make surethat the end of the film does not protrudebeyond the spool groove. Otherwise theframe spacing may not be uniform.

8 Focus and adjust the apertureiris diaphragm

Focus on the specimen and adjust thecondenser aperture iris diaphragm so thatsuitable contrast is achieved. The aperturediaphragm is ordinarily set at 60% to 80%of the objective numerical aperture. (refer topages 24-25)

3 Confirm the winding of the fit

• OLYMPUS :3"l.D2 _ " -'

Wind the film onto the empty take-up spooland advance it to frame no. 1. To confirmthat the film is winding, be certain that thefilm rewinding crank is rotating. If it is not,rotate the crank 2 or 3 times in the directicof the dotted arrow to pick up the slack ofthe film inside the camera, and again checithe rewinding crank to confirm the windingof the film.

9 Adjust shutter speed

Check the shutter speed. Adjust the speedto between 0.01 and 0.5 seconds by usingND filters.

5--------------~--Set the characteristics forreciprocity failure

Set the speed for the film used.

11 Focusing the image---- 12Exposl:.lr:eadjostment

Exposure adjustment is based on the totaldistribution of the specimen. (refer topages 42-43)

.- ..,&;1 - ~-- ~ i

Set the characteristics for reciprocity failurefor the film used.

Focus is adjusted either through the focusingtelescope of the PM-1OAD/PM-1OADS or thebinocular tube of the microscope. (refer topages 26-27)• The focusing telescope of the PM-10ADSI

PM-1OAD includes different photo framesfor different types of film .

• The type of finder eyepiece varies withfilm size and each type of finder eyepieceincludes several photo frames indicatingdifferent magnifications of NFK photoeyepieces.

6 Measure color temperature n~=~H~Set the voltage above 6V. photography)

Color photographySet the color temperature to match the typeof color film used. Measure the temperatureat a blank area not covered by the specimen.

Light Dial posi-Type of film balancing tion on CTRfilterDaylight film LBD-2N DTungsten film LBT T

The voltage position when not using the colortemperature meter CTR is:8.5-9.5V. for models BHS and4 or 6V. for models BHT and BHTU

Black-and-white photographySet the voltage above 6V. Normally, a greenfilter is used.(For emphasizing particular colors referto page 57)

Press the shutter release button. Aftercompleting exposure, check for the soundof film winding.

Magnification of the photographic equipmentPhoto magnification on the film surface varieswith the projection length of the photographic Photographic equipment FK, NFK·type P·typeequipment and the type of photo eyepiece

PM-1OADused. In all cases photo magnification is 1 X 0.5 Xcomputed by multiplying the objective mag- PM-10ADS

nification and the photo eyepiece magnification PM-10M 1 X 0.5 Xwith the coefficient listed in the table onthe right.

PM-6 0.9X 0.45X

BH2-PM-6 0.8X 0.4 X

When using models PM-1OAD or PM-1OADS; 10X objective and 2.5X NFK photo eyepiece:Photo manification = 10 x 2.5 = 25

Even though when the overall photo magnifica- Example: Photo magnification of 100Xtion is the same, resolution varies dependingon the combination of objective and photoeyepiece. The resolution on the film planeis improved if the magnification of the photoeyepiece is low and that of the objective high.As the photo shows, a combination of objec-tive 40X and NFK 2.5X provides a cleardisplay of even minute image detail. Focaldepth, however, becomes shallow.

Differences in resolution according to thecombinations of objectives and eyepieces

Objective S Plan 20X and photo eyepiece NFK 5X

to

Objective S Plan 40X and photo eyepiece NFK 2.5X

Setting of photographic magnification(effective magnification)

en taking pictures, you have to first decideon the desired enlargement ratio. The mag-nification on the film plane is generallydetermined by objective magnification timesphoto eyepiece magnification. But the effec-

Objective 4X 10X 20X 40X 100Xmagnification

Type of S Plan S Plan S Plan S Plan S Plan S Plan S Plan S Plan S Plan S Planobjective Achromat Apochromat Achromat Apochromat Achromat Apochromat Achromat Apochromat Achromat Apochromat

N.A. •• 0.13 0.16 0.30 0.40 0.46 0.70 0.70 0.95 1.25 1.30

Effective 65-130 80 -160 150-300 200-400 230-460 350-700 350-700 475-950 625-1250 650-1300magnification

tive magnification of the picture changes *The effective magnification is based on thewith the numerical aperture of the objective, assumption that the picture is viewed withinand is normally based on the following rela- the closest distance affording distinct vision.tionship, which must be taken into account The values listed below do not apply whenwhen enlarging the photomicrograph. trying to project 35mm format slides.

500N.A. <M < 1000N.A. N.A. =numerical aperture of the objective M = effective magnification

Framing of the specimenring photography, the specimen oftens not fit into the photo frame. This problem

can be overcome by rotating the camerasection in relation to the specimen, though .

operation of the microscope becomes moredifficult. Here, the framing of the specimenis carried out the preferred way, by rotatingthe stage.

Checking of the finished photomicrographsPhotomicrography involves scientific photo-graphy, which makes it imperative that thephotographer accurately records his findingson film. Additionally, the photograph shouldconvey a strong esthetic impression to theviewer. Valuable records should not bedocumented with run-of-the-mill photos.

To further improve your results in photo-micrography, it is important that you alwayscheck your own photos. By referring to thefollowing checkpoints, you can pinpoint anyproblems concerning your photos. Then pro-ceed to the second half of this sectiontitled Photomicrography Techniques.

1. Questions relating to a" photography(1) Is the image in focus (refer to pages 26-27)

and the exposure properly adjusted?(refer to pages 42-46)

(2)Have dirt or dust specks on the specimenfound their way onto the photograph?(refer to pages 28·33)

(3) Is the specimen properly stained?(4)Is there uneven illumination? (refer to

pages 22·25)2. Color photography (refer to pages 47-54)(1)ls the background (empty space) white or

of light grey tone?(2) Is the color of the specimen accurately

reproduced?

