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

Brief History of the Microscope What‘s a microscope? Definition of Magnification Conventional Viewing Distance Leeuwenhoek > Compound > Stereo Microscope The “Telescope”, a simple detour How to make the specimen visible – Contrast! Definition of Contrast Techniques:  Brightfield  Phase  Darkfield  Pol  DIC (Differential Interference Contrast)  Fluorescence  Optical Sectioning – an expansion of Fluorescence Setting up the Microscope (Lab) Koehler Illumination Resolution & Empty Magnification Agenda

Other things you may wish to know – but there won‘t be enough time today: Components of the Microscope Different Light Sources Field / Aperture Diaphragms Condensers – why Objectives – what to consider in the selection process Adapting Cameras Understanding Numerical Aperture Diffraction Experiment

Objects appear to the eye at different magnifications, depending on their distance from the eye. Accommodation (lens) will make it possible. M B ~ 2x M A A B What is “Magnification”?

Objects appear to the eye at different magnifications, depending on their distance from the eye. Accommodation (lens) will make it possible. M B ~ 2x M A A B What is “Magnification”?

Conventional Viewing Distance 250 mm 1x ?

“Magnification” 1x f = 250 mm 1x

Magnification via Single Lens f = 250 mm 1x Example: f=50mm 5x Magnifying Glass (Loupe)

The simple microscope Leeuwenhoek Microscope

The  -corrected Compound Microscope Tube lens (Zeiss: f=164.5mm) Objective Eyepiece

Q: What happens if we take the objective away? Tube lens (Zeiss: f=164.5mm) Objective Eyepiece Tube f 250mm f M  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 TypeTelescope Type Introduced first by Zeiss Introduced first by Zeiss

Greenough Stereo Microscopes SteMi DV4

Greenough Stereo Microscopes SteMi 2000 (2000-C, 2000-CS)

Research Stereo Microscopes SteREO Discovery V12SteREO Lumar V12

Short Break Short Break