ECE1466 Modern Optics Course Notes Part 7

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ECE-1466Modern OpticsCourse NotesPart 7

• Prof. Charles A. DiMarzio
• Northeastern University
• Spring 2002

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Some Transmission Techniques
Brightfield, Koehler Illumination
Darkfield
Fluorescence
Polarimetric
3
Some Reflection Techniques
Simple Reflection
Retroreflection
Epi-Fluorescence Dichroic Mirror
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Digital Imaging with CCDs

• Light Collection and Photodetection
• Charge Transfer
• Resolution in Time, Space, and Wavelength
• Noise, Digitization, Dynamic Range
• Pixelation and Digitization

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Semiconductor Detector
Conduction Band
Emission
Absorption
10057p1-2 here
Valence Band
hn
e-
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Remember N-FET Structure?
Channel Length 1 to 10 mm
Metal-Oxide-Semiconductor
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Charge-Coupled Device (CCD)
G
S
S
D
D
B
B
10 mm X nRows
Channel Length 1 to 10 mm
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CCD Charge Transfer
V
One Line
V
Clock Signals
time
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Formats
Frame Transfer
Line Transfer
Collection Frame
Transfer Frame
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Signals and Noise
10057p1-3 and 4 on this page
(Gaussian)
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Linearity and AGC

• Automatic Gain Control (AGC)
• Feedback
• Control G
• Based on...
• Peak Signal
• Average Signal
• Peak in a Region
• Not Desirable for Quantitative Work

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Pixelation and Digitization
Count
255
0
Brightness
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Digitization and Dynamic Range
2N-1
Saturation
Maximum Signal
Minimum Signal
Step Size
Pedestal
Signal Voltage
0
Dark
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Some Standard and Extreme Parameters

• VGA Frame Size 640 by 480
• Up to 4k Square?
• Standard Update Rate 30 Hz. Interlaced
• Up To few kHz.
• Standard Digitization 8 Bits
• Up To 12.
• Pixel Size 10 micrometers.
• Color Camera 3 Channels, 8 Bits Each

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CCD and Microscope
x
x
Place Camera Image Plane at intermediate Object
Location.
Pixel Size on Specimen is Pixel Size on Camera
Divided by m
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Camera Port and Eyepiece
F
F
F
F
A
A
Options Beamsplitter, Movable Mirror, Movable
Beamsplitter/Mirror Lens in Camera Port (relay or
magnifier)
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Resolution

• Resolution is l R / d
• Approximation
• l f / d0l( f )
• Typical Range of Values
• 1.2 l to 22 l
• Smears Image over a Fraction of a Pixel to Many
  Pixels Depending on f
• Pixel Acts as Anti-Aliasing Filter for Image

x
x
x
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Confocal Microscopy

• Confocal Concept
• Reflectance Imaging
• Fluorescence Imaging
• Details of the Optics

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Basic Concept
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Confocal Sensing
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Confocal Microscopy
Laser
Detector
Polygonal Mirror Scanner
Sample
Galvo Scanner
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Hints on Designing A Scanner

• Place the mirrors at pupils

Put Mirrors Here
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Research Confocal Microscope
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Confocal Reflectance Images of Oocytes
pxl
pxl
pxl
pxl
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Commercial Confocal Microscopes
VivaScope 2000 - imaging ex vivo
VivaScope 1000 - imaging in vivo
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Fluorescence Microscopy
Excitation Light
Emitted Light

• Main Advantages
• Specificity Fluorescent substances are usually
  very specific about excitation emission
  wavelengths, and only fluoresce when the
  excitation is ON.
• We attach fluorescent molecules (fluorochromes)
  to the molecules we want to study.
• Sensitivity Using highly sensitive detectors and
  carefully chosen filters, one can image as few as
  50 molecules per square micrometer.

this slide borrowed from Prof. Badri Roysam, RPI
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Fluorescence Confocal Imaging A Great
Combination!
Alexa Dye Injected Neuron Image Dimensions
512x480x301
this slide borrowed from Prof. Badri Roysam, RPI