3. Black-and-white photography (referpages 55·57)

(1)Does the finished photograph show clearfgraded black and white tones without excessively dark (solid black) shadow areas ancwashed-out (completely white) highlights?

Date {q84 Dec. 1Purpose Film Kodachrome 25

No. Objective Photo eyepiece Specimen Filter Voltage Aperture iris Exposure Remarksdiaphragm adjustment1 S Plan lOX NFK 2.5X KIDNEY LBD IOV FULL lX2 'I 'I 'I " 'I 70% I,

3 " " 'f LBD+CCOSM " FULL "4 'I f, I, " 'I 70% "56789 ~

, 1011121314 .15

Purpose Film Date

No. Objective Photo eyepiece Specimen Filter Voltage Aperture iris Exposure Remarksdiaphragm adjustment12

~ •,"456789101112131415161718192021222324

I"l ~5~627282930313233343536

Photomicrography techniques

Exposure adjustment techniquesWhen taking photomicrographs, exposurecompensation is necessary depending on thedistribution of the specimen in the field.But if the specimen is evenly distributed

1. PM·10AD(60% Average metering with modelPM·10AD)

Average metering 60%

within the integrated or spot metering range,no compensation is required (in case of 1X).

..(

: '

Adjustment 0.25X

Adjustment 1X

Adjustment 4X

-,-,Adjustment 0.5X

Adjustment 2X

M·10ADS(Model PM·10ADS can be used for lnteq-rated metering of 30% and spot meteringof 1%.)

Integrated metering 30%

Measuring area

Set the recipro-city failurescompensationvalue for the

--------------jr----------j film being used.

Joint use ofThe dark background is dotted the ISO/ASAwith specimens sensitivity dial

Specimen condition within Exposure adjust- Reciprocity failurethe 1% metering area men! dial setting characteristics

About half (50%) of thespecimen is distributed within 2Xthe dark background

About one-fourth (25%) of thespecimen is distributed in the 4Xdark background

Brightfield background dottedwith fairly dense specimens

Brightfield background con-taining scattered specimens

Specimen is evenly distributedwithin the 1% metering area.

0.25X

0.5X

1X

AE lock operation

~~ ••. Use of the AE lockBy pressing this button during the automaticexposure mode, you can adjust the exposuretime indicated on the display panel (expectedexposure time, actual exposure time).

1. Taking panoramic pictures using theAE lock

For panorama pictures where any number ofcopies is taken from different sections of thesame specimen or where several photos arepatched together, perfect panoramic photo-graphs with uniform density can be taken bylockinq- in a fixed exposure for all the photo-micrographs.

PM·10ADS (integrated metering 30%,spot metering 1%) PM·10AD (average metering 60%)

When pressing the AE lock button, thewarning light above it flashes to indicateAE lock mode. The unit is now ready forphotographing. To cancel this functionpress the AE lock button again. Thisswitches the light off, and the device re-verts to normal automatic exposure mode.

After positioning the specimen to bephotographed and pressing the AE lockbutton, the warning light above it flashesto indicate AE lock mode. Then, aftertaking the first photograph, exposuretime is locked-in.

Tocancel this function press the AE lockbutton again. This switches the warninglight off, and the device reverts to thenormal automatic exposure mode.

Eliminating density variations by AE lock

Panorama photography. ,

Example of using the AE lock on modelPM·10ADS

(1)/ncombination with spot meteringIf specimen areas requiring spot meteringare not centered because of framing prob-lems, they are first positioned in the centernd the AE lock is activated. Then the

specimens are moved back to their originalposition and photocraphed .

•

(2)Photographing within a range closeto maximum exposure time

If exposure display is close to the maximumexposure time, for example during fluores-cence photomicrography, the photographicequipment might display an underexposurewarning during automatic exposure. This canbe prevented by using the AE lock, resultingin long-time exposure.

1 The specimen detail is at the locationwhere it is to be photographed, butexposure cannot be measured.

2 After moving the specimen detail tothe measuring area and activatingthe AE lock, the specimen detail cannow be moved back to its originalposition and the photograph is taken.

Compensating for a film'sreciprocity failure

With normally used photographic emulsionsthere is a rule (the reciprocity law) thatdetermines the luminance of the light strikingthe film surface. According to this rule, thetotal amount of exposure is defined as theproduct of the luminance and the exposuretime. For example, the amount of exposurewith 1/60 sec. exposure at f8 is the sameas for 1/30 sec. at f11. But for longer exposuretimes this rule no longer applies, leading tounder exposure and changes in color re-production. This phenomenon is known asreciprocity law failure. But since, in photo-micrography, exposure compensation cannotbe carried out via the aperture diaphragm,exposure time is lengthened or shortenedto obtain a suitable exposure level. If thereciprocity dial is set on models PM-10ADSand PM-1OAD, compensation is carriedout automatically, resulting in proper

Example: Data for the characteristics of reciprocity law failure (Kodak color film DKD-1 I

Exposure time

Kodachrome 25 1/10,00011/1,000 11/100 1 1/10 1 10 100ASA 25

No exposure compensation required +1/2 +2 +3aperture aperture aperture

No filter required no filter CC10B CC20Brequired

exposure. Uneven color reproduction mustbe compensated for with a CC filter.The above chart lists the compensation datafor reciprocity law failure characteristics whenusing Kodachrome 25 film for general photo-micrography. The chart shows that for longexposure times exceeding 1 sec., bothexposure time compensation and color

Example of compensation (when using Fujichrome RD100)

No com-pensation j

0.02 see

Compen-sation

0.02 see

0.2 see

0.2 see

compensation by filter are necessary. Foradditional data on reciprocity law failurecharacteristics contact the film manufacturer.

