Basic Microscopy – An Overview – October 2005 Protistology Course MBL, Woods Hole, MA

Basic Microscopy Basic Microscopy – An Overview –– An Overview –

October 2005October 2005

Protistology CourseProtistology Course

MBL, Woods Hole, MAMBL, Woods Hole, MA

Brief History of the MicroscopeBrief History of the Microscope

What‘s a microscope?What‘s a microscope?

Definition of MagnificationDefinition of Magnification

Conventional Viewing DistanceConventional Viewing Distance

Leeuwenhoek > Compound > Stereo Leeuwenhoek > Compound > Stereo Microscope Microscope

The “Telescope”, a simple detourThe “Telescope”, a simple detour

How to make the specimen visible – How to make the specimen visible – Contrast!Contrast!

Definition of ContrastDefinition of Contrast

Techniques: Techniques: BrightfieldBrightfield PhasePhase DarkfieldDarkfield PolPol DIC (Differential Interference Contrast)DIC (Differential Interference Contrast) FluorescenceFluorescence Optical Sectioning – an expansion of FluorescenceOptical Sectioning – an expansion of Fluorescence

Setting up the Microscope (Lab) Setting up the Microscope (Lab)

Koehler IlluminationKoehler Illumination

Resolution & Empty MagnificationResolution & Empty Magnification

Age

nda

Objects appear to the eye at different magnifications, depending on their distance from the eye. Accommodation (lens) will make it possible.

MB ~ 2x MA

A B

What is “Magnification”?

Conventional Viewing Distance

250 mm

1x

?

“Magnification” 1x

f = 250 mm

1x

1x

Magnification via Single Lens

f = 250 mm

1x

Example: f=50mm

5x

Magnifying Glass (Loupe)Lensf

mmM

250

The simple microscope

Leeuwenhoek Microscope

The -corrected Compound Microscope

Eyepiece

Tube

Objective f250mm

250mm

f

f250mm

M

EyepieceObjectiveMicroscope Compound M MM

Tube lens

(Zeiss: f=164.5mm)

Objective

Eyepiece

EyepieceObjective

Tube

f250mm

ff

M

Q: What happens if we take the objective away?

Tube lens

(Zeiss: f=164.5mm)

Objective

Eyepiece

Eyepiece

Tube

f

fTelescopeM

Eyepiece

Tube

f250mm

250mm

fM

Answer: We have created a “Telescope”

∞

∞

AxioImager Upright Research Microscope

Axiovert 200 Inverted Research Microscope

The basic light microscope types

Upright microscope.

Inverted microscope

Illumination via Transmitted Light

The specimen must be transparent !

Upright microscope.

Inverted microscope

Illumination via “Reflected” (Incident) Light

Eg. Fluorescence, Opaque Samples

Upright microscope.

Inverted microscope

Upright microscope.

Inverted microscope

Mixed Illumination

Other Ways to Illuminate

Reflectors

Ring Lights

Fiber Optics

LED’s

Etc.

Which Microscope types are typically illuminated this way?

Reflectors

Ring Lights

Fiber Optics

LED’s

Etc.

“Couldn’t one build a microscope for both eyes, and thereby generate spatial images?”

Question addressed to Ernst Abbe in 1896

by Horatio S. Greenough

1896: Drawing by Horatio S. Greenough

1897 – the first Stereo Microscope in the world, built by Zeiss, according to the “Greenough” principle

Greenough Type

Telescope Type Introduced first by Zeiss - 1946

Introduced first by Zeiss - 1897

Greenough Stereo Microscopes

SteMi DV4

Greenough Stereo Microscopes

SteMi 2000

(2000-C, 2000-CS)

Research Stereo Microscopes

SteREO Discovery V12 SteREO Lumar V12

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