LIGHT MICROSCOPYBY: NICOLE SULLIVAN

MODERN LIGHT MICROSCOPE

• OPERATE WITH VISIBLE LIGHT

• TWO MAGNIFYING LENSES AND A VARIETY OF CORRECTING LENSES

• HIGH MAGNIFICATION AND CLARITY

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.

TYPES OF LIGHT MICROSCOPES

Bright-Field Microscope

Dark-Field Microscope

Phase-Contrast

Microscope

Differential-Interface-Contrast

Microscope

Fluorescence Microscope

Confocal Microscope

BRIGHT-FIELD MICROSCOPE

• LIGHT IS TRANSMITTED THROUGH SPECIMEN

• LITTLE CONTRAST• STAINING IMPROVES

CONTRAST, BUT REQUIRES FIXED CELLS, WHICH CAN ALTER COMPONENTS

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

PHASE-CONTRAST MICROSCOPE

COMPONENTS OF MICROSCOPE BRING LIGHT WAVES OUT OF PHASE. THIS PRODUCES DIFFERENCES IN CONTRAST AND BRIGHTNESS WHEN LIGHT WAVES RECOMBINE

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.

FLUORESCENCE MICROSCOPE

• FLUORESCENT STAINS ABSORB LIGHT AT ONE WAVELENGTH AND EMIT IT AT ANOTHER

• FILTERS TRANSMIT ONLY THE EMITTED LIGHT

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

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.