2.4 see

3.2 see

28 see

52 see

Color photomicrography

Color filmny different types of color film are offered

on the market today, leaving the user at aloss as to which brand to use for photomicro-graphy. Normally, daylight-type reversal filmwith an ISO/ASA speed of 50-100 is used,while microscopes require a light balancingfilter (LBO-2, 2N).

Daylight type

Ektachrome 64Ektachrome 100Kodachrome 25Kodachrome 64Agfachrome CT-18Agfach rome 50 Type-SAgfachrome 100Fujichrome 500Fujichrome 1000

Ektachrome 50Kodachrome 40Agfachrome 50 Type-L

Tungsten type

Requirements for the selection of color film(1)Film with high resolving power, since photo-

micrography requires display of detailedstructu res.

(2)The display must be able to discriminatebetween the fine color differentiations con-tained in a single specimen.

(3)Faithful reproduction of specimen colorswithout background discoloration.

AGFACHROME

In view of these factors, the following condi-tions can be set for photomicrography:(1)Fine grain(2)Good color contrast(3)Good color rendition

For normal photomicrography in trans-mittedlight bright field illumination color filmwith an ISO/ASA speed between 50 and 100 willassure satisfactory quality. For special casessuch as printed publication or enlargements,Kodachrome film offers good quality.When photographing dark specimens (phase

m

contrast, polarized light, fluorescence),exposure time increases. Although theremay be excessive grain, for cases requiringfast shutter speeds use film with a highISO/ASA speed.

Types of filtersFiltersSelection of filters for use in photomicro-graphy is based on the type of film used.Use LBO-2N for daylight films and LBT filtersfor tungsten-type film. For changing lightintensity, use of a neutral density filter (N0)is recommended. The number on the filterrim gives the transmission value. In caseof filter N06, 94% of the total illuminatinglight is absorbed and only 6% of the lightis transmitted.

Types of filter and their functionsFilters for use in color photomicrographycomprise the following main types:

CD Light balancing filter

®ND (neutral density) filter

® CC (color compensating) filter

To convert the color temperature of the micro-scope light source to the film being used.(refer to page 49)

To reduce the light intensity without affecting colortemperature, when illumination is too bright.

For color rendition problems such as slight dif-ferences in color hue or fading in developed photos.(refer to page 53)

® Didymium filter (marked FFon Olympus models)

oTo emphasize color, i.e. when trying to compensatefor the insufficient intrinsic red color rendition ofa film or to enhance the red color of the specimenor the color contrast of Polaroid color film.(refer to page 58)

® Heat absorbing filterTo absorb heat rays emanating from the lightsource of the microscope to prevent damage toor destruction of live specimens. Since this filtertransmits a small quantity of blue light, it maybecome necessary to use a color-compensatingfilter (CC10Y or CC10M) when taking color photos.(refer to page 53)

Differences in color rendition dependingon the combination of film and light source

re are two types of color films-daylight-type (balanced for sunlight) and tungsten-type (balanced for artificial light), the selectiondepending upon the light source. VirtuallyIIusers have experienced the disappointment

of their photos appearing excessively redwhen they used daylight film under incandes-cent light conditions.

Similarly, when using the tungsten-type filmoutdoors, a bluish photograph is obtained.The fault in both cases is failure to usethe proper film to match the light source.

Color rendition depending on the combination of film and light source

LBD

light balancing filter fordaylight-type color film.(sunlight)

LBT

light balancing filter fortunqsten-type color film.(artificial light)

.... o

x

o

Differences in color reproduction dependingon differences in color temperature

Color temperatureColor temperature designates the propertiesof the light source. A blackbody radiator,when heated, emanates light of different colordepending on the temperature. The propertiesof the light source can be indicated by referringto the temperature of the blackbody at a fixedtemperature level. This feature is called colortemperature, the numerical unit being ex-pressed either in absolute temperature orin degrees Kelvin (K).

Color film for use in photomicrography isnormally daylight type, but since the lightsource of the microscope is a tungsten type,its color temperature is low (2800-3400K)and as such it is unsuitable for daylight filmwith its color temperature of 5500-6000K.In order to achieve the proper color rendition,a light balancing filter is used, and thetungsten light is converted to daylight.

Differences in color reproduction depending on differences in color temperature

4000K

5000K

4500K

6000K 6500K(If color temperature is low, the photo shows a "red shift", and if color temperature is high, it exhibits a "blue shill

~

Differences in color rendition dependingon the type of film

llor rendition for different types of film fromthe same manufacturer tends to vary, andeven different production lots of the sametype show slight differences.

When you start to take color photos, youshould first determine the optimum conditionsfor the film through test photographs, matchingthem with both microscope and specimen.(For the method of taking test photosrefer to page 54)Differences In color developmentdepending on the type of filmThe six photos on the right were taken withfilms of different brands, and with the excep-tion of the ISO/ASA speed all photographicconditions were identical. This example clearlyshows that properties such as color rendition,contrast, clarity of backqrouno, etc. are all dif-ferent because 01 the various brands of film.

Photographic conditionsBHS, PM-10AD, LBD-2N (color temperature 5500K), LBT (color temperature 34OOK),shutter speed 0.1-0.4 secDaylight type

A

E

o

Purchase of color film1. What to watch for when buying color film• Color film is a highly sensitive substance,

and environmental factors such as heatand humidity easily cause chariges in filmspeed and color rendition. When buyingcolor film, avoid camera stores in whichthe shelves storing color film are exposedto sunlight.

• Choose only color film with a sufficientlylong period before the expiration dateas marked on the package.

• Color rendition of the same film type maydiffer, if it comprises different productionlots. If you frequently use color film, youcan use it under identical photographicconditions and avoid variations in the colorof the film if you buy large quantities fromthe same production lot.

