Microscopy

Microscopy allows us to view small objects as if they we large

Simple optical systems were used in early microscopes to view objects in the visible spectrum

Today, microscopists employ complex optics, specialized photons, and detectors to image objects in fascinating ways

How small can we see?

Magnification Optics

Assyrians noted magnifaction of small glass spheres (B.C.)

Ptolemy wrote about magnification and refraction (2nd century A.D.)

Spectacles (1300 A.D.) More detailed investigation in 16th century Galileo is credited with the first biological

observations, using the Galilean microscope

Properties of lenses

Spherical Bi-convex Bi-concave Plano-convex Plano-concave

Contrast in Microscopy

Most thin objects are transparent Need objects as thin as possible, but this can be quite

challenging and does not allow easy 3D analysis Stains can be used to differentiate features

Resolution in Microscopes

Human eye (unaided) is about 0.1 mm at 30 cm Limited by the nature of light

Red light has a wavelength of 700 nm Violet light has a wavelength of 430 nm Optical limits are roughly 0.21-0.35 μm

Limitations of early magnifiers

Distortion Imprecise focus Chromatic abberation Light loss

Simple Microscopes

Simple microscopes consist a specimen holder A single lens

Disadvantages include chromatic aberration Inflexibility in viewing fields

Compound Microscopes

Compund microscopes employ two or more lenses

Compund microscopes can reduce chromatic aberration and increase flexibility

More complex to design

Early Optical Microscopes

Janssen created a microscope capable of magnifying images between 3 and 10 times

Bi-convex eyepiece Plano-convex objective

Leeuwenhoek's Microscope

Magnification between 70x to 250x Small lenses (mm size) Resolution of roughly 1 micron

Robert Hooke's Microscope

Compound design Bi-convex objective, eyepiece, and field lens Significant spherical and chromatic aberrations Internal diapragm Ingenious light source

Optical Microscopes

By 1900, microsopes had reached the limits determined by optical light Optical theory says smallest clearly objects visible are

no smaller than ½ wavelenth of light Specimen preparation Optical properties including light polarization were

routinely used Photographic techniques allowed for easy

dissemintion of findings

Polarization

In polarizing microscopy, polarized light is reflected by or transmitted through a specimen

Some light changes polarization depending on the specimen properties

A second polarizing filter removes light unchanged in the specimen

Helps distinguish similar materials with Isotropy (minerals, crystals, glass, ceramics, DNA,

etc.) Boundaries of materials

Phase-contrast

Contrast is enhanced by exploiting sublte differences in refractive indices

Similar to polarization A phase shift of the light waves can be introduced

which causes interference Allows contrast between living cells and solution

by removing background light

Chilodonella uncinata (parasite) (100x)

A fibroblast in tissue culture visualized with four types of light microscopy. The image in (A) was obtained bythe simple transmission of light through the cell, a technique known as bright-field microscopy. (B) Phase-contrastmicroscopy; (C) differential-interference-contrast microscopy; and (D) dark-field microscopy.

Fluorescence Microscopes

Contrast is obtained by staining a specimen with a dye (fluorochrome) which absorbs UV light and re-emits it in the visible spectrum

Stained objects appear bright with respect to unstained features

New techniques allows a green fluorescent protein from a jellyfish to be combined with proteins to undestand cellular function at the protein level.

Human umbilical vein (120X)

Bovine Pulmonary artery endothelium cells (400x)

Confocal Scanning Microscopy

Confocal miscropscopy gives a way to preserve the three-dimensional structure of a specimen

A focussed laser beam sweeps across a specimen at a specific depth

Features at other depths have very low contrast An electronic slice is obtained and can be

combined with others to study the 3D nature of the specimen's features

Resolution is around 0.2 microns

HeLa (cancer) cells in culture (1,350x)

Pollen (40x)

2 micron diameter spheres as a volume showing how a 3D image can be generated.

Electron Microscopes

Electrons behave just like light photons, except they have a much smaller wavelength (0.05 nm) than light

Electrons can not be directly viewed with the human eye

Electrons can only travel in a vacuum Electrons are emitted from a cathode ray gun,

interact with the specimen, and are detected Electrons are focussed using magnets

Transmission Electron Microscopes

An image is created by measuring the number of electrons passing through the specimen at each image location

Contrast is obtained due to differential electron interactions with materials Large atoms attract and stop more electrons Small atoms attract fewer electrons

A phosphor screen converts the transmitted electrons into visible light

TEM Staining

As in light microscopy, objects can be treated to give more contrast

Specimens can be fixed with a heavy metal salt such as Osmium Tetroxide

Specimens can be directionally sputter coated with gold or metallic salts

The metals absorb more electrons, providing increased contrast in the specimen

Transmission Electron Microscope image of cytomegalovirus (CMV) infection in the lung. Clusters of coated virions are present in membrane-limited vesicles. Single, coated virions are also seen in the cisternae of the endoplasmic reticulum (arrow).

High magnification transmission electron micrograph of an ultrathin section through a mammalian mitochrondia. The inner and outter mitochondrial membranes as well as the intralumenal space are clearly visible at the periphery of this organelle. Numerous cristae formed by foldings of the inner mitochondrial membrane are also visible throughout the organelle's lumen.

Scanning Electron Microscopes

Developed in the 1940's Perfected in the 1960's Produces 3D images from shadows Has a large depth of field An electron beam is swept across the specimen

and the electrons scattered onto a detector An electron beam inside a CRT is swept

synchonously with the miscrocope using its intensity to create an image

Scanning Tunneling Microscopes

Acoustical Microscopes

Atomic Force Microscopy