May Chuck DiMarzio, Northeastern University ECE-1466 Modern Optics Course Notes Part 7 Prof. Charles A. DiMarzio Northeastern University Spring 2002
May Chuck DiMarzio, Northeastern University Some Transmission Techniques Brightfield, Koehler Illumination Darkfield Fluorescence Polarimetric
May Chuck DiMarzio, Northeastern University Some Reflection Techniques Simple Reflection Retroreflection Epi-Fluorescence Dichroic Mirror
May Chuck DiMarzio, Northeastern University Digital Imaging with CCD’s Light Collection and Photodetection Charge Transfer Resolution in Time, Space, and Wavelength Noise, Digitization, Dynamic Range Pixelation and Digitization
May Chuck DiMarzio, Northeastern University Semiconductor Detector Valence Band Conduction Band h e-e- EmissionAbsorption 10057p1-2 here
May Chuck DiMarzio, Northeastern University Remember N-FET Structure? Channel Length 1 to 10 m B, Body n+n+ n+n+ S, SourceG, GateD, Drain P-Type Material SiO 2 Insulator m Channel: 2 to 500 m into page NMOS Metal-Oxide-Semiconductor
May Chuck DiMarzio, Northeastern University Charge-Coupled Device (CCD) Channel Length 1 to 10 m B S G D S D B ~10 m X n Rows
May Chuck DiMarzio, Northeastern University CCD Charge Transfer Clock Signals time One Line V V
May Chuck DiMarzio, Northeastern University Formats Collection FrameTransfer Frame Frame Transfer Line Transfer
May Chuck DiMarzio, Northeastern University Signals and Noise 10057p1-3 and 4 on this page (Gaussian)
May Chuck DiMarzio, Northeastern University Linearity and AGC Input Voltage Output Voltage Automatic Gain Control (AGC) Feedback –Control G –Based on... Peak Signal Average Signal Peak in a Region Not Desirable for Quantitative Work
May Chuck DiMarzio, Northeastern University Pixelation and Digitization “Brightness” Count 0 255
May Chuck DiMarzio, Northeastern University Digitization and Dynamic Range Signal Voltage 2 N -1 0 Pedestal Saturation Step Size Dark Minimum Signal Maximum Signal
May Chuck DiMarzio, Northeastern University 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
May Chuck DiMarzio, Northeastern University CCD and Microscope s’s x’ x Pixel Size on Specimen is Pixel Size on Camera Divided by m Place Camera Image Plane at intermediate Object Location.
May Chuck DiMarzio, Northeastern University Camera Port and Eyepiece F’ F A’ A F’ F Options: Beamsplitter, Movable Mirror, Movable Beamsplitter/Mirror Lens in Camera Port (relay or magnifier)
May Chuck DiMarzio, Northeastern University Resolution Resolution is R / d Approximation: – f / d 0 = f #) –Typical Range of Values 1.2 22 –Smears Image over a Fraction of a Pixel to Many Pixels Depending on f# –Pixel Acts as Anti-Aliasing Filter for Image x’ x R d0d0
May Chuck DiMarzio, Northeastern University Confocal Microscopy Confocal Concept Reflectance Imaging Fluorescence Imaging Details of the Optics
May Chuck DiMarzio, Northeastern University Basic Concept
May Chuck DiMarzio, Northeastern University Confocal Sensing
May Chuck DiMarzio, Northeastern University Confocal Microscopy Polygonal Mirror Scanner Galvo Scanner Laser Sample Detector
May Chuck DiMarzio, Northeastern University Hints on Designing A Scanner Place the mirrors at pupils Put Mirrors Here
May Chuck DiMarzio, Northeastern University Research Confocal Microscope
May Chuck DiMarzio, Northeastern University Confocal Reflectance Images of Oocytes pxl
May Chuck DiMarzio, Northeastern University VivaScope imaging ex vivoVivaScope imaging in vivo Commercial Confocal Microscopes
May Chuck DiMarzio, Northeastern University Fluorescence Microscopy 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. Excitation Light Emitted Light this slide borrowed from Prof. Badri Roysam, RPI
May Chuck DiMarzio, Northeastern University Fluorescence & Confocal Imaging: A Great Combination! Alexa Dye Injected Neuron Image Dimensions: 512x480x301 this slide borrowed from Prof. Badri Roysam, RPI