Production lot

~

Emulsion number

~g~sA;:~6••~~~;)o~LOt 1$0 64/19·

I36 EXPOSURES24 6 mm ASA 64 19 OINREVERSALFILM· ROCES5

EllUL. NO. to' ~ .., i~_;~_;PROCESS "'_BEFORE f ~:l

EPR1~36 Cr1602754 "~~r. ~IIIU.S. by~ EA6TllA.N K COMPAHY •. RochHtet. N.'I', I46SO

Tefilitl of walidllty (explratlon dafe)

2. How to store filmFor the basic principles of composition andcolor rendition check the technical literatureon the subject. As has been pointed out,performance changes according to the con-ditions under which the film is stored.

Color film is normally kept in the refrigeratorto protect it from the effects of heat andhumidity. Remove the film from the refrigerator1 hour before use and allow it to reach roomtemperature. Do not remove the film fromthe roll immediately after taking it out of the

refrigerator to prevent condensation on thefilm surface.

3. Points calling for special attentionwhen using the film

• Do not leave the film in the camera longerthan necessary.

• Develop exposed film as soon as possible.

Summer

• Even though other photographic conditionsmay be identical, color rendition mey varywith low or high ambient temperatures.

• Do not use the film in a gaseous en-vironment (such as formalin), since colorrendition will be adversely affected.

(Red background)

Use of color-compensating (CC) filterswhen taking color photos

or-compensating filters used(Blue background)

/,

CCY filter

(Green background) (Yellow background)

Since color-compensating filters are highlysensitive to heat, do not use them closeto the light source .

• CC filter is the abbreviation for color-compensating filter, and the color of theparticular filter is marked in capital letterson its rim. Six different colors are available,each color being offered in six differentdegrees of density. NO.5 and No. 10 aremost widely used in photomicrography.

.Color filters are made by several manu-'facturers. Check your Olympus dealer ora reputable camera store for availability.

Color to be reduced Color-compensating filter requiredBLUE Yellow CCY

CYAN Red CCR

GREEN Magenta CCM

YELLOW Blue CCB

RED Cyan CCC

MAGENTA Green CCG

Test photography1. The need for test photographyEven if you use automatic photographic equip-ment and set both the ISO/ASA speed andthe reciprocity law failure characteristics,you cannot be sure that you will alwaysachieve the perfect exposure for every typeof specimen. The reason for this is thatphotographic equipment is manufactured tomatch distribution state, color, and exposurestandard of average specimens. In order toobtain better results, you should first taketest photographs to match the conditions ofthe specimen, film, microscope, filters, etc.

3. Evaluating the test photosObservation of reversal film transparenciescan be carried out either with a light boxor a slide projector, but the display of colorvaries widely with the color temperature ofthese light sources. As a standard for evaluat-ing the proper color, use a light box witha color temperature close to 5000K.

The following types of light boxes areavailable on the market:o Fuji film color box 5000o Macbeth Proofliteo Durotest Color Classer 50o General Electric Chroma 50

2. Method for test photography(when using models BHS and PM-10ADand reversal film)

(1)Prepare one roll of film (36 exposures) anda frequently used specimen.

(2)Set the test conditions (see chart 1) andprepare a data chart (see chart 2). Listingall the conditions given in chart 1 will resultin a data chart 2. Take your photos basedon this chart and determine the optimumphotographic conditions on the basis of theresults obtained on the developed film.

•For color compensation, first take photoswithout a compensating filter and thenchoose the proper compensating filteron the basis of the finished p'hoto.

Chart 1: Test conditions

Chart 2: Data chart Q0Color Exposure Color corn-

temperature pensatlon

1 0.8Xf-- No com-2 1Xr-- pensation3 1.25X

I--4 5900K 0.8X

r--

5 or CC5G 1XI--10V6 1.25X

f--

7 0.8X!--

8 CC5M 1Xf--

9 1.25X

10 5500K 0.8XI-- No corn-11 or pensation 1X- 9V12 1.25X

Measuring with CTR 170 mired, 180 mired, 190 mired,5900K 5500K 5260KColor temperature

No CTR

CC5G, no compensation, CC5M

Exposure Exposure adjustment

Color compensation Color-compensating filter

Voltage WV, 9V, 8V

0.8X, 1X, 1.25X

4. Organizing photographing dataMake it a habit to record all data relatingto both test photography and regular' photo-graphy. If the photographic conditions thatresulted in good photos are retained on file,troubleshooting and correction of problemscan be carried out qulckly and efficiently.(Pages 40-41 give an example of a datareport form for your reference)

5. Storing developed filmWhen storing film for a long period of timthe most important precaution is protectiofrom light. Since mold is prevalent in hot andhumid places, it is advisable to keep the filmtightly sealed, together with a desiccant,such as silica gel.We recommend storing film in an environ-ment with a retetive humidity of 15-40% andan ambient temperature below 21°C (71°F).(from Kodak Color OKP 141 instructions).

Black-and-white photography

Black-and-white filmce the conditions differ for photomicro-

graphy and general photography, it isnecessary to select the proper film for eachtype of photography. In photomicrography,high contrast film with fine grain is used inorder to document minute structures ofbiological specimens and to achieve sharpphotographic reproduction. Examples of thisfilm type are Kodak Panatomic X, Agfapan 25,IIford Pan F, and Fuji Neopan F.

Use of black-and-white filmThere are many types of black-and-white film,and the key to obtaining good photographs liesin selecting the right film for a particular job.

Specimen conditions Film brand ISO/ASA Remarks

Normal or high contrast Kodak Panatomic-X 32 The use of contrast filtersfor the stained section of IIford Pan F 50 depends on the color of thegeneral pathological Agfapan 25 25specimens Fuji Neopan F 32 specimen.

