Bioimaging Lecture 2

Time taken for excitation to an excited state, the occurrence of fluorescence emission

10 ^-15 seconds

~10 ^-9 seconds

What happens to an excited electron before fluorescence emission

It rapidly loses some energy as vibration/heat and reaches the lowest vibrational level of S1

Why is emitted fluorescence usually longer in wavelength than excitation light ?

Some energy is lost before emission, so the emitted photon has less energy and a longer wavelength

Why are fluorescence timescales important ?

They affect how soon a molecule can be excited again and can vary with the fluorophore’s local environment

How is an excitation spectrum measured ?

Fix the emission wavelength and scan excitation wavelengths, noting where a signal appears

How is an emission spectrum measured ?

Fix the excitation wavelengths and scan emission wavelengths, noting where a signal appears

Fluorophore structural features

Several combined aromatic groups, or planar or cyclic molecules

Three fluorescence labelling/tagging approaches

Immunofluorescence

Fluorescent-protein tags

Small molecule tags

What is the purpose of genetic fluorescent reporters ?

To use genetically encoded fluorescent signals to report biological features or activity

Cellular structures/compartments listed for fluorescence cell profiling

Early and late endoscopes

Lysosomes

Peroxisomes

Mitochondria

Endoplasmic reticulum

Microfilaments

Microtubules

Cell surface

What is coloured filter glass (absorption glass)

A filter that attenuates light by absorption; its spectral performance depends on the glass thickness

Problems with coloured filter glass

Absorbed light energy becomes heat

Thicker glass improves blocking but also reduces peak in-band transmission

Broad thin-film coating categories

Metallic coatings (mirrors and neutral-density filters)

Thin-film interference coatings

Thin film interference coatings and wavelengths

Reflections at interfaces between thin layers combine through wave interference, reflecting some wavelengths and transmitting others

Thin film interference example

Swirls of colour on a soap bubble

Limitations of thin-film interference ?

Blocking occurs only in specific wavelength ranges; transmission, absorption and reflection elsewhere must be considered; performance is sensitive to angle of incidence

Long pass, short pass and band pass filters transmit

Long pass - longer wavelengths

Short pass - shorter wavelengths

Band pass - a selected wavelength band

Dichroic beamsplitter designed to do in fluorescence microscopy

Reflect the excitation band and transmit the emission band, improving signal and reducing background noise

Modern dichroic beamsplitter performance

Over 95% reflectance in the excitation band and average transmission over 95% in the emission band

Filter cube in fluorescence microscopy

To hold the appropriate filters and dichroic mirror for selecting excitation and emission light