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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 fluorescently labeled Dextran 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 fluorescently labeled Dextran to various light intensities through multiple objectives. This increase will be used to gauge the physical impact of light onto the fluorescent 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:

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. Do the following for the Andor camera (and steps 3 - 8 with the Hamamatsu).
  2. Obtain one imaging slide that contains agarose pads embedded with fluorescently labeled dextran. 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).
  3. Using the low NA objective, find the initial focal plane of one of the agarose pads by using PFS.
  4. Once the surface is found, lower the LED illumination intensity to 5% and illuminate your sample with the GFP channel. Adjust the focus, exposure and EMCCD gain until a clean, non-saturated, image is observed.
  5. Once the appropriate imaging conditions are found, acquire a 5x5 Large Image around this central field of view. Do not Stitch the images
  6. Then, using the ND Acquisition Window, acquire a 180s time lapse of this field of view, acquiring one image every 5 seconds. Be sure to uncheck the box that says "Close Active Shutter". When this step is repeated (see below), you will be asked to increase the LED power
  7. After the time lapse, acquire another 5x5 Large Image around this central field of view.  Do not Stitch the images; Make sure the LED power is at 5%.
  8. Repeat Steps 3 - 7 on a new location on the agarose pad using 25% LED power for the time lapse (step 6). This new XY location should be far enough from the initial position that your large image will not overlap the initial position. When the timelapse is repeated acquire a new 60s time lapse. Return to the original imaging conditions and acquire a new 5x5 Large Image.
  9. Repeat Step 8, but at a new XY location and at 50% LED power.
  10. Repeat Steps 2 - 9 using a high-NA objective.

A1R

  1. Obtain one imaging slide that contains agarose pads embedded with fluorescently labeled Dextran. 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 (40x or 100x Oil).
  2. Using the low NA objective, find the initial focal plane of one of the agarose pads by using the PFS.
  3. Once the focus is found, select the 4 Ch + DIC optical configuration, disabling all of the lasers except the GFP channel. Under Nyquist sampling conditions, reduce the laser power to 1 and put the HV to 20 (use 1024 steps). Adjust the focus and slowly increase the Gain and laser power until a clean image is observed.
  4. Once the appropriate conditions are found, acquire a 5x5 Large Image around this central field of view. Do not Stitch the images
  5. Within the ND Acquisition window, acquire a 180s time lapse of the central field of view. When this step is repeated (see below), you will be asked to increase the laser power
  6. After the time lapse, acquire a 5x5 Large Image around this central field of view. Do not Stitch the images.
  7. Repeat Steps 2 - 6 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. Acquire a new 5x5 Large Image then increase your laser power by 10%. Acquire a new 180s time lapse at the increased laser power. Return to the original illumination power and acquire a new 5x5 Large Image.
  8. Repeat Step 7, but at a new XY location and increase the laser power by another 10%.
  9. Repeat Steps 2 - 7 using a high-NA objective.

SDC

  1. Obtain one imaging slide that contains agarose pads embedded with fluorescently labeled Dextran. 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 PFS.
  3. Once the surface is found, select the GFP optical configuration. Lower the laser power to 10%. Adjust the focus, exposure and EMCCD gain until a clean, non-saturated, image is observed.
  4. Acquire a 5x5 Large Image around this central field of view. Do not Stitch the images.
  5. Then, using the ND Acquisition Window, acquire a 180s time lapse of this field of view, acquiring one image every 5 seconds. Be sure to uncheck the box that says "Close Active Shutter". When this step is repeated (see below), you will be asked to increase the laser power
  6. After the time lapse, acquire a 5x5 Large Image around this central field of view. Do not Stitch the images.
  7. Repeat Steps 2 - 6 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. Acquire a new 5x5 Large Image. Then, increase the laser power to 50%, and capture one image. Adjust the Gain and Exposure Time if necessary to avoid saturation. Once the new imaging conditions are found, acquire a new 180s time lapse. Return to the original imaging conditions (Power, Exposure Time) and acquire a new 5x5 Large Image.
  8. Repeat Step 7, but at a new XY location and increase the laser power to 100%.
  9. Repeat Steps 2 - 8 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 agarose with more power? You should be able to generate a table showing the illumination power, the objective, and the decay rate of the fluorescence intensity.
  2. Using your Large Images, how does the size of your initial Field of View compare to the bleached area (A ratio of the pre- and post-bleach large images may help here)? Can you overlay your central Field of View onto the 2D plot, showing where you observed vs. where you illuminated?  What affect may this have on your ability to image other regions of interest?
  • 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?

 

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