L11: Instrumentation for Fluorescence spectroscopy

Instrumentation for fluorescence

Filter cube

dichromatic mirror - only transmits light above a certain wavelength

Inner filter effect

light is absorbed by a material, which reduces the intensity of emitted light

high concentration - absorbs more than emits

can impact the accuracy of techniques like fluorescence spectroscopy

minimized by diluting the sample.

Stimulated emission and LASER

Light Amplification by Stimulated Emission of Radiation

Spontaneous vs stimulated emission

Stimulated emission is

1. Monochromatic (one colour/wavelength/energy)

2. Directional (perpendicular to excitation)

3. Polarized (travel in same plane)

4. Coherent (release at same time, travel in phase)

Fluorescence Spectrometer

Excitation source (lasers, xenon arc lamps, LEDs)

• Xenon arc lamps (broad spectrum, general use)

• Lasers (high intensity, monochromatic, ideal for single-molecule studies)

• LEDs (very common, cheap, stable, useful for portable sensors)

pulse width - structural or even quantum properties of a system - cyclic excitation

photochemistry - adjust pulse width to reaction time to

Excitation monochromator/filter (selects excitation wavelength)

extract specific wavelengths from broad-spectrum composite light source, i.e. prism

diffraction grating - smooth material with indented patterns; different wavelengths split/bend at different angles

Sample chamber (holds fluorophore or biomolecule)

90° from excitation direction

absorbance spectra - stops here

Emission monochromator/filter (selects emitted wavelength)

measure all wavelengths one at a time

Detector (PMT, CCD, or avalanche photodiodes for lowlight detection)

historically - eye

photo multiplier tubes - produce electrons in response to photons

spectrum - wavelength vs. # of photons

Importance

high sensitivity - look at things which are active

molecular specificity - molecular beacons (fluorophores, dyes, fluorescent proteins) = macromolecules or small molecules which you can attach to your biological molecule of interest AT SPECIFIC LOCATIONS

temporal dynamics from about 100 microseconds all the way to seconds

Spectral shifts

observed upon changes in solvent environment

Applications in biochemistry

sensing, tracking, reaction

Proteins

• location

• conformation

  • open/closed

  • folded/unfolded

  • binding

Chemical sensing of the environment

Kinetics

localization studies or binding studies also as a function of time

Physical sensing

dying cell - enzymes fall off

Advanced applications

Chronic pain is a huge burden on global health.

Sodium channels regulate pain through poorly known pathways (Nav1.7, Nav1.8, Nav1.9, etc)

• sodium channels are too active - chronic pain

• sodium chennels aren’t active enough - CIPA

Why do Nav1.7 molecules cluster? Are there other pain signaling proteins in the cluster?

Why is there energy loss before fluorescence?

Energy loss is due to a variety of dynamic processes that occur following light absorption

The excess vibrational energy is rapidly lost to the solvent.

If the fluorophore is excited to the second singlet state (S2), it rapidly decays to the S1 state in 10–12 s due to internal conversion.

Solvent effects shift the emission to still lower energy due to stabilization of the excited state by the polar solvent molecules.

Solvent relaxation

Rotational motions of small molecules in fluid solution are rapid, typically occurring on a timescale of 40 ps or less.

Fluorescence lifetime is ns or more

This allows for the solvent molecules to reorient around the excited-state dipole, which lowers its energy and shifts the emission to longer wavelengths.

dipole moment - not the same in ground and excited

• different attractions

• different energy - minimisation

This occurs within 10^–10 s in fluid solution.

very sensitive - can result in substantial Stokes shifts.

Other solvent factors

1. Solvent polarity and viscosity

2. Rate of solvent relaxation

3. Probe conformational changes

4. Rigidity of the local environment

5. Internal charge transfer

6. Proton transfer and excited state reactions

7. Probe–probe interactions (collision)

8. Changes in radiative and non-radiative decay rates (light intensity)