Low contrast for the Since the contrast of the filmstained sections of Kodak Technical Pan 64 is very high, the range of pro-pathological specimens, 2415 per exposure is very narrow.as well as those of other Agfaortho 25 100 Unless exposure is set withinareas. When shape is Fuji Minicopy HR-II 25 ± 1/3 step of the optimum,more important than the photo will be either over-structure details. 32 or underexposed.

Kodak Tri-X Pan 400Dark specimens, and Agfapan 400 400 Use these types when youwhen long shutter speed Ilford HP5 400 want to shorten exposure timeis required Ilford XP1 50-1600 at the expense of film grain.

Fuji Neopan 400 400

Comparison of different film brandsTypes of film providing a wide gradation fromshadow to highlights and featuring goodcontrast are Fuji Neopan F and KodakPanatomic X. Technical Pan offers slightlyhigher contrast, while Mini Copy eliminatesgrey tone details.Neopan SS and Tri X, on the other hand,have fairly low contrast.

Fuji Neopan F

Kodak Technical Pan 2415

Neopan SS Kodak Trt-X

Filterse of contrast filters

Contrast filters are used to control the contrastof black-and-white photos.For black-and-white photomicrography, greenfilters are normally used, but choice of themost effective filter depends on the type ofspecimen. Filters that enhance the colorsand contrast of the specimen are:

Color of thespecimen Color of the filter

Red/yellow GreenYellow/orange Blue

Blue OrangeFor reducing contrast, use a filter of the samecolor as the specimen.

Reasons for using a green filterThere are two reasons why contrast improveswhen a green filter is used.(1)Since objective aberrations are most

effectively compensated near the greenwavelength, loss of image clarity due tochromatic aberration is averted by a greenfilter.

\ ) Dyes such as hematoxylin and eosinabsorb green light well, resulting in highercontrast when a green filter is used.

Effects of various contrast filters

•

Green filter:the red portion is enhanced

Without filter

•Orange filter:the red portion is reduced

Photography with Polaroid" filmOne of the advantages of Polaroid film is thatit can be quickly viewed as a finished photo,but it is seldom possible to obtain colorreproduction comparable to that of 35mmreversal color film,

1. Types of film used in photomicrography41/4" x31/4" 4" x 5" (actual size 9cm x 11.5cm)(actual size 7.3cm x 9.5cm)

BType ISO/ASA Pack containing Type ISO/ASA Sheet Pack containing

I speed 8 photos speed 8 photosac 107 3000 0 52 400 0k -I 667 3000 0 57 3000 0a -n

552 400 0d -I 665 50 0w 55 50h (Printlt negative)

(Printl 0 -

e negative)

C 108 80 0 58 80 0 -0I 668 80 0 59 80 0 -0r 669 80 0 559 80 - 0

.4" x 5" film holders differ according to the type of film

Sheet film-545 type film holder

"Polaroid" is a trademark registered by the Poraloid Corporation, Cambridge, Mass., U,S,A. ~

Packed fllm-550 type film holder

2. Photographic techniques(1)Black-and-white photographyUse a green filter to obtain good contrast.(2)Color photographySince the overall color hue tends to be eitherlight green or blue, use CC1 0 - 20M orCC1 0 - 20Y for compensation, If you wantto enhance color contrast, use the FF filteravailable from your Olympus dealer.

Since Polaroid film is more easily affectedby reciprocity law failure than normal film,exposure time should be adjusted to between0,05 and 0,5 seconds,

Color temperature 4500K-LBD·2N plus FF filter

••••••••...•0". ...•.•

Section 3How to obtain good prints

How to obtain good color prints 60-61• Preparing black-and-white prints 62

Marks on the photo 63How to avoid marks during development 64- 65

How to obtain good color printsThe following diagram shows how to getgood color prints (positive prints).

Color negative film

Color printColor reversal film

Polaroid

In photomicrography, the color reproductionof finished prints taken with negative filmdiffers from the observed color-a fact manyusers are probably very much aware of.Possible reasons for this are that the technicianwho made the prints did not view the speci·men color through a microscope and thusdid not know the actual color, or the photo-grapher made a mistake when he used a filterand failed to notice that the film qualitydeteriorated. In order to get better results,you should first photograph the specimenfrom which you want to get prints on reversalfilm, and attach the finished photo as colorreference sample to the negative film. Printscan also be made from color reversal film,but the process does not match the printquality as obtained from negative film.

Color negative film~

Reversal film

Color print with poor color reproduction

•

Reversal film with optimumcolor rendition

Color print with good color reproduction

Preparing black-and-white printsThere are many technical publications on themarket that explain in detail how to developfilm. If you plan to do your own film pro-cessing, use these books as reference.But since contrast in photomicrography islower than with normal photographic subjects,you should pay particular attention to under-development. If you make a positive printfrom an underdeveloped, low-density negative,an even greater reduction in contrast willoccur, leading to a poor result.

1. How to obtain a good negative(1)Use a developer that matches the film(2)Use the developer at the temperature

specified by the manufacturer(3)Adhere to the specified development time

that matches film sensitivity(4)Agitate gently and frequently to eliminate

uneven development(5)Make sure that there is neither too much

nor too little fixing(6)Follow the paper specifications when

washing the print(7)Handle the negative carefully and protect

it from fingerprints, scratches and dust

2. Selection of photographic paperWhen making prints, you can vary th-econtrastdepending on the type of printing paper.Select the type that best suits both thedensity of the negative and specimen contrast.The examples on the right show prints madefrom the same negative when using differenttypes of printing paper.

Grade no. ofphotographic paper

Grade 2-low contrast

Grade 3-normal contrast

••

Grade 4-high contrast

Marks on the photorks on the print tend to stand out more 1. Marks as a result of negative development 2. Marks occurring during print development

prominently in photomicrography than onnormal photos of people and landscapes.Since photomicrography employs film withfairly high contrast, even the most minute dif-ferences in brightness show up in the picture.Marks resulting from improper developmentare also very conspicuous. This section dealswith marks resulting from development.

