Biology 150 Estonilo | Gascon

property of some atoms and molecules to absorb light of a particular wavelength and after a brief interval (fluorescence lifetime) to re-emit light at longer wavelengths

Allows quantitative spatial and temporal visualization of fluorescent material in microscope specimens that are either intrinsically fluorescent (primary or autofluorescence) or which have been coupled to extrinsic fluorescent molecules (secondary fluorescence)

To deliver excitation energy to the fluorescing species in the specimen and to separate the much weaker emitted fluorescence light from the brighter excitation light. (filter fluorometer)

Specificity Sensitivity Environmental sensitivity High Temporal and Spatial Resolution

The mercury lamp emits ultraviolet (UV) light

o directed to a specific fluorescence filter cube located in the mirror

turret unit.

The exciter filter in the cube is designed to allow specific wavelengths of UV light to pass though
o those that are absorbed by the dye in use o reflected by the dichroic mirror o pass through the objective lens to illuminates the specimen

(violet rays).

The light emitted from the specimen (multiple color rays) is directed upwards through the objective and pass through the dichroic mirror
o removes the incident UV light o allows the longer wavelength visible light to pass through

The light emitted is viewed after passing through the emission filter
o selects which wavelength of light passes through to the eyepieces

(only the green ray). o Bright green glowing dye-stained cell or organelle against a dark background.

Excitation light sources Wavelength selection devices Objectives Detectors

- consist of tungsten or halogen lamps (incident illumination) and mercury, xenon or metal halide arc lamps or lasers (epi-illumination) - selection depends on the fluorescent probes being used.

o Include tunable lasers, monochromators,

AOTF and LCTF o Used to select a certain part of the electromagnetic spectrum for transmission, while preventing the rest of the electromagnetic spectrum from passing through the filter o Excitation filter and barrier filter

o Transmitted-light and reflected-light

versions o Depends on wavelength of light o Quartz objectives for use in UV; Fluars for wide spectral range

o Allow visualization of low levels of emitted

fluorescence without photobleaching to the specimen. o Allow realtime recording of living cell and tissue physiology

TIRFM (Total Internal Reflection Fluorescent Microscopy) uses a completely different concept. Very thin layer of the specimen (around 200 nm) is used to create the image. Therefore, TIRFM is ideal to analyse e.g. single molecule interactions or membrane processes. To achieve this target, a light beam is directed within a critical angle towards the cover slip. Because of the higher refractive index of the cover slip compared to the specimen,

total internal reflection occurs

o almost no direct light enters the specimen o evanescent wave travels in the specimen direction o only strong enough to excite fluorochromes within the

first few hundred nanometres close to the cover slip. o The fluorescent image is restricted to this small depth
cannot be driven into deeper areas of the specimen, and also does not contain out of focus blur from deeper areas.