Polarization, DIC, fluorescencejokr7175/docs/Lesson 12.pdfPolarization, DIC, fluorescence • Last...
Transcript of Polarization, DIC, fluorescencejokr7175/docs/Lesson 12.pdfPolarization, DIC, fluorescence • Last...
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Polarization, DIC, fluorescence
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• Last class• Morphological operators• Matlab morphological image processing
• This class• Polarization microscopy• DIC• Intro to fluorescence
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Diffraction limited spot vs resolution limitd = λ/2*NARadius of spot
r = 1.22 λ /2*NARayleigh criterion
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Magnification
Imagine 10x objectiveImage on the camera, would be 10x larger
Cell that is 20 um in diameter, would appear 200 um diameter on camera
Conversely, a 10 um pixel on the camera would represent 1 um on the sample
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Polarization microscopy
• Another way to add contrast to samples
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Properties of light
𝑦𝑦 𝑥𝑥, 𝑡𝑡 = 𝐴𝐴 cos(𝑘𝑘𝑥𝑥 − 𝜔𝜔𝑡𝑡 + 𝜑𝜑)
𝑦𝑦 𝑥𝑥, 𝑡𝑡 = 𝑨𝑨 cos(𝑘𝑘𝑥𝑥 − 𝜔𝜔𝑡𝑡 + 𝜑𝜑)
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Properties of polarization
Linear
x
Linear
x + y
Circular
x + (y+φ)
UnpolarizedElliptical
ax + b(y+φ)
Eyes and cameras can not detect polarization.Only able to detect intensity.
x2 + y2 = 1
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Vertebrate vs cephalopod eyes
• Invertebrate eyes are different than our own
• Some invertebrates can detect polarization of light
First step is isomerization from 11 cis to all trans
Cephalopods can sense polarization
Us
Them
Dipole
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Manipulating polarization
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Manipulating polarization• Half wave plate, converts to
linear polarization• Quarter wave plate, converts
to circular polarization
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Linear polarizers
Polaroid filters
Light that comes through is polarized parallel to filter
I = I0cos2(θ)Malus’ Law
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Cross polarizer puzzle
Have to apply Malus’ law twice
Forms the basis for polarization microscopy
I2 = I1cos2(θ)
Then
I3 = I2cos2(θ)
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Polarization microscopy
Birefringent – refraction is different for different polarizations
Biological samples:-Mitotic spindles-Actin filament bundles-Condensed DNA-Helical strands of cellulose-Some lipid bilayers
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Strain induced bi-refringence
• Birefringence: refractive index depends on polarization of light
• Useful for detecting strain in extra cellular matrix (ECM)
• Label free technique
Relaxed
Strained – non-isotropic
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Differential Interference Contrast
• Interference technique similar to phase contrast
• The magic starts with a Wollaston prism
• Wollaston prism is birefringent, and rays will exit at different points in space
Wollaston splits polarization
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DIC microscopeBeams are split by small amount (nanometers)
If there is a difference in optical path between them, they will negatively interfere at camera
Only get contrast at the edges
Form what looks like a 3d image on the camera
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DIC vs phase contrast Characteristic Phase Contrast DICImage Brightness
1.3 Percent 0.36 - 2.3 Percent(Brightfield = 100 Percent)
Epi-Fluorescence Light Loss28 Percent 73 Percent
(Brightfield = 0 Percent)
Lateral ResolutionCondenser
SuperiorAnnulusRestricted
Axial ResolutionPoor Superior
(Depth Discrimination)
Illuminating Aperture10 Percent of
VariableObjective NA
Phase Shift< l/100 < l/100
Detection LimitUtility at High Phase Shifts Not Useful Useful
Azimuthal Effects No YesHalos and Shade-Off Yes NoStained Specimens Not Useful Useful
Birefringent Specimens Useful Not UsefulBirefringent Specimen
Yes NoContainers
Brightfield ImageSlight None
DeteriorationCost Moderate High
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And on to Matlab…
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Optical coherence tomography• Another interference
based technique• Non destructive, and
no labelling• Limited resolution,
but high applicability
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OCT contrast• When light in sample goes through index change, the reflected light
interferes with reference arm light – giving rise to bumps in the signal
Particularly useful in ophthalmology.Also used in the brain (rats)