Both upper and lower portions of the photo arebrighter than the central section.

How to avoid creating marks(1)Confirm the degree of exhaustion of the

developer.Carefully note production date and fre-quency of use of the developer.

(2)Agitate the developer thoroughly before use.(3)Use the developer at the specified

temperature.(4)Do not work with an exposure that results

in a development time of less than 5 minutes(the result will be extremely desensitizeddevelopment).

(5)Agitate the bath thoroughly during develop-ment.

(6)Maintain the specified fixing time.(7)Wash the film thoroughly.

Marks with irregular brightness show up.

If you use an automatic processor to developthe printing paper no marks will occur, butsince marks can easily show up whendeveloping in a tray, the following pointsshould be noted:How to avoid creating marks(1)Agitate the developer thoroughly.(2)Keep the temperature of the developer at

20°C, or as specified by the manufacturer.(3)Since marks can easily occur if develop-

ment time is too short, set the exposureof the enlarger at such a level that develop-ment time is between 1 min 30 sec and2 min.

(4)Agitate the bath thoroughly during develop-ment.

(5)Continue to agitate thoroughly even whilestopping and fixing.

(6)Wash the print thoroughly in running water.Regular-base paper-30-60 minResin-coated paper-4-5 min

How to avoid marks during development..•• • • • • • •. .s., .•

2Development of film

Since marks on the film occur easily whenusing a reel-type tank, development shouldbe done in the following manner in thedark-room.

1

Immerse the reel in the developer, and afterturning it 2-3 times, tap it another 2-3 timesagainst the bottom of the tank to removeair bubbles sticking to the film surface.Complete this process within 5-6 seconds.

Now remove the reel from the developer andimmediately immerse it again. After repeatingthis procedure, turn the reel immersed inthe developer 2-3 times, then pause for30 seconds.

Development of pfiAting paper

This section deals with development usinga tray.

1

.As shown in the picture, do not immersethe printing paper into the developerparallel to the developer surface, but tiltit into the developer bath.

While holding the edge of the exposed printingpaper lightly with a pair of tongs, quicklyplace it into the developer.

4

By removing the reel from the developer, thedeveloper can reach the spaces between the rolledfilm, preventing the generating of spots.

x

The developer will not be suf-ficiently agitated simply byturning the reel, and the resultwill be spots on the film.

Perform the operation shown in picture 2 on the left in 30-second intervals till the completionof development work.

o After completing development, follow thesequence of stopping, fixing, and washingat the proper time.

2 ----~--------------------~- 3• Make sure that the photographic paper

does not float up to the surface of thedeveloper.

To agitate the photographic paper, hold itat a corner with a pair of tongs and moveit back and forth while it is submerged in thedeveloper. Repeat this operation in 15-secondintervals until development work is completed.

After completing development work on theprinting paper, quickly perform the sequenceof stopping, fixing, and washing .• Make sure that separate sheets of printing

paper do not stick together in the fixer .• Do not use the same pair of tongs during

stopping and fixing that you used duringdevelopment work.Make sure not to contaminate the develop-er with either stop bath or fixer liquid.

Section 4•• ~ ••••• ~ •• Trouble-shooting

Problems in finished photes and their correctionPoor color reproduction 68- 71Blurred image 72- 73The image is in focus but not sharp 74- 79Objects other than the specimen image appeared

on the film 80-81Uneven brightness 82-83

Problems in finished photos and their correction

-Poor color reproduction••••••••_,,,. -.2_

1. The background is colored (redlblue)

Cause Refer topage

The color temperature of the illurnl-nation is not matched to that ofthe film.eLight balancing filter specified by

manufacturer is not used.

Correction

eUse filters specified by the manu-facturer.

Remarks

eDaylight type: LBD-2N, 5500K,D setting

eTungsten type: LBT, 3400K,T setting

Page 48/page 50

Lamp voltage is too low (too high). eRaise (lower) lamp voltage

9V-correct value

eBHS above 8.5VeBHT and BHTU at 5-6Velf you have reached the desired

color temperature, do not changethe voltage position. If you wantto change intensity, use an NDfilter.

eWe recommend that for testphotography the voltage shouldbe set to the standard position± 1V when first determiningthe optimum color temperature.

Page 501page 54

Blackening of lamp due to pro-longed use eReplace with a new lamp

he background is colored (green/magenta)

Cause correctlon Remarks Refer topage

eA film different from your usual eSince color rendition varies with Page 51film type was used. different film types even from the

same maker, choose a film withcoloring suitable for your typeof work.

eA film of the same type but with elf possible buy film with the eln order to maintain the perter- Page 52different emulsion number was same emulsion number in large mance quality of the film, storeused. quantities. it in a refrigerator and remove

it one hour prior to use, allowingit to reach room temperature.

eEven for film of the same typethere will be slight variations incolor reproduction depending onlaboratory,developmentconditions,and staining of the specimen.

eUse a color-cornpensatino (CC) *Color-compensating (CC) filters Page 53filter. Buy from your photo dealer.

eToo much greenUse CCM (magenta)

eToo much pinkUse CCG (green)

For photomicrography, use CCfilters 05-10.For further compensa-

• tion use CC05and CC10together.• Greenish background CC05M-optimum compensation

•CC10M-over·compensated

3. Incorrect color rendition

Cause Correction Remarks Refer topage

eAn excessively long exposure eSet shutter speed at 0.01-0.05sec. eWith long exposure times, even Page 46time has been used and the char- Adjust shutter speed uniformly as with the exposure time compen-acteristics of reciprocity law far as possible by using an NOfilter. sated, the color rendition changesfailure lead to incorrect color eWhen using long exposure times as a result of film properties.rendition. (above 0.5 sec) with models Use a cOlor-compensating filter

PM-10AD and PM-10ADS,set the specified by the film manu-characteristics number of the film facturer.used on the dial for reciprocitylaw failure.

eAutomatic exposure has been eWhen using models PM-1OADSI elf you change the magnification Page 421used without adjustment. PM-10ADset the exposure adjust- of the objective, the light distribu- page 43

ment dial to: tion within the visual field will0.8-0.25Xfor bright background also change.specimens and to 1.25-4Xfor darkbackground specimens.

eCarry out exposure adjustmentsBright background: ISO/ASA 100 to 50Dark background: ISO/ASA 100 to 200

eActual and nominal sensitivity eVary the ISO/ASA speed setting. eNominal speed may differ from Page 42of the film differ. actual light sensitivity as much

as 1/3 to 1/2 aperture.

