|
|
| (24 intermediate revisions by 3 users not shown) |
| Line 3: |
Line 3: |
| | [[Category:Optical Microscopy Lab]] | | [[Category:Optical Microscopy Lab]] |
| | {{Template:20.309}} | | {{Template:20.309}} |
| − | <br />
| |
| − | <center>
| |
| − | {|
| |
| − | ! style="color: black; background-color: black; width: 210px;" |
| |
| − | [[Image:Galileo Galelei.jpg|center|200px]]
| |
| − | ! style="color: black; background-color: black; width: 210px;" |
| |
| − | [[Image:George Stokes.jpg|200px]]
| |
| − | ! style="color: black; background-color: black; width: 210px;" |
| |
| − | [[Image:Fluorescent Gameroom.jpg|200px]]
| |
| − | |-
| |
| − | !style="color: blue;background-color: black; width: 210px;" |
| |
| − | <blockquote>
| |
| − | <div>
| |
| − | ''It must be explained how it happens that the light is conceived into the stone, and is given back after some time, as in childbirth''
| |
| − | </div>
| |
| − | <blockquote>
| |
| − | <div>
| |
| − | ''— Galileo Galilei on the unusual properties of the stone ''lapis solaris''. ''
| |
| − | </div>
| |
| − | </blockquote>
| |
| − | </blockquote>
| |
| − | <div>
| |
| − | !style="color: red;background-color: black; width: 210px;" |
| |
| − | </div>
| |
| − | <blockquote>
| |
| − | ''It was certainly a curious sight to see the tube instantaneously lighted up when plunged into the invisible rays: it was literally ''darkness visible''. Altogether the phenomenon had something of an unearthly appearance.''
| |
| − | </blockquote>
| |
| − | <div>
| |
| − | <blockquote>
| |
| − | ''— Sir George Stokes from his article ''On the Change of Refrangibility of Light''
| |
| − | </blockquote>
| |
| − | </div>
| |
| − | !style="color: green;background-color: black; width: 210px;" |
| |
| − | <blockquote>
| |
| − | <div>
| |
| − | ''Whoa. Those little soccer dudes are like ... ''TOTALLY GLOWING BRIGHT GREEN''.''
| |
| − | </div>
| |
| − | </blockquote>
| |
| − | <blockquote>
| |
| − | <div>
| |
| − | ''— unknown''. ''
| |
| − | </div>
| |
| − | </blockquote>
| |
| − | |}
| |
| − | </center>
| |
| − | <br/>
| |
| | | | |
| − | ==Overview==
| + | This assignment has 3 parts. |
| − | [[Image:Fluorescent minerals.jpg|right|thumb|Naturally fluorescent minerals exposed to ultraviolet light.]] | + | * [[Assignment 2 Part 1: Noise in images|Part 1: Noise in images]] |
| − | The [https://www.youtube.com/watch?v=9FoCIkAXYhE phenomenon] of fluorescence has beguiled both sober and inebriated witnesses since at least the [https://www.fluorescence-foundation.org/lectures%5Cmadrid2010%5Clecture1.pdf late sixteenth century]. Some of the earliest observers noticed an unusual blue tint in water that had been infused with the wood of a Mexican tree called Coatli. Other naturally-occurring fluorescent and phosphorescent materials such as Bologna Stone (baryte), fluorite, and quinine sparked considerable interest from some the most preeminent scientists in all of history. Galileo Galilei, Robert Boyle, David Brewster, John Herschel, and that trippy dude from your high school all spent time contemplating the curious nature and simple beauty of fluorescence.
| + | * [[Assignment 2 Part 2: Fluorescence microscopy|Part 2: Fluorescence microscopy]] |
| | + | * [[Assignment 2 Part 3: Build an epi-illuminator for your microscope|Part 3: Build an epi-illuminator for your microscope]] |
| | | | |
| − | [[File:Tonic water fluorescence.jpg|right|thumb|Blue fluorescence of quinine excited by UV light..]]
