Epi-Fluorescence Illumination
Definition
Fluorescence excitation through the objective lens from above the specimen, with the same objective collecting the emission. A dichroic mirror directs excitation down and passes emission up, separating the two signals.
- Path
- Excitation and emission through the same objective
- Key component
- Dichroic mirror separating excitation and emission
- Direction
- Illumination from above the specimen
Common questions
Why is epi-illumination preferred over transmitted-light fluorescence for many forensic samples?+
Illuminating and collecting through the same objective avoids needing light to pass through the specimen, which works for opaque or thick samples such as fibres and questioned documents where transmitted light would not reach the detector cleanly.
What role does the dichroic mirror play specifically?+
It reflects the shorter-wavelength excitation light down onto the sample while transmitting the longer-wavelength emitted fluorescence back up to the detector, keeping the two signals from mixing despite sharing the same optical path.
Related terms
- Autofluorescence
- Native fluorescence of endogenous biological molecules (collagen, elastin, NADH, flavins) that produces background signal in fluorescence microscopy, potentially mimicking specific probe signals....
- Birefringence
- The difference between the highest and lowest refractive indices of an anisotropic mineral, seen as a characteristic interference colour between crossed polars....
- Conoscopic Illumination
- A convergent polarised beam mode in which a range of angles is passed through the specimen simultaneously, producing an interference figure (optical...
- Crossed Nicols
- The configuration in which the polariser (below the condenser) and the analyser (above the objective) are oriented at 90 degrees to each...
- DAPI
- 4',6-diamidino-2-phenylindole; a fluorescent dye that binds double-stranded DNA and emits blue (~460 nm) under UV excitation (~360 nm). Standard nuclear counterstain; used...
- Dichroic Mirror
- A spectrally selective beam splitter that reflects shorter (excitation) wavelengths at 45 degrees and transmits longer (emission) wavelengths, forming the core element...
- Extinction Angle
- The angle, measured in degrees on the rotating stage of a petrographic microscope, between a crystallographic reference direction (cleavage trace or crystal...
- Michel-Lévy Chart
- A birefringence-thickness diagram correlating retardation values with the interference colour seen at that retardation under white-light crossed polars. Standard reference for identifying...
- Photobleaching
- The irreversible photochemical destruction of a fluorophore by repeated excitation. Limits the observation time for low-copy forensic fluorescence samples. Mitigated by antifade...
- Retardation
- The path difference (in nm) introduced between the fast and slow ray components as they traverse an anisotropic specimen of given thickness...
- Stokes Shift
- The wavelength difference between the excitation peak and the emission peak of a fluorophore. A positive Stokes shift (emission at longer wavelength...