the light microscope

Activity 1Activity 1

Light MicroscopyLight Microscopy

Bright Field MicroscopeBright Field MicroscopeGeneral PerspectiveGeneral Perspective

Schematic PerspectiveSchematic Perspective

AdvantageAdvantage

Simplicity of setup Simplicity of setup Allows viewing of live cellsAllows viewing of live cells Dark or highly coloredDark or highly colored

Specific UseSpecific Use

Suited for utilizationSuited for utilization Viewing stained or naturally Viewing stained or naturally

pigmented specimenpigmented specimen Useless for some living specimensUseless for some living specimens

Parts of Bright Field MicroscopeParts of Bright Field Microscope

BaseBase – – supports the structure supports the structure

Objective lenses- magnify the image.Objective lenses- magnify the image.

Ocular Ocular - - magnify the image from the magnify the image from the objective lens. objective lens.

Body Tube – Body Tube – send light to the ocular send light to the ocular lens. lens.

Condenser Lens- Condenser Lens- directs light to directs light to pass through the pass through the

specimen. specimen.

Stage Stage - platform that allows - platform that allows mechanical movement of a mechanical movement of a

microscope slide. microscope slide.

Adjustment Knob – course and fine Adjustment Knob – course and fine adjustmentadjustment

Arm – support structureArm – support structure Iris Diaphragm Lever- controls the Iris Diaphragm Lever- controls the

amount of light entering the condenser amount of light entering the condenser and to specimenand to specimen

Nosepiece – which are found the Nosepiece – which are found the objectives. objectives.

Each objectives can be rotated into Each objectives can be rotated into place by simply rotating the place by simply rotating the nosepiecenosepiece

Dark Field MicroscopeDark Field MicroscopeGeneral PerspectiveGeneral Perspective

Accessory PartsAccessory Parts

AdvantageAdvantage

Quality of image is impressiveQuality of image is impressive Raised featuresRaised features


Live and biological samplesLive and biological samples Study of crystals and crystal defectsStudy of crystals and crystal defects

Phase Contrast MicroscopePhase Contrast MicroscopeGeneral PerspectiveGeneral Perspective

AdvantageAdvantage

Does not require stainingDoes not require staining living cells can be examined in their living cells can be examined in their

natural state without being killed, natural state without being killed, fixed, and stainedfixed, and stained


Transparent specimensTransparent specimens Cell parts are transparentCell parts are transparent

Fluorescent MicroscopeFluorescent MicroscopeGeneral PerspectiveGeneral Perspective

AdvantageAdvantage

Detect structures and molecules Detect structures and molecules within the cellwithin the cell


• Living cells and internal components Living cells and internal components are contrasted against the are contrasted against the background giving greater definition background giving greater definition and detail of cell structureand detail of cell structure

• Cells need not be fixed or stainedCells need not be fixed or stained• Absorb light at one wavelength and Absorb light at one wavelength and

emit light at anotheremit light at another

Differential Interference Contrast Differential Interference Contrast MicroscopeMicroscope

General PerspectiveGeneral Perspective

Light Pathway in DIC MicroscopeLight Pathway in DIC Microscope

Inverted micoscopeInverted micoscope General PerspectiveGeneral Perspective

Light pathways of Inverted Light pathways of Inverted MicroscopeMicroscope

Dissecting MicroscopeDissecting Microscope

Pathway of LightPathway of Light

Stereo MicroscopeStereo Microscope General PerspectiveGeneral Perspective

Types of Types of Light Light

MicroscoMicroscopepe

AdvantageAdvantage Specific Specific UseUse

Bright Bright Field Field

MicroscoMicroscopepe

Simplicity of Simplicity of setup with only setup with only

basic equipment basic equipment required. required.

No sample No sample preparation preparation

required, required, allowing viewing allowing viewing

of live cellsof live cells

Sample Sample illuminatiillumination is via on is via

transmittetransmitted white d white lightlight..