| + | Submit your work on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment2.pdf. |
| − | The brilliant physicist and mathemetician George Stokes conducted a particularly clever experiment in which he used a prism to separate sunlight into its component colors. As he moved a vial of quinine sulfate from color to color, Stokes noted that quinine fluoresces only when illuminated by ultraviolet light — the invisible part of the rainbow just beyond the majestic blue band. The vial of quinine did not glow when Stokes placed the it any other part of the rainbow. Because ultraviolet illumination gave rise to blue light, Stokes concluded that fluorescent emission has a ''longer'' wavelength than the excitation. This observation turns out to be spot on, and in his honor the difference in wavelength between the excitation and emission peak a fluorophore's spectrum is called the ''Stokes shift''.
| + | |
| | | | |
| − | Reconciling Stokes' observation with the prevailing theory of light at the time of his experiment proved to be extraordinarily difficult. In classical electromagnetic theory, the power of a wave depends on its ''intensity'', not its frequency. Bright light has a larger electric field than dim light, so classical theory predicts that bright light should excite electrons more vigorously than dim light. But that prediction is at odds with the results of Stokes' experiment. Quinine is excited by UV light and none of the other colors, regardless of how bright the light is.<ref>This is not perfectly true. We will talk about two-photon excitation later on.</ref> In order to explain this, Albert Einstein had to dream up a whole new branch of physics: the quantum theory. Einstein introduced the basic that light comes in discrete packets in one of his "miracle year" papers in 1905 with the dense title of ''Concerning an Heuristic Point of View Toward the Emission and Transformation of light''. This is a lovely paper, and you should put it on your reading list. In it, Einstein posited that light is made of discrete packets of energy (which we now call photons). The energy of photon depends only on its color and is given by the equation:
| + | {{Template:Assignment Turn In|message= Here is a comprehensive list of what you need to turn in: |
| | + | Part 1 |
| | + | # Turn in your plot of simulation results |
| | + | #* Find the interval [ <math>\mu - s</math>, <math>\mu + s</math> ] that contains about 68% of the simulation results. |
| | + | #* Turn in the code you used to find the interval. |
| | + | # On the same plot, plot the PDF of a normal distribution with the same mean and standard deviation as the simulation results. Multiply the PDF by a constant so that it has the same vertical scale as the histogram. |
| | + | # Is the Poisson distribution a good approximation of shot noise? |
| | + | #* What percentage of the results fall with one, two, and three standard deviations? |
| | + | #* Produce a log-log plot of standard deviation of the number of photons emitted as a function of the average number of photons emitted for probability of photon emission equal to the values: 10<sup>-6</sup>, 10<sup>-5</sup>, 10<sup>-4</sup>, 10<sup>-3</sup>, and 10<sup>-2</sup>. |
| | + | #* Hints: Use a nested loop. Use the poissrnd function instead of the line containing rand. Speed things up by getting rid of the plotting inside the loop and only run 100 simulations for each probability. |
| | + | #* What is the relationship between the number of photons detected and the noise (standard deviation)? |
| | + | #* Turn in the code you used to generate the plots |
| | + | # On one set of axes, plot the variance vs. mean for exposure times of 10<sup>-4</sup>, 10<sup>-3</sup>, 10<sup>-2</sup>, 10<sup>-1</sup>, and 10<sup>0</sup> seconds. |
| | + | # Using the camera measurements provided: |
| | + | ## Plot the raw data from the static scene measurements and the model best fit on one set of axes. |
| | + | ## Calibrate the gain setting: make a plot of the actual camera gain in electrons per ADU versus the software setting. |
| | + | ## Provide a formula for converting the camera gain setting to the actual gain value. |
