BRIGHT FIELD MICROSCOPESREPORTER:CASIDO, NICASIO JR. S.SCHEDULE: MWF 3:00pm-4:00pm
Importance of the Microscope• Important for hematology, microbiology, TB, andmalaria testing• Compound microscope used in bacteriology,biology, and medicine to examine minute objectssuch as bacteria, other unicellular organisms, andplant and animal cells and tissue• Advances in fluorochrome stains and monoclonalantibody techniques caused growth in use offluorescence microscopy in both biomedicalanalysis and cell biology
ADVANTAGES Brightfield microscopy is very simple to use withfewer adjustments needed to be made to viewspecimens. Some specimens can be viewed without staining andthe optics used in the bright-field technique don’t alterthe color of the specimen. It is adaptable with new technology and optionalpieces of equipment can be implemented with bright-field illumination to give versatility in the tasks it canperform..
DISADVANTAGES Certain disadvantages are inherent in anyoptical imaging technique. By using an aperture diaphragm for contrast,past a certain point, greater contrast addsdistortion. However, employing an irisdiaphragm will help compensate for thisproblem. Bright-field microscopy can’t be used to observeliving specimens of bacteria, although whenusing fixed specimens, bacteria have anoptimum viewing magnification of 1000x.
DISADVANTAGES Bright-field microscopy has very low contrastand most cells absolutely have to be stained tobe seen; staining may introduce extraneousdetails into the specimen that should not bepresent. Also, the user will need to be knowledgeable inproper staining techniques. Lastly, this method requires a strong light sourcefor high magnification applications and intenselighting can produce heat that will damagespecimens or kill living microorganisms.
TERMINOLOGIES Resolution Ability to distinguish (resolve) two close-together points as separate. Contrast Differences in intensity between two objects,or between an object and background Important in determining resolution Staining increases contrast
DESCRIPTION Brightfield microscopy is the mostelementary form of microscope illuminationtechniques and is generally used withcompound microscopes. The name "brightfield" is derived from thefact that the specimen is dark and contrastedby the surrounding bright viewing field.Simple light microscopes are sometimesreferred to as brightfield microscopes.
WHEN TO USE BRIGHT FIELD MICROSCOPY Bright field microscopy is best suited toviewing stained or naturally pigmentedspecimens such as stained prepared slides of tissue sections orliving photosynthetic organisms. It is useless for living specimens of bacteria,and inferior for non-photosynthetic protists ormetazoans, or unstained cell suspensions ortissue sections.
OBSERVED USING BRIGHT-FIELD MICROSCOPY,AND APPROPRIATE MAGNIFICATIONS Prepared slides, stained - bacteria (1000x), thicktissue sections (100x, 400x), thin sections withcondensed chromosomes or specially stainedorganelles (1000x), large protists or metazoans(100x). Smears, stained - blood (400x, 1000x), negativestained bacteria (400x, 1000x). Living preparations (wet mounts, unstained) - pondwater (40x, 100x, 400x), living protists or metazoans(40x, 100x, 400x occasionally), algae and othermicroscopic plant material (40x, 100x, 400x). Smallerspecimens will be difficult to observe withoutdistortion, especially if they have no pigmentation
STEPS Mount the specimen on the stage Optimize the lighting Adjust the condenser Think about what you are looking for Focus, locate, and center the specimen Adjust eyepiece separation, focus Select an objective lens for viewing Adjust illumination for the selected objectivelens
In bright-field microscopy a specimen isplaced on the stage of the microscopeand incandescent light from themicroscope’s light source is aimed at alens beneath the specimen. This lens iscalled a condenser. The condenser usually contains anaperture diaphragm to control and focuslight on the specimen; light passes throughthe specimen and then is collected by anobjective lens situated in a turret abovethe stage.
