Fluorescence, Absorbance & Microscopy

What is the relationship between energy and wavelength? Can you give an example of this relationship?

The higher the energy absorbed, the shorter the wavelength will be. The lower the energy absorbed, the longer the wavelength will be. This can be quantified as an inverse relationship. For example, the color purple has a short wavelength, and the color red has a long wavelength. From this information you can infer that the color purple has higher energy than the color red.

What is the relationship between absorbance and concentration? What does the formula look like? What is the law called?

The relationship between absorbance and concentration is directly proportional. The formula is A = kc, where A = absorbance, k is a constant that is specific to that solute, and c is the concentration of that solute. The name of this law is Beer's Law.

Why is ethidium bromide orange on a gel electrophoresis?

Because it emits 595 nm of light, which we can see.

Why do we care about concentration?

Because we also care about fluorescence.

What are covalent bonds?

Bonds that connect atoms: They share electrons

What would it take to get rid of the normal conditions?

The electron would have to absorb a photon of light

What would it take to get back to normal conditions?

The electron would have to emit a photon of light.

How many peaks are there in a graph of fluorescence and what does it mean?

There are two peaks: one for excitation, and one for emission. both are on the y axis. on the x axis is the wavelength. Since excitation is caused by high energy the peak will be on the short wavelength. Since emission is caused by low energy the peak will be on the long wavelength.

How can you convert absorbance into concentration?

You need to measure out a standard curve. 1. Determine absorbance readings for known concentrations. 2. Plot them on a graph. x axis = concentration, y axis = absorbance. 3. Find k using linear regression analysis. 4. Use this to find the concentration of unknown substances (C=A/k).

What is the light that enters the sample called?

incident light

What is the light that exits the sample called?

transmitted light

What are wavelengths less than 350 nm called? What are wavelengths greater than 800 nm called?

ultra violet, gamma, & x-rays are less than 350 nm. Infrared, radio, & microwaves are greater than 900 nm.

How does a spectrophotometer work?

1. We have a source of white light. 2. We have a wavelength band filter. We select different filters, so that only narrow bands of wavelength can enter. 3. We have an exit slit, for the wavelengths to exit. 4. A photoelectric censor detects the intensity of the light. 5. The digital display compares the intensity of the light entering and exiting the cell. If the amount of light entering the cell is greater than the amount of light exiting the cell, then we know that some of the light is absorbed. The wavelength will match the gap between energy levels. Optical Density/Absorbance is recorded.

What are some of the ways to see things within a cell?

- Light microscopy (can identify organelles) - Absorbance and Fluorescence (biological processes)

What are some instruments that measure fluorescence?

1. Fluorescence Spectrometer: Detects photons emitted by the fluorophore. 2. Fluorescence Microscope: contains an emission filter, so you can only see light on sample (electrons emitted). 3. Flow Cytometry. Measures fluorescence of individual cells and you can compare them to nonfluorescent cells. (Useful in immunology).

What are some ways to detect molecules of interest and how do they work?

1. GFP: Linked to a protein that is naturally fluorescent; genetically engineer a single protein to GFP. 2. Antibodies: They detect antigens. We can link an antibody to a fluorophor so we can specifically see the antibody binding to the molecule of interest (we can quantify the molecule of interest in our sample). 3. Staining (least specific): It stains organelles. DAPI staining stains DNA, but it stains all of the DNA - it can't locate a specific sequence. Ethidium Bromide Stains DNA in gel electrophoresis.

What is fluorescence? How does it work?

1. The electron absorbs a high-energy photon. It has so much energy that the electron can move past the first excited state and move onto a higher state, like the second excited state, and so forth. 2. When the electron is ready to emit a photon, it will first go to back to the previous excited state. However, no energy is going to be emitted here. 3. It will finally go back to the ground state. The now low-energy photon will be emitted. It has a longer wavelength and a lower energy than the photon that was initially absorbed. ~Fluorescence looks for light emitted directly by the sample~

Generally, how does the process of absorbance & fluorescence work?

1. The electron is ready to absorb a photon of light 2. The electron moves up from the ground state to the excited state 3. The amount of energy it took to move from the ground state to the excited state is equivalent to the energy absorbed from the photon. In other words, energy from photon = energy level difference. 4. The electron is ready to emit energy in the form of a photon. 5. The electron moves down from the excited state to the ground state. The amount of energy it took to move from the excited state to the ground state is equivalent to the energy emitted by the photon. In other words, energy from photon = energy level difference.

What are the normal conditions for electrons?

The electron exists in the ground state

What are the quantum mechanical requirements for this absorbance/emission process in electrons?

1. e- energy levels are discrete. There are only specific energy levels for a given electron. Example: If the energy level difference between the ground state and the excited state is 1 eV, and the amount of energy in the ground state is 1 eV, then your electron has to absorb energy in the equivalent of 1 eV in order to move to the first excited state. From there, the amount of energy in that level is 2 eV. You cannot absorb 0.5 eV and be at a "1.5 eV," because no such level exists that contains that amount of energy. 2. The photon of light absorb by the e- has to absorb enough E corresponding to change in E level in order to be promoted from 1 level to another. Example: If the energy level difference is 1 eV, the photon has to have enough energy to the equivalent of 1 eV. If you absorb 0.5 eV, your electron will not be promoted to the next energy level.

What is a blank sample and why do we need to use it?

A blank sample is a sample that is identical to the experimental sample except that it doesn't contain a test solute. The solute = Amino Acid Tyrosine in a Phosphate Buffer The blank = Phosphate Buffer. The spectrophotometer automatically subtract's the blank's absorbance from the experimental sample, so that we do not measure the number of photons absorbed by the phosphate buffer or the cuvette.

What is a spectrophotometer?

A device that measures the amount of light at a given wavelength absorbed by a solution.

What is the electromagnetic spectrum?

A spectrum which defines ranges of wavelengths

Why would we want to link fluorophore to our molecule of interest?

Since most biochemical molecules are not fluorescent (they don't emit light), we can use it to detect molecules of interest.