| | + | ## Plot dark current versus exposure time and determine the value of ID in units of electrons per pixel per second. |
| | + | ## Determine the read noise standard deviation. |
| | + | ## Under what circumstances is each of the three noise terms is dominant? |
| | | | |
| − | :<math>E=h\nu=\frac{hc}{\lambda}</math>,
| + | Parts 2 & 3 |
| | + | # Draw a block diagram of the LED epi-illumination path. Indicate the focal lengths of all lenses, the correct lens orientation, and all important distances between components. |
| | + | # Lenses L3 and L4 make an image of the LED. Assuming the initial size of the LED is 1.3 mm, what is the size of the LED image made by lens L3? |
| | + | # For each bead sample, include the original, reference, and flat-field corrected images in your lab report. In the caption note the exposure and gain settings used for each image. |
| | + | # For one set of images (either the 0.84 or 3.6 μm beads and their corresponding dark and reference images), include the MATLAB code you used to calculate the flat-field correction. |
| | + | }} |
| | | | |
| − | where ''ν'' is wavelength and ''h'' is the Planck constant approximately equal to 6.626070040(81)×10<sup>−34</sup> J⋅s. In Section 7 of the paper, Einstein offered a persuasive theoretical argument for the correctness of Stokes' rule: "If the fluorescent substance is not a perpetual source of energy, the principle of conservation of energy requires that the energy of an emitted energy quantum cannot be greater than of the incident light quantum.."<ref>http://www.colorado.edu/physics/phys2170/phys2170_sp15/Library_files/A.%20Einstein,%20Ann.%20Phys.%2017,%20132%201905.pdf</ref>
| + | {{Template:Assignment 2 navigation}} |
| − | | + | |
| − | ===Mechanism of fluorescence===
| + | |
| − | The diagram below depicts the sequence of events that gives rise to fluorescence. The process begins when a molecule absorbs a photon. Absorbance increases the amount of energy stored in the molecule. Most of the energy goes into raising the energy level of an electron above its ground state. The remainder into increasing the vibrational energy of the molecule. Very rapidly, the molecule sheds some of the energy through the processes of vibrational relaxation and internal conversion. After that, the molecule returns to its ground state by emitting a photon.
| + | |
| − | | + | |
| − | <center>
| + | |
| − | [[File:Fluorescence sequence.gif]]
| + | |
| − | </center>
| + | |
| − | | + | |
| − | In 1933, Aleksander Jablonski published a simple diagram similar to the one below. The diagram shows the details of the energy transitions that that take place during fluorescence and phosphorescence. The vertical axis of the diagram represents energy. Electrons confined to the molecule can only occupy certain discrete energy levels. Allowable electronic energy levels are indicated by thick horizontal lines. The ground state S<sub>0</sub> appears at the bottom of the diagram. Two excited states are shown: S<sub>1</sub> and S<sub>2</sub>. As energy increases, the allowable states get closer together. Above each electronic energy level, fine horizontal lines represent energy stored in vibrational modes of the molecule. (Vibrational energy is also discrete.) Thus, each line represents a possible value for the sum of electronic and vibrational energy stored in a molecule.
| + | |
| − | | + | |
| − | <center>
| + | |
| − | [[File:Jablonski diagram.png|Jablonski diagram.]]
| + | |
| − | </center>
| + | |
| − | | + | |
| − | The possible energy states are grouped into two columns. Each columns represents a different molecular spin state. (numbers of unpaired electron spins). Singlet states (denoted by the letter "S") have no unpaired spins while triplet states (denoted by "T") have two unpaired spins. Don't sweat the details too much. the bottom line is that transitions ''within'' a column are much more likely than transitions that cross columns. Fluorescence does not require an electron to change spin. It is a fast phenomenon that takes place entirely in the lefthand column. Phosphorescence involves an electron changing its spin. The probabilities of the transitions are much lower and hence phosphorescence occurs over a much longer timescale (up to hours).
