Download - Light Microscopy
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LIGHT MICROSCOPYBY: NICOLE SULLIVAN
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MODERN LIGHT MICROSCOPE
• OPERATE WITH VISIBLE LIGHT
• TWO MAGNIFYING LENSES AND A VARIETY OF CORRECTING LENSES
• HIGH MAGNIFICATION AND CLARITY
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COMPOUND MICROSCOPE
• FIRST LENS FOCUSES THE IMAGE ONTO THE SECOND LENS• SECOND LENS MAGNIFIES THE IMAGE AND FOCUSES IT ON THE BACK OF
THE EYE• A COMPOUND MICROSCOPE IS ONE THAT MAGNIFIES IN STAGES USING
SEVERAL LENSES
• NOTE: COMPOUND MICROSCOPES CAN RESOLVE STRUCTURES THAT ARE SEPARATED BY AT LEAST 200 NANOMETERS.
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TYPES OF LIGHT MICROSCOPES
Bright-Field Microscope
Dark-Field Microscope
Phase-Contrast
Microscope
Differential-Interface-Contrast
Microscope
Fluorescence Microscope
Confocal Microscope
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BRIGHT-FIELD MICROSCOPE
• LIGHT IS TRANSMITTED THROUGH SPECIMEN
• LITTLE CONTRAST• STAINING IMPROVES
CONTRAST, BUT REQUIRES FIXED CELLS, WHICH CAN ALTER COMPONENTS
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DARK-FIELD MICROSCOPE
• LIGHT IS DIRECTED AT AN ANGLE TOWARD SPECIMEN
• A CONDENSER LENS TRANSMITS ONLY LIGHT REFLECTED OFF SPECIMEN
• FIELD IS DARK, SPECIMEN IS LIGHT
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PHASE-CONTRAST MICROSCOPE
COMPONENTS OF MICROSCOPE BRING LIGHT WAVES OUT OF PHASE. THIS PRODUCES DIFFERENCES IN CONTRAST AND BRIGHTNESS WHEN LIGHT WAVES RECOMBINE
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DIFFERENTIAL-INTERFERENCE-CONTRAST MICROSCOPE
POLARIZED LIGHT IS SPLIT INTO TWO BEAMS THAT HAVE SLIGHTLY DIFFERENT PATHS THROUGH THE SAMPLE. COMBINING THESE TWO BEAMS PRODUCES GREATER CONTRAST.
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FLUORESCENCE MICROSCOPE
• FLUORESCENT STAINS ABSORB LIGHT AT ONE WAVELENGTH AND EMIT IT AT ANOTHER
• FILTERS TRANSMIT ONLY THE EMITTED LIGHT
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CONFOCAL MICROSCOPE
• LIGHT FROM A LASER IS FOCUSED TO A POINT AND SCANNED ACROSS THE FLUORESCENTLY STAINED SPECIMEN IN TWO DIRECTIONS.
• PRODUCES CLEAR IMAGES OF ONE PLANE OF THE SPECIMEN
• OTHER PLANES ARE EXCLUDED TO PREVENT BLURRING
• MULTIPLE PLANES CAN BE USED TO RECONSTRUCT A 3-D IMAGE
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KEEP IN MIND…
LIGHT MICROSCOPES ARE NOT POWERFUL ENOUGH TO RESOLVE MANY OF THE STRUCTURES WITHIN CELLS. THIS IS DUE TO THE FACT THAT WHEN TWO OBJECTS ARE CLOSER THAN A FEW HUNDRED NANOMETERS, THE LIGHT BEAMS REFLECTING FROM THE TWO IMAGES START TO OVERLAP EACH OTHER.