The objective magnifies the light andtransmits it to an oracular lens or eyepieceand into the user’s eyes. Some of the light isabsorbed by stains, pigmentation, or denseareas of the sample and this contrast allowsyou to see the specimen. For good results with this microscopictechnique, the microscope should have alight source that can provide intenseillumination necessary at high magnificationsand lower light levels for lower magnifications
BASIC COMPONENTS OFTHE MICROSCOPEAND THEIR FUNCTIONS
Power switchLight intensity controlCondenserStageObjectivesEyepiece lensStage motioncontrol knobsCourse and fineadjustments knobsField diaphragm ringAperture diaphragm
Care and Use of the BrightField MicroscopeMalaria parasiteMycobacterium tuberculosis
CARE AND MAINTENANCE OF THEMICROSCOPE Good preventive maintenance and care includes: Regular cleaning of oculars and objectives Avoid damaging oculars and other optics with eyemake-up or other debris Careful handling to avoid abrupt motions Protect from direct sunlight, high temperature,humidity, dust and vibration Use appropriate materials to clean the lenses Cover when not in use with vinyl or plastic dust cover
CLEANING THE MICROSCOPERoutine Cleaning Supplies: Commercial lens tissue for optics Caution: Do not use paper towels orother rough paper products Cotton swabs with wooden shaft(optics) 70% isopropyl alcohol Dilute methanol is satisfactory Mild detergent and soft cloth for stageand base of microscope
CLEANING OCULARS AND OBJECTIVES Unplug the microscope Wash hands Remove dust from optical glasssurfaces Carefully remove eyepieces,objectives, condenser, and filters–oneat a time Excessive rubbing can cause damageto iridescent coating on lens Clean and replace as completed Do Not take eyepiece or objectivesapart
Unplug microscope and allow bulb tocool Carefully place microscope on itsside Open bulb house; use tissue toremove bulb Use tissue (to avoid fingerprints) topick up new bulb Insert new bulb and close bulbhouseReplacing Microscope Bulb
Plug in microscope and turn on illuminator.Rotate nosepiece to lock 10X objective in place Place smear on stage and center it under the10X objective Open the field diaphragm all the way and closecondenser diaphragm all the way Move up (rack up) stage to its highest position Adjust the oculars for interpupillary distance sothat only one circle of light is seen Rack up condenser as high as possibleSetting the KoehlerIllumination
• Close field diaphragm half way and focus smear at10X• Close field diaphragm until diameter of illuminatedimage is smaller than the field of view• Lower condenser with positioning knob until you havea sharp, focused image of the edges of the fielddiaphragm• Adjust condenser using centering screws so that thecircle of light is centered in field• Open field diaphragm until illuminated image is justlarger than the field of view. If more light is needed,use the transformer.• Koehler illumination is now set. It is important not tomove the condenser up or down or change the fielddiaphragm.Setting the Koehler Illumination(continued)
OPERATION OF THE MICROSCOPE –EXAMINING SMEARS Put smear on stage and center it under the 10Xobjective Adjust intensity of the light to a comfortable levelwith the transformer Open condenser diaphragm about 70% toachieve a good balance of resolution andcontrast Adjust oculars for interpupillary distance so thatwhen looking with both eyes only one circle oflight is seen
Examining Smears (continued)• Adjust sharpness of image by movingadjustment ring on adjustable ocular• Once 10X focus is achieved, rotate nosepieceso that the 40X objective is in place• Readjust the intensity of light to a comfortablelevel using the transformer• Use the fine adjustment knob to focus up anddown through the different planes of the field
Microscope Problems –Troubleshooting 1Problem: Black FieldPossible Causes:• Microscope not plugged in• Power not available at outlet• Illuminator not turned on• Bulb burned out• Objective not clicked into place• Condenser too low withdiaphragms closed
Microscope Problems –Troubleshooting 2Problem: Field only partiallyilluminatedPossible Causes:• Objective not clicked into position• Condenser not centered correctly• Condenser too low• Field diaphragms closed too much
Problem: Difficulty focusing with10XobjectivePossible Causes:• Wrong objective in place• Objective not screwed into place• Not in correct plane of focusMicroscope Problems –Troubleshooting 3
Microscope Problems –Troubleshooting 4Problem: Difficulty focusing with 40XobjectivePossible Causes:• Not in correct plane of focus• Not initially focused at 10X
Microscope Problems –Troubleshooting 5Problem: Blurry image at 10X or40XPossible Causes:• Dirty objective• Dirty slide• Dirty coverslipProblem: Ground glassappearancePossible Causes:• Condenser too high
REFERENCES: http://www.microscopemaster.com/brightfield-microscopy.html Advanced Light Microscopy vol. 1 Principles and Basic Propertiesby Maksymilian Pluta, Elsevier (1988) Advanced Light Microscopy vol. 2 Specialised Methods byMaksymilian Pluta, Elsevier (1989) Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle,BIOS Scientific Publishers (1998) Microbiology: Principles and Explorations by Jacquelyn G. Black,John Wiley & Sons, Inc. (2005) Microscopy and Imaging Literature http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html