Lecture Notes on Summarization, Biomedical Engineering, Leaves, Jablonssky Diagrams, Fluorescence, Quenching and lifetime

Summarization Techniques

  • When summarizing, focus on the 'why' something was done and the problem it solves, rather than just the details of 'what' was done.
  • Analogy: Summarizing highway construction can be done by describing the trucks and shovels (details) or by explaining the traffic problem it solves (purpose).

Biomedical Engineering's Impact on Society

  • Consider how biomedical engineering technologies can aid society and improve human treatment.
  • Example: Cardiac arrest is a significant problem, and technology that can distinguish between mild and major repercussions could allow for targeted drug administration to alleviate inflammation and damage.

Translating Biomedical Technologies to Real-World Use

  • Key factors for technology translation:
    • Improved survival rates
    • Cost-effectiveness
    • Improved quality of life
  • These factors are often interlinked.
  • Academic research may prioritize solving riskier problems and assume technology costs will decrease over time, while companies often avoid very expensive technologies.

Why are Leaves Green?

  • Leaves are not black to avoid overheating by absorbing all photons.
  • Plants in jungles with tree cover are darker green to absorb as many photons as possible, while plants in direct sunlight have strategies to reduce photon absorption.
  • Green color results from chlorophyll absorbing blue and red light, reflecting green light.
  • Different types of chlorophyll exist, leading to different shades of green.
  • Carotenoids are orange pigments also present in nature (e.g., sweet potatoes, carrots).

Deciduous Trees and Leaf Color Changes

  • Deciduous trees lose leaves in colder temperatures because their structure doesn't respond well to the cold.
  • Before leaves fall, trees expend energy to create new molecules with different color structures.
  • The exact purpose of these color changes isn't fully understood, but theories include light protection, delaying leaf fall, and structuring leaf decay for soil health.

Molecular Structure and Energy States (Jablonsky Diagram)

  • Molecules with more structured aromatic hydrocarbons have distinct energy states.
  • Distinct energy states absorb wavelengths in narrower bands, often resulting in color if the bands are in the visible spectrum.
  • Every bond contains two electrons with opposite spins.
  • When a photon is absorbed, one electron (often described as 'spin up') gains energy and occupies a larger probability density cloud (orbit).
  • Only distinct orbits exist, requiring specific amounts of energy for electron transitions.

Fluorescence

  • The bottom curves in a Jablonsky diagram represent ground states, while higher-level curves relate to internuclear separation.
  • Energy is required to pull atoms apart or push them together.
  • Lines represent vibrational states within the bond.
  • If the next ground state exceeds the vibrational state cap, the system releases energy through photon emission or transfer to a nearby molecule.
  • In the excited state, nuclei are not at optimal separation, causing them to spread out and fall back to the ground state, emitting fluorescence.

Red Shift

  • Fluorescence involves less energy release than excitation, leading to longer wavelengths (red shift).
  • Red shifted doesn't necessarily mean red, just lower energy.
  • Fluorescent imaging filters out excitation light to only show emitted fluorescent light.

Phosphorescence

  • Phosphorescence involves a spin flip in the excited electron, making the transition forbidden.
  • Energy release occurs via a rare, slow process (hours), like with glow-in-the-dark stars.
  • Fluorescence typically occurs in nanoseconds, making it nearly instantaneous for practical purposes.

Energy Spectrum of Fluorescence

  • The energy spectrum represents all possible transitions between energy states.
  • Absorption spectrum shows energy absorbed to reach excited states, while fluorescence spectrum shows energy released when returning to ground states.
  • A single photon has a single wavelength and energy, but a population of molecules absorbs and emits photons across a range of energies.

nano = 10^{-9}

  • A nanomole, though seemingly small, contains approximately 10^{14} molecules, each absorbing and emitting photons within an energy range.

Stokes Shift

  • Stokes shift is the difference in peaks due to internuclear separation in ground and excited states.
  • Absorption and fluorescence spectra often exhibit a mirror-image relationship due to vibrational state separation.

Quantum Yield

  • Quantum yield indicates the efficiency of photon release as fluorescence after absorption.
  • High quantum yield means a higher percentage of absorbed photons are released as fluorescence.
  • In fluorescent imaging, aim for the highest quantum yield possible to maximize brightness.

Factors Affecting Brightness of Fluorescent Molecules

  • Brightness depends on:
    • Light source intensity and color matching absorption spectrum.
    • Fluorophore concentration.
    • Absorption efficiency of the molecule.

Excitation Light Spectrum

  • A bright light source is more effective if its color aligns with the absorption spectrum of the fluorophore.
  • The spectrum of released light is independent of the excitation light used.

Efficiency of Light Absorption

  • A subtle change in a molecule's chemistry can affect its efficiency in absorbing light, changing the shape of the absorption peak.