Dark field Dark field MicroscopeMicroscope

Quality of Quality of image is image is impressiveimpressive

Raised Raised featuresfeatures

Live and Live and biological biological samplessamples

Study of Study of crystals and crystals and crystal defectscrystal defects

Phase Phase Contrast Contrast MicroscopeMicroscope

Does not Does not require require stainingstaining

living cells can living cells can be examined be examined in their in their natural statenatural state

Transparent Transparent specimensspecimens

Cell parts are Cell parts are transparenttransparent

Fluorescent Fluorescent MicroscopeMicroscope

Detect Detect structures structures and and molecules molecules within the within the cellcell

Living cells Living cells and and internal internal componentcomponents are s are contrasted contrasted against the against the background background giving giving greater greater definition definition and detail and detail of cell of cell structurestructure

Differential Differential Interference Interference

Contrast Contrast MicroscopeMicroscope

Do not mask Do not mask or otherwise or otherwise obstruct the obstruct the objective and objective and

condenser condenser apertures apertures

Allows the Allows the specimen to specimen to be seen more be seen more

clearlyclearly

Inverted Inverted MicroscopMicroscop

ee

Allow to Allow to observe a observe a

living cells or living cells or organisms at organisms at the bottom of the bottom of

a large a large container container

Allow the Allow the specimen to specimen to

remain active remain active for long for long

periods. periods.

Dissecting Dissecting MicroscopMicroscop

ee

Gives true Gives true color with 20x color with 20x and 40x power and 40x power

using 10x using 10x eyepieces and eyepieces and

rotating rotating turret of 2x turret of 2x

and 4x and 4x objectives objectives

Used for Used for dissection to dissection to get a better get a better look at the look at the

larger larger specimen. specimen.

Stereo Stereo MicroscopeMicroscope

Light in Light in weight, fixed weight, fixed achromatic achromatic objectives 2x objectives 2x or 3xor 3x

designed long designed long working working distance distance objectives & objectives & wide field wide field eyepieces eyepieces produce an produce an extremely extremely large, large, brilliant & brilliant & bright bright images. images.

FormulasFormulas

R = (0.61 x λ) / N.A.R = (0.61 x λ) / N.A. λ – Light Wavelengthλ – Light Wavelength N.A. – Numerical ApertureN.A. – Numerical Aperture

Wavelength of ROYGBIVWavelength of ROYGBIV

Numerical ApertureNumerical Aperture

N.A. = n sin θN.A. = n sin θ• n : the refractive index of the media at n : the refractive index of the media at

d-line (587m)d-line (587m)• for dry objective n = 1.000 airfor dry objective n = 1.000 air• for oil objective n = 1.515 oilfor oil objective n = 1.515 oil• for water objective n = 1.333 waterfor water objective n = 1.333 water• θ = half angle of incident rays to the top θ = half angle of incident rays to the top

lens of the objective.lens of the objective.

SubstanceSubstance Refractive IndexRefractive Index Air Air 1.000293 1.000293 IceIce 1.31 1.31 WaterWater 1.33 1.33 Ethyl alcoholEthyl alcohol 1.36 1.36 FluoriteFluorite 1.431.43QuartzQuartz 1.54 1.54 SaltSalt 1.54 1.54 TourmalineTourmaline 1.62 1.62 GarnetGarnet 1.73-1.89 1.73-1.89 Cubic Zirconia Cubic Zirconia 2.14 - 2.20 2.14 - 2.20 Diamond Diamond 2.41 2.41

Steps in Preparing the Steps in Preparing the SpecimenSpecimen

Fixation – it is done by putting chemicals Fixation – it is done by putting chemicals that preserve material in a lifelike that preserve material in a lifelike condition, thus, the specimen can’t be condition, thus, the specimen can’t be distorted distorted

Dehydration – water is removed from Dehydration – water is removed from the specimen using ethanol the specimen using ethanol

Staining – most of biological material is Staining – most of biological material is transparent and needs staining to transparent and needs staining to increase the contrast between different increase the contrast between different structuresstructures

Mounting – mounting on the slide Mounting – mounting on the slide protects the material so that it is protects the material so that it is suitable for viewing over a long suitable for viewing over a long period. period.