| + | |
| − | | + | |
| − | It's not a coincidence that fluorescence takes place for the most part in visible wavelengths. Einstein
| + | |
| − | When it comes to
| + | |
| − | As summarized in the diagram below, single photons with wavelengths longer than about 700 nm have energies less than about 1.8 electron volts (2.88×10<sup>-19</sup> J), which roughly corresponds to the energy range of vibrational modes of molecules. Visible photons (λ= 400 - 700 nm) have energies in the same range as the differences between in energy levels of electrons bound to a molecule. X-rays (0.01 - 10 nm) have more than enough energy to completely remove an electron. Ultraviolet light bridges the gap.
| + | |
| − | <center>
| + | |
| − | [[File:Energy levels.png|400 px]]
| + | |
| − | </center>
| + | |
| − | | + | |
| − | | + | |
| − | [[20.309_130828_FluoAbsorbEmitSpectra2.png]]
| + | |
| − | | + | |
| − | Some naturally fluorescent molecules like fluorescein and quinine have important biological applications on their own. But a key breakthrough in the early 1940s dramatically increased the utility and importance of fluorescence in biology. The key idea was to attach a fluorescent molecule to an antibody. The combination of the two offers spectacular contrast and specificity in microscopic studies of biological samples. Today, fluorescent immunostaining is one of the most important and frequently used techniques in all of biological research.
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | [[Image:Alexa 568 and filters.png|thumb|260px|right|Excitation and emission spectra of Alexa 563. Green stripe indicates excitation laser wavelength of 532 nm. Shaded area shows passband of emission filter.]]
| + | |
| − | In this part of the lab, you will add the hardware necessary to make epi-illuminated fluorescent images with your microscope and make a few test images of plastic, fluorescent beads. A typical spectrum is shown on the right. The excitation light source is a 5 mW diode laser with a nominal wavelength of 532 nm — a striking, brilliant green. Emission is in the red/orange range. To make the correction for nonuniform illumination, you will also make images of a uniform fluorescence reference slide and a dark image with the illuminator turned off.
| + | |
| − | | + | |
| − | [[Image:20.309 130911 YourMicroscope.png|thumb|260px|right|Fluorescence microscope block diagram.]]
| + | |
| − | As shown in the block diagram, the major components required for fluorescence imaging are an illuminator (laser, L3, L4, and L5), dichroic mirror (DM), and emission filter (BF). The illuminator provides light in the appropriate wavelength range to excite the fluorescent molecules in the sample. Fluorescence microscopes that use broadand light sources such as arc lamps require an additional filter called an excitation filter to limit the wavelengths in the illumination to the proper range. Because lasers emit light in a very narrow range of wavelengths, an excitation filter is unnecessary.
| + | |
| − | | + | |
| − | [[Image:20.309Hw3Imagespectra.JPG|right|thumb|260px|Transmission spectra for the 565DCXT dichroic and the E590LPv2 barrier filter]]
| + | |
| − | Excitation light comes from beneath the sample, through the objective lens. A dichroic mirror directs the excitation toward the objective and sample. The mirror must reflect wavelengths in the excitation range and pass the longer wavelengths of the emitted fluorescence. In fluorescence imaging, illumination intensity is typically 5 or 6 orders of magnitude greater than emitted fluorescence, so it is crucial to filter out excitation photons as completely as possible. The dichroic mirror passes a substantial amount of green light, on the order of five percent. The barrier filter does a much better job of removing the green light, attenuating the excitation wavelengths by about 5 orders of magnitude. The barrier filter is essential for making crisp, high-contrast images.
| + | |
| − | | + | |
| − | To provide collimated illumination in the sample plane, light from the illuminator is focused at the back focal point of the objective.
| + | |
| − | | + | |
| − | ==Assignment details==
| + | |
| − | | + | |
| − | This assignment has 2 parts:
| + | |
| − | | + | |
| − | # [[Assignment 2: epi illuminator for fluorescence microscopy|design and build an epi illuminator for your microscope]]
| + | |
| − | # [[Assignment 2: resolution and noise|develop a software simulation to explore the concepts of resolution and noise]]
| + | |
| | | | |
| | {{Template:20.309 bottom}} | | {{Template:20.309 bottom}} |
