Difference between revisions of "Assignment 3, Part 2: experimental design with fluorescence"

From Course Wiki
Jump to: navigation, search
(Matching fluorescent dyes, light sources, filter sets, and detectors)
Line 3: Line 3:
 
{{Template:20.309}}
 
{{Template:20.309}}
  
This is Part 2 of [[Assignment 3 Overview| Assignment 3]].<br>
+
This is Part 2 of [[Assignment 3 Overview| Assignment 3]].<br/>
 +
 
 
Let's explore fluorescence in contexts beyond your 20.309 specific design.
 
Let's explore fluorescence in contexts beyond your 20.309 specific design.
  
Line 10: Line 11:
 
The excitation source is a laser with wavelength 395 nm that produces a flux density of 6 W/cm<sup>2</sup> in the sample plane.  The extinction coefficient of EGFP at this wavelength is about &epsilon;  = 55,000 M<sup>-1</sup>cm<sup>-1</sup>, and its quantum yield is 0.6. <br>
 
The excitation source is a laser with wavelength 395 nm that produces a flux density of 6 W/cm<sup>2</sup> in the sample plane.  The extinction coefficient of EGFP at this wavelength is about &epsilon;  = 55,000 M<sup>-1</sup>cm<sup>-1</sup>, and its quantum yield is 0.6. <br>
 
Recall that the Beer-Lambert law states that A = &epsilon;&#8467;c = -log<sub>10</sub>(I/I<sub>0</sub>), where &epsilon; is the molar absorption coefficient, &#8467; is the path length, c is the concentration, I is the transmitted light intensity, and I<sub>0</sub>  is the incident light intensity.<br>
 
Recall that the Beer-Lambert law states that A = &epsilon;&#8467;c = -log<sub>10</sub>(I/I<sub>0</sub>), where &epsilon; is the molar absorption coefficient, &#8467; is the path length, c is the concentration, I is the transmitted light intensity, and I<sub>0</sub>  is the incident light intensity.<br>
{{Template:Assignment Turn In|message=Turn in your answers to the following questions.}}
+
{{Template:Assignment Turn In|message=<br/>
 
# How many photons per second are incident on a single pixel’s image in the sample plane?
 
# How many photons per second are incident on a single pixel’s image in the sample plane?
 
# What fraction of the incident photons passing through the center of the cell is absorbed? Based on the amount absorbed, what fraction of incident photons will give rise to fluorescence emission?
 
# What fraction of the incident photons passing through the center of the cell is absorbed? Based on the amount absorbed, what fraction of incident photons will give rise to fluorescence emission?
 
# To make an image with a signal to noise ratio of > 100, what is the minimum exposure time? (Assume shot noise predominates.)
 
# To make an image with a signal to noise ratio of > 100, what is the minimum exposure time? (Assume shot noise predominates.)
 
<br>
 
<br>
 +
}}
  
 
==Matching fluorescent dyes, light sources, filter sets, and detectors==
 
==Matching fluorescent dyes, light sources, filter sets, and detectors==
Line 22: Line 24:
 
# Visit the [http://searchlight.semrock.com/ Semrock website] and its SearchLight tool to pick out adequate filters, dichroic mirrors, illumination sources, and light detectors to support your experimental design.
 
# Visit the [http://searchlight.semrock.com/ Semrock website] and its SearchLight tool to pick out adequate filters, dichroic mirrors, illumination sources, and light detectors to support your experimental design.
 
{{Template:Assignment Turn In|message=Turn in a screenshot of your Semrock SearchLight choices to justify your approach.}}
 
{{Template:Assignment Turn In|message=Turn in a screenshot of your Semrock SearchLight choices to justify your approach.}}
Back to [[Assignment 3 Overview| Assignment 3]]<br>
 
On to [[Assignment 4 Overview| Assignment 4]]
 
  
 +
{{Template:Assignment 3 navigation}}
 
{{Template:20.309 bottom}}
 
{{Template:20.309 bottom}}

Revision as of 22:59, 11 September 2017

20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg


This is Part 2 of Assignment 3.

Let's explore fluorescence in contexts beyond your 20.309 specific design.

Beer-Lambert law

You are trying to image a 10 micron thick cell that expresses Enhanced Green Fluorescent Protein (EGFP) at a concentration of 1 μM using a Nikon Plan Apo 100X objective with an NA = 1.0 and a choice of a CCD camera with a pixel size of 7.5μm x 7.5μm. The camera has a quantum efficiency of 0.8.
The excitation source is a laser with wavelength 395 nm that produces a flux density of 6 W/cm2 in the sample plane. The extinction coefficient of EGFP at this wavelength is about ε = 55,000 M-1cm-1, and its quantum yield is 0.6.
Recall that the Beer-Lambert law states that A = εℓc = -log10(I/I0), where ε is the molar absorption coefficient, ℓ is the path length, c is the concentration, I is the transmitted light intensity, and I0 is the incident light intensity.

Pencil.png


  1. How many photons per second are incident on a single pixel’s image in the sample plane?
  2. What fraction of the incident photons passing through the center of the cell is absorbed? Based on the amount absorbed, what fraction of incident photons will give rise to fluorescence emission?
  3. To make an image with a signal to noise ratio of > 100, what is the minimum exposure time? (Assume shot noise predominates.)



Matching fluorescent dyes, light sources, filter sets, and detectors

Design a specimen labeling protocol you would use to image the reorganization of the cytoskeleton during cell migration on a surface. Specifically, you would like to simultaneously image (1) tubulin (a cytoskeletal protein) and (2) the cell nucleus.

  1. Select fluorescent probes from the Invitrogen website .
  2. Visit the Semrock website and its SearchLight tool to pick out adequate filters, dichroic mirrors, illumination sources, and light detectors to support your experimental design.


Pencil.png

Turn in a screenshot of your Semrock SearchLight choices to justify your approach.


Navigation

Back to 20.309 Main Page