4. Poor color of a print enlarged from a negative ~Cause Correction Remarks R~~&Jo

eThe printing technician in the eAdd a transparency of the same eSetter color is achieved when Pageslaboratory is not sure of the specimen with correct color as shooting a transparency with 60-61subject's correct color. a sample. reversal color film (but the cost

of prints goes up and qualitydecreases).

·'oor color of Polaroid film

Cause Correction Remarks Refer topage

eWrong color temperature. eThe film is a daylight type, but elf you set color temperature be- Page 58because of the film characteris- tween 4000K and 5000K andtics color temperature should be use an Olympus FF filter, colorset lower than for 35mm film. contrast will be enhanced.

eAbnormal color characteristics. elf the abnormalities are within eSince Polaroid film is easily Page 54/the adjustment range for color affected by adverse storage page 58temperature and within the conditions as far as color charac-operational range of the color- teristics are concerned, protect itcompensating filter, determine from heat and humidity by storingthe conditions by test photography. it in a refrigerator.

eTerm of validity of the film has eUse film whose term of validityexpired. has not yet expired.

elf room temperature is abruptly eFor development time and colorlowered (raised), sensitivity is reproduction, refer to the filmaffected and print color turns instructions.towards blue (red).

•

Blurred image1. The overall focus of the picture is blurred

Cause

eFinder eyepiece or focusing tele-scope is not properly adjusted.

IIII

eSlur and drift resulting fromvibrations.

Image affected by vibrations

Correction

eAdjust the diopter until the doublecross lines are clearly visible.

II

II

eUse a vibration-proof table.

No vibrations

eUse an ND filter and increaseshutter speed (1/2 sec to 1 sec).

eUse a stand for the photographicequipment to separate photo-graphic equipment and microscope.

Remari<:s

eSince most people do not havethe same visual acuity in botheyes, determine which eye youalways use for focusing.

Refer topage

Pages26-27

Page 12page 48

Focusing error occurs when you use a low-rnaqnlcatlon objective of less than 4X

Cause Correction Remarks Refer topage

elf magnification is low, focal eMount a focusing magnifier to elncreasing the magnification Page 27depth at the film plane becomes the finder eyepiece orthe focusing eliminates focusing errors.shallow, easily causing errors telescope, and after focusing onin focusing. the double cross lines, fine focus

the specimen.

3. The periphery is uniformly blurred

Cause Remari<s Refer topageCorrection

eAn Achromat type objective wasused.

D Ach 10X NFK 2.5X

eUse a Plan Achromat typeobjective

D Plan 10X NFK 2.5X

Page 14

eObjective and photo eyepiecewere used in the wrong com-bination.

eLB series (long barrel)-NFKphoto eyepiece

eShort-barrel series-FK photoeyepiece

Page 13

The image is in focus but not sharp1. Inadequate resolving power

Refer topageCause

eUse of a combination of low-magnification objective and high-magnification photo eyepiece.

S Plan 20X NFK 5X

eUse of the condenser with theaperture iris diaphragm fullyopened.

Correction

eStop down theaperture iris dia-phragm to 60-80%of the numericalaperture of theobjective.

Remarks

eln order to obtain a magnificationof 100X on the film plane, useeither an objective 40X with aphoto eyepiece of 2.5X or anobjective 20X with a photo eye-piece 5X. To increase resolvingpower, the combination of objec-tive 40X and photo eyepiece 2.5Xis preferable. (Focal depth,however, will become shallow.)

-Vary the amount by which youreduce the aperture iris diaphragmaccording to the magnificationof the objective and the contrastof the specimen.

Page 38

Pages24-25

eThe field iris diaphragm was fullyopened.

-Use of thick cover glass.

@) -In order to reducestray light, stopdown the field irisdiaphram to anarea only slightlylarper than theframe reticle.

-Use a cover glass with a thick-ness of 0.17mm.

Cover glass O.17mm

-Do not reduce the diameter of thefield iris diaphragm to such anextent that it touches the framereticle because the actual areaphotographed is always slightlylarger than the area within theframe reticle.

-Olympus objectives for biologicalspecimens have been designedin such a way that optimum re-solving power is obtained whena cover glass with a thicknessof 0.17mm is used.

Page 23

CauseRefer to

page

Brightfield

-The specimen stain is too weak,resulting in lack of contrast.(Photographing a specimen thatcannot be stained)

(I

Correction

-Use a denser stain.-Use a contrast filter (for black-and-white photography only).-If the specimen cannot be stained, use phase contrast, differential inter-

ference contrast or darkfield to create contrast optically.

Phase contrast

Differential interference contrast

Darkfield

o

Paqe 57

Cause Refer tpage

Black-and-white photographyeVariations in the spectral sen-

sitivity of the film affect "thecontrast.

Correction Remarks

Color photography

eUse of a low contrast film.

Ektachrome 200

eUse a high contrast film.

Kodachrome 25

Page 51

Page 57eNormally, a green filter is used,

but if you want to emphasize aspecific portion of the specimen,use another contrast filter.

elf a filter complementing the colorof a specimen is used, it willemphasize the contrast.

