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Lab 2: Effects of Light: Photobleaching

Lab 2.  Effects of Light: Photobleaching

Lab 2 will examine the effects of light on living samples. Light sources are very powerful, with the capability of inducing blindness if one is not careful. However, in fluorescence microscopy, one must use a light source in order to induce a fluorescent response. In Lab 2, the effect of light exposure onto fluorescent cyanobacteria will be investigated, and the resulting photoresponse determined. In live cell imaging, too much light exposure can lead to phototoxicity, which often results in cell death. Fluorophores (in live or dead cells) can also photobleach, which is the phenomenon whereby a fluorophore is fundamentally destroyed when exposed to an overabundance of light.

In lab 2, you will expose fluorescent cyanobacteria to various light intensities through multiple objectives. These bacteria have the interesting property that their fluorescence will increase with light exposure. This increase will be used to gauge the physiological impact of light onto the sample. Further, by acquiring a large image around your central illuminated field of view, you will be able to calculate the actual exposure area and compare this to your field of view. Using time-lapse imaging, you will record the fluorescence intensity as a function of time, and relate that to illumination intensity and the objective. Using Matlab you will plot out and fit each intensity curve.

Useful Reading:

Yokoo, et al, discuss live cell imaging of cyanobacteria. Please read through page 35 and look at Figure 1c.

Shaner, et al, discuss the need to develop quantifiable properties of fluorescent proteins, including photostability.

Dean, et al, discuss the creation of a microfluidic device aimed at identifying and selecting photostable fluorescent proteins.

Ishikawa-Ankerhold, et al, give a very nice overview of several F-techniques of fluorescence microscopy.

Song, et al, takes a deep dive into the mathematics that describe fluorescein photobleaching.

To do:

NSTORM

  1. Obtain one imaging slide that contains agarose pads with colonies of cyanobacteria. Two objectives should be used for this experiment, preferably one with a low NA (such as a 10x or 20x air) and one with a high NA (60x Water or 100x Oil).
  2. Using the low NA objective, find the initial focal plane of one of the agarose pads by using the Brightfield optical configuration (make sure the light path is to the Eye port) and the PFS to locate the bacteria.
  3. Once the bacteria are found, push in the two ND filters for the LED light source. With the illumination attenuated, start a live image under the TRITC channel. Adjust the exposure and EMCCD gain until a clean image is observed.
  4. Once the appropriate conditions are found, acquire a 60s time lapse of this field of view.
  5. After the time lapse, acquire a 5x5 Large Image around this central field of view.  Do not Stitch the images.
  6. Repeat Steps 2 - 5 on a new location on the agarose pad. This new XY location should be far enough from the initial position that your large image will not overlap the initial position. Further, remove one of the ND filters (note which one), and capture one image. Adjust the exposure time and Gain if necessary. Once the new imaging conditions are found, acquire a new 60s time lapse. Return to the original imaging conditions and acquire a new 5x5 Large Image.
  7. Repeat Step 6, but at a new XY location and remove the final ND filter.
  8. Repeat Steps 2 - 7 using a high-NA objective.

A1R

  1. Obtain one imaging slide that contains agarose pads with colonies of cyanobacteria. Two objectives should be used for this experiment, preferably one with a low NA (such as a 10x or 20x air) and one with a high NA (60x Water or 100x Oil).
  2. Using the low NA objective, find the initial focal plane of one of the agarose pads by using the Brightfield optical configuration (make sure the light path is to the Eye port) and the PFS to locate the bacteria.
  3. Once the bacteria are found, select the 4 Ch + DIC optical configuration, disabling all of the lasers except the TRITC channel. Under Nyquist sampling conditions, reduce the laser power to 1 and put the HV to 120 (you may need 1024 steps). Adjust the focus and slowly increase the laser power until a clean image is observed.
  4. Once the appropriate conditions are found, acquire a 60s time lapse of this field of view.
  5. After the time lapse, acquire a 5x5 Large Image around this central field of view. Do not Stitch the images.
  6. Repeat Steps 2 - 5 on a new location on the agarose pad. This new XY location should be far enough from the initial position that your large image will not overlap the initial position. Further, increase the laser power by 5%, and capture one image. Adjust the HV if necessary. Once the new imaging conditions are found, acquire a new 60s time lapse. Return to the original imaging conditions and acquire a new 5x5 Large Image.
  7. Repeat Step 6, but at a new XY location and increase the laser power by another 10%.
  8. Repeat Steps 2 - 7 using a high-NA objective.

SDC

  1. Obtain one imaging slide that contains agarose pads with colonies of cyanobacteria. Two objectives should be used for this experiment, preferably one with a low NA (such as a 10x or 20x air) and one with a high NA (60x Water or 100x Oil).
  2. Using the low NA objective, find the initial focal plane of one of the agarose pads by using the Brightfield optical configuration (make sure the light path is to the Eye port) and the PFS to locate the bacteria.
  3. Once the bacteria are found, select the TRITC optical channel. Reduce the laser power to 10%. Adjust the focus and slowly increase the exposure time (and laser power if needed) until a clean image is observed.
  4. Once the appropriate conditions are found, acquire a 60s time lapse of this field of view.
  5. After the time lapse, acquire a 5x5 Large Image around this central field of view. Do not Stitch the images.
  6. Repeat Steps 2 - 5 on a new location on the agarose pad. This new XY location should be far enough from the initial position that your large image will not overlap the initial position. Further, increase the laser power by 10%, and capture one image. Adjust the Gain if necessary. Once the new imaging conditions are found, acquire a new 60s time lapse. Return to the original imaging conditions and acquire a new 5x5 Large Image.
  7. Repeat Step 6, but at a new XY location and increase the laser power by another 10%.
  8. Repeat Steps 2 - 7 using a high-NA objective.
All
  1. For each imaging condition (illumination power and objective), measure the change in fluorescence intensity over time. Try to fit the curve to some function (it may be an exponential, or it could be linear, I do not know). What happens as you illuminate the bacteria with more signal?
  2. From your Large Images, plot out the intensity of each bacteria with respect to its XY location (this should be a 3D plot). Have fun plotting out your data. Extra imaginary points will be awarded to those that can make the plot look pretty. How does the size of your initial Field of View compare to the intensity of the bacteria? Can you calculate the area of the intense bacteria (in other words, your illumination area)? Can you overlay your central Field of View onto the 3D plot, showing where you observed vs. where you illuminated?  Does your central field of view directly overlay the area of intense bacteria? What affect may this have on your ability to image other regions of interest?
  3. In groups, summarize your results and discuss the implications of your findings (no more than a couple of pages including the figures).
  • For each objective and illumination intensity, provide an example intensity curve and a fit indicating equation and the R2 value. Ideally your equation would take time into consideration.
  • Use a table to summarize your equation and the R2 for all of the conditions.
  • How comparable in size (or XY dimensions) was your bleached area to the acquired area?
  • Discuss the importance of the NA on your illumination area. Remember, your photon flux, I, is proportional to the NA and objective magnification (M) through the following equation: I ∝ (NAobj)4/M2 (If you would like, try making a plot of this. Do your intensity rates generally follow the change in photon flux?).

 

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