the light microscope
TRANSCRIPT
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
Schematic PerspectiveSchematic Perspective
Accessory PartsAccessory Parts
AdvantageAdvantage
Quality of image is impressiveQuality of image is impressive Raised featuresRaised features
Specific UseSpecific Use
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
Schematic PerspectiveSchematic Perspective
Accessory PartsAccessory Parts
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
Specific UseSpecific Use
Transparent specimensTransparent specimens Cell parts are transparentCell parts are transparent
Fluorescent MicroscopeFluorescent MicroscopeGeneral PerspectiveGeneral Perspective
Schematic PerspectiveSchematic Perspective
Accessory PartsAccessory Parts
AdvantageAdvantage
Detect structures and molecules Detect structures and molecules within the cellwithin the cell
Specific UseSpecific Use
• 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
Schematic PerspectiveSchematic 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