A. By staining the specimen B. By changing how the microscope interacts with the light that passes through the specimen C. By setting Köhler illumination D. All of the above
Problem with stains • Specimens prepared with stains usually are killed. • Stains may add artifacts to cell structures that don’t normally exist in the live cells.
The Phase Contrast Microscope to the rescue… • Pros: – Contrast is produced in apparently transparent cells without killing cells. • Cons: – Phase contrast condensers and objective lenses add considerable cost to a microscope
Phase Contrast Microscope Light passing through a transparent part of a specimen travels slower and is shifted compared to the uninfluenced light. This difference in phase can be increased by a phase-plate. This makes the transparent object shine out in contrast to its surroundings.
Human glial cells grown in culture Brightfield Phase Contrast Phase Contrast Microscopy
Fluorescence Excitation Emission For fluorescence to occur , a molecule must be capable of absorbing light of a relatively short exciting wavelength and emitting some of this energy as longer wavelength fluorescent light. We need molecules that can fluoresce! Absorbance
Another way of staining a cell… • A fluorophore is a molecule which has the property of fluorescence. • A fluorophore absorbs electromagnetic radiation of some specific wave length and then emits radiation of some slightly longer, lower energy wave length. • A fluorophore can mark the location of the structure of interest.
Fluorescent Labels Fluorophores (or fluorochromes) are used as stains in fluorescent microscopy. These are molecules that “fluoresce”, i.e. they absorb a relatively short wavelength of light, become excited, and decay to the ground state by emitting a slightly longer wavelength of light.
One way it can lose this energy is to emit a photon of slightly lower energy (i.e., longer wave length)light. DAPI (4',6-diamidino-2-phenylindole) Emitted light 420 nm Absorbed UV 365 nm