Problems Encountered and Problems Encountered and Corrective MeasuresCorrective Measures

When using compound microscope and you come When using compound microscope and you come with very high magnifications with transmitted light, with very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by point objects are seen as fuzzy discs surrounded by diffraction rings or the so called airy-disc. diffraction rings or the so called airy-disc.

Limitations of lens design which can result in Limitations of lens design which can result in increased magnification without increased resolution increased magnification without increased resolution resulting to image that is larger but not clearer and resulting to image that is larger but not clearer and could not present more detailed information.could not present more detailed information.

Two objects must be 0.1 mm apart so that Two objects must be 0.1 mm apart so that they will be perceived as two, there are lens deign they will be perceived as two, there are lens deign that even though objects appears 0.1 mm apart, the that even though objects appears 0.1 mm apart, the edges becomes blurry that we detect two objects as edges becomes blurry that we detect two objects as single. single.

Hypothetical ProblemsHypothetical Problems

Using the oil immersion objective of Using the oil immersion objective of a bright-field microscope, an object a bright-field microscope, an object is seen and measured as 1.2cm long. is seen and measured as 1.2cm long. What is the actual size of the What is the actual size of the observed object as expressed in observed object as expressed in nanometers?nanometers?

1.2cm1.2cm = = yy

1000cm 1y1000cm 1y

y= y= 1.2cm x 1y1.2cm x 1y = 0.0012 cm = 0.0012 cm

1000cm1000cm

ReferencesReferences Davidson, M. & Abramowitz, M. 2003. Davidson, M. & Abramowitz, M. 2003. Brightfield Microscopy Brightfield Microscopy

Digital Image GalleryDigital Image Gallery. MolecularExpressions,. MolecularExpressions,http://micro.magnet.fsu.edu/primer/anatomy/brightfieldgallehttp://micro.magnet.fsu.edu/primer/anatomy/brightfieldgallery/index.htmlry/index.html..

Caprette, D.2005. Caprette, D.2005. Bright Field MicroscopyBright Field Microscopy. Experimental . Experimental Biosciences, Biosciences, http://www.ruf.rice.edu/~bioslabs/bios211/index.html.http://www.ruf.rice.edu/~bioslabs/bios211/index.html.

P. Hirsch, A. Howie, R. Nicholson, D. W. Pashley and M. J. P. Hirsch, A. Howie, R. Nicholson, D. W. Pashley and M. J. Whelan.2008. Whelan.2008. Dark field microscopyDark field microscopy. Wikipedia the free . Wikipedia the free encyclopedia,encyclopedia,http://en.wikipedia.org/wiki/Dark_field_microscopyhttp://en.wikipedia.org/wiki/Dark_field_microscopy..

Douglas B. Murphy, Ron Oldfield.2003. Douglas B. Murphy, Ron Oldfield.2003. Principles of phase Principles of phase contrast microscopy.contrast microscopy.MicroscopyU,MicroscopyU,http://www.microscopyu.com/articles/phasecontrast/phasehhttp://www.microscopyu.com/articles/phasecontrast/phasehome.htmlome.html..

Frängsmyr,T. & Ekspång,G.1993.Frängsmyr,T. & Ekspång,G.1993.The Fluorescence The Fluorescence Microscope- Preparation of a Microscope- Preparation of a SpecimenSpecimen.Nobelprize,http://nobelprize.org/educational_gam.Nobelprize,http://nobelprize.org/educational_games/physics/microscopes/fluorescence/preparation.htmles/physics/microscopes/fluorescence/preparation.html

the light microscope

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