Challenges of Light Absorption

  • Determining the amount of excitation light that reaches the fluorophore can be complex, especially within biological tissue.

Key Factors for Brightness

  • Key factors for Brightness:
    • Excitation light wavelength.
    • Absorption peak alignment.
    • Absorption efficiency.
    • Quantum efficiency.
    • Likelihood of fluorescence reaching the camera.
    • Coefficient of absorption ($\mu_a$):
      • Indicates the likelihood of a photon being absorbed by a molecule.
    • Quantum Efficiency ($\eta$):
      • The likelihood of releasing absorbed energy as a photon.

Quantification of Fluorescence

  • Systems quantify fluorescence by reconstructing an image proportional to the absorption coefficient of the fluorophore at each location, multiplied by its quantum efficiency ($\eta \mu_a f$).

Quenching

  • As fluorophore concentration increases, brightness increases linearly until quenching occurs.
  • Quenching is when molecules get too close together and find non-radiative ways to release energy, reducing fluorescence.

Reabsorption

  • Reabsorption is when your fluorescence will actually be reabsorbed by the molecule and then can actually cause secondary fluorescence.

Practical Implications of Quenching

  • If the density of molecules is too high, they will quench, resulting in no signal.

Nonlinear Effects

  • Engineers can use these nonlinear effects to their advantage.
    • Example:
      • Nanoparticles containing a drug and quenched fluorophores can release the drug and produce a fluorescent signal upon lysing in targeted cells.

Photo Bleaching

  • Bleaching agents, like sunlight, break bonds in colorful molecules, causing them to lose color.
  • Aromatic hydrocarbons are often responsible for color and fluorescence, and breaking these bonds eliminates both.
  • Photo bleaching is a concern in fluorescence microscopy, especially with raster scanning lasers.

Fluorescence Lifetime

  • Fluorescence lifetime is the time a fluorophore stays in an excited state before releasing a photon (typically nanoseconds).

Phosphorescence Lifetime

  • Phosphorescence lifetime is much easier to measure.

Measuring Light Using Stars

  • Measure the light emitted from stars as an example to see how florescence works.

Exponential Decay

  • Exponential decay that, the rate of change of signal is directly proportional to the signal itself for, this these two are just constants.

\frac{dy}{dt}=k*y

  • Solving for the equation:

\int \frac{1}{y} dy = \int k dt
\int{y0}^{y} \frac{1}{y} dy = \int{0}^{t} k dt \ln{y} |{y0}^{y} = kt |{0}^{t}

\ln{\frac{y}{y_0}} = kt

\frac{y}{y_0} = e^{kt}

y = y_0 * e^{kt}

Understanding the Formula

  • Since k is negative it means that it's a decaying exponential.

Important Considerations about Lifetime Curves

  • Reasons:
    • Individual molecules have distinct excitation times.
    • Probability density function that results in a decaying exponential.
      • The likelihood of the molecule to release energy quickly.

The Effect of Non-radiative means

  • Signal loss due to non-radiative energy release affects the time curve.

Microenvironment influence

  • Fluorescence lifetime depends on, is the likelihood of releasing that photon, The environment is very key.
  • Tumors might reduce the PH. Change this = different environment.

Understanding the Microenvironment Influence on Fluorescence

  • Because tumors might grow fast. They have less blood in there. Anaerobic glycolysis.

Benefits of Understanding Fluorescence

  • Benefits:
    • In mole something can block the light
    • Blood blocks light

Spectrophotometry

  • If you can tell the absorption spectra. What would you do?
    • Mix the powder
    • Solution
      • Into a fluids

Cuvette Measurement

  • Cuvette:
    • Rectangular shaped thing. Depth of it, It's always 1cm.

Prism Consideration

  • If I tilt the prism, I can let out distinct colors of light.

Beers Law

  • Beers lambert law.

Absorbance

  • Absorption:

    log(\frac{I}{I_0})

  • The solvent of the cuvette is what helps

  • It's how much signal did you get with stuff?

Absorbance Meaning

  • A: Epsilon cl = The meaning.
    • L = Length of the cuvette.
    • C = Concentration. Higher the concentration, hired of light of absorption.

Units

  • Log of something with new units is new units
  • The thing must have nanomolar centimeter

Concentration of absorber

  • The rate of change of the intensity, its proportional to how much you stuff you have

Absorption Factor

  • Likelihood of absorbing the light.

Correct Formula of Light absorbed

  • Likelihood of absorbing light.
  • Likelihood, it is something can absorb the light do anything

\frac{1}{I} = \int -c \epsilon dx
\int -c \epsilon dx |{I0}{I} = \int 0 ^ L log (\frac{I}{I0}) = \epsilon * I
log I\frac{I}{I0}) = \epsilon I log I\frac{I0}{I} = \epsilon I

  • If I switch over is gonna go into a negative