2. The image appears hazy

Cause Refer topageCorrection Remarks

eThe correction collar of the ob-jective is not adjusted to thethickness of the cover glass.

eAdjust the correction collar whileexamining the specimen and setit at a position providing a clearimage.

eOn the LB objective a correctioncollar is mounted on the S PlanApo 40X, S Plan 100X dry andD Plan Apo 60X.

Page 16A

Cause Refer topageCorrection Remarks

eAn objective normally used withcover-glassed specimens wasused on a specimen without coverglass (or vice versa).,

eFingerprints, an oil film or dirtparticles in the optical system(objective front lens, photo eye-piece, prism, specimen, etc.)

Oil film

Fingerprints

eUse a no-cover objective.

eClean the optical system.eAlways cover the microscope with

a dust cover when not in use.

Clean the optical system.

LB objectiveseNC S Plan 40XeNC D Plan FL 60XeNC S Plan Apo 100X oileNC S Plan 100X dry

elf you use dry 40X or 60Xobjectives together with an oil-immersion objective, the oil on thespecimen may soil the frontlenses of the dry objectives.

Page 17

Pages26-32

Cause

3. No sharp image is obtained with a 100X ell-immersion objective

Correction Remarks Refer topage

-No immersion oil was used. -Use oil specified by the manu-facturer. Page 18

Page 18-Unsuitable oil was used. -Use specified oil, since the typesused for fluorescence and normalwhite light often differ.

-The oil contained air bubbles. Page 18-Apply the oil after you haveremoved bubbles in the bottle.

- Remove the eyepiece beforeexamination and look through theeyepiece sleeve.

-Using oil in a room with unsuitabletemperatures (too high or low) Page 12

-If room temperature is either toohigh or too low, or if the air is toohumid, the diffraction indexchanges, causing changes in theimage. Use the oil at a roomtemperature of 22-25°C and ata humidity of about 55%.

-The specimen is too thick.

4-51'

-It is advisable to use a specimenwith a thickness of 2-3",.

21'

~. Entire roll of black-and-white film is not sharp

Cause Correction Remarks Refer topage

-Possible causes are: type of film, -Check these possibilities and Pagesemulsion, overexposure, over- correct them. 55-57development, improper handling Pagesor accidents during development,etc. 64-65

5. The finished print appears grainy

Cause Correction Remarks Refer topage

-A film with coarse grain was used. -Use a fine grain film. -Kodak Panatomic X-Kodak Technical Pan 2415 Page 55-Fuji Neopan F-Agfapan 25-liford Pan F

-A standard developer was used. -Use a fine grain developer. -Kodak MicrodolD-23D-25

-Fuji Micro-Fine

-Magnification ratio was too high. -Use a large-format film. -4" x5" sheet film.

Objects other than the specimen imageappeared on the film

1. Shadow-like image

Cause Refer topage

eOptical path selector of thephotographic attachment or trino-cular tube was interrupted atsome stage.

Correction

eEngage the optical path selectorat its proper position.

Remarks

eOpen the field iris diaphragm alittle wider than the photographedarea of the finder eyepiece ofthe focusing telescope.

Page 23

eTiny bits of film, dirt, etc. stuckto the prism of the photographicequipment or to the large-formatrelay lens.

eCheck for, and remove, dirt fromthe prism of the photographicattachment while the shutter isopen (Time setting).

eRemove the larqe-torrnat relaylens and clean it.

ePeriodic checks are recom-mended if a large number ofphotographs is taken.

Pages ""29-3.

Cause Refer topage

eDirt in the optical system

S Plan FL ix

Correction

eLocate the dirt and remove it.

Remarks

eYou can locate the dirt by movingand rotating each checkpoint,alternately looking through thebinocular tube, the focusingtelescope of the photographicattachment and the film plane(by placing a piece of frostedglass in the camera body.)

Page 28Pages

30-31

2. Sparks

Cause Correction Refer topage

eWhen moving the film or when eDo not rewind the film too rapidly.unrolling the backing paper of eKeep humidity at 45% minimumthe film, static electricity causes in the room where you handlesparks. the film.

eMake sure that the camera backand the darkroom are free of dust.

Cause

. Reflection of window or room illumination

Correction

eStray light enters from the eye-pieces or the focusing telescope.

eMove the optical path selectorof the trinocular tube to theCamera 100% position and coverthe focusing telescope of thephotographic attachment witha cap.

ePut caps on both eyepieces andthe focusing telescope of thephotographic attachment.

eSet up the microscope in adifferent location.

Refer topage

Page 12

Uneven brightness

Cause

1. Uneven areas occur on one side of the frame, in the center, and under the perforation of the film

Remarks

eThe microscope light source isnot properly centered.

Correction

eProperly adjust the light source.

Refer topage

eThe field iris diaphragm is offaxis. Page 21eOlose the field iris diaphragm so

that it appears in the visual fieldand adjust the condenser tocenter the diaphragm.

eThe optical system is contarni-nated by dirt.

eClean the optical system. Pages29-31

eDevelopment problems (on black-and-white print)

eDevelop the film properly. eBe aware that a change in tem-perature occurs between thecenter and the periphery of thetank as a result of heat conduc-tion by the steel tank.

Pages64-65

.•../!JIarkson the negative

Cause Correction Remarks Refer topage

eFixing time was too short or ex· elncrease fixing time or use ahausted fixer was used. new fixer.

j I I

OLYMPUS~Match your skills with ours.OLYMPUS OPTICAL CO., LTD.San-Ei Building, 22·2, Nishi Shinjuku t-chome. Shinjuku-ku, Tokyo. JapanOLYMPUS OPTICAL CO. (EUROPA) GMBHPostlach 104908, wencenstrasse 14-16, 2 Hamburg 1. west GermanyOLYMPUS CORPORATION4 Nevada Drive, Lake Success, N.Y. 11042-1179, U.S.A.

j

Printed in Japan M132E-1284T