THREE GOALS: •produce a magnified image of the specimen, •separate the details in the image, •render the details visible to the human eye or camera.
The History • Hans and Zacharias Janssen of Holland in the 1590’s created the “first” compound microscope • Anthony van Leeuwenhoek and Robert Hooke made improvements by working on the lensesAnthony van Leeuwenhoek Hooke Microscope Robert Hooke 1632-1723 1635-1703
The History Zacharias Jansen The “First” Microscope 1588-1631
Types of microscopes Light microscopes Bright field microscope Dark field microscope Phase contrast microscope Fluorescent microscope Electron microscopes Transmission electron microscope Scanning electron microscope
Magnification• To determine your magnification…you just multiply the ocular lens by the objective lens• Ocular 10x Objective 40x:10 x 40 = 400 So the object is 400 times “larger” Objective Lens have their magnification written on them. Ocular lenses usually magnifies by 10x
RESOLVING POWER/ RESOLUTION• ability of a lens to separate or distinguish small objects that are close together• wavelength of light used is major factor in resolution shorter wavelength ⇒ greater resolution Actual What We Might See
WAVE LENGTH• Wave length of light – used for illumination – Visible range – 400 – 750nm (Shorter wave length – high resolution) E.g. Blue light has a shorter wave length than red light
NUMERIAL APERTURE• NA – of the objectives (property of lens that decides the quantity of light enter into it) – Two factors 1) Refractive Index 2)Angle
REFRACTION• The bending of light as it passes from one medium to another of different density• The bending of the light ray gives rise to an angle of refraction, the degree of bending• Index of refraction: A measure of the speed at which light passes through the material
ILLUMINATION• To collect and reflect the light – two devices – 1) Mirror 2) artificial Light• 1) Mirror – natural source or artificial source – Two side – reflective- plain – artificial light – Concave – natural source
Properties of Light:Light & Objects • Reflection: If the light strikes an object and bounces back (giving the object color) • Transmission: The passage of light through an object • Absorption: The light rays neither pass through nor bounce off an object but are taken up by the object
How a Microscope WorksConvex Lenses arecurved glass used tomake microscopes(and glasses etc.) Convex Lenses bend light and focus it in one spot.
How a Microscope WorksOcular Lens Objective Lens(Magnifies Image) (Gathers Light, Magnifies And Focuses Image Body Tube Inside Body Tube) (Image Focuses)•Bending Light: The objective (bottom) convex lensmagnifies and focuses (bends) the image inside thebody tube and the ocular convex (top) lens of amicroscope magnifies it (again).
Caring for a Microscope• Clean only with a soft cloth/tissue• Make sure it’s on a flat surface• Don’t bang it• Carry it with 2 HANDS…one on the arm and the other on the base
Using a Microscope• Start on the lowest magnification• Don’t use the coarse adjustment knob on high magnification…you’ll break the slide!!!• Place slide on stage and lock clips• Adjust light source (if it’s a mirror…don’t stand in front of it!)• Use fine adjustment to focus
I. Dark field contrast microscopy takes advantage ofobjects that “scatter” light - this requires a specialcondenser that can “angle” the incident lightII. Phase contrast microscopy takes advantage ofobjects that alter the phase of incident light - Thisrequires “phase rings” in the condenser and in theobjective lensIII. Fluorescence microscopy take advantage ofinherently fluorescent Material of biologicalobjected that can be fluorescenlty labeled.
DARK FIELD MICROSCOPE Contrast Bright Field Phase Contrast Dark-Field
Dark field contrast microscopytakes advantage of objects that“scatter” light - this requires aspecial condenser that can“angle” the incident light
FLUORESCENT MICROSCOPEOptical System of a FluoresceinFluorescence -excited by blueMicroscope light (450-490) -emits green light (520-560)
[INSERT FIGURE 4.9]Fluorescence microscopy take advantage of inherentlyfluorescent Material of biological objected that can befluorescenlty labeled.