1. May 02002 Chuck DiMarzio, Northeastern University 10100-6-1 ECE-1466 Modern Optics Course Notes Part 7 Prof. Charles A. DiMarzio Northeastern University Spring 2002

2. May 02002 Chuck DiMarzio, Northeastern University 10100-6-2 Some Transmission Techniques Brightfield, Koehler Illumination Darkfield Fluorescence Polarimetric

3. May 02002 Chuck DiMarzio, Northeastern University 10100-6-3 Some Reflection Techniques Simple Reflection Retroreflection Epi-Fluorescence Dichroic Mirror

4. May 02002 Chuck DiMarzio, Northeastern University 10100-6-4 Digital Imaging with CCD’s Light Collection and Photodetection Charge Transfer Resolution in Time, Space, and Wavelength Noise, Digitization, Dynamic Range Pixelation and Digitization

5. May 02002 Chuck DiMarzio, Northeastern University 10100-6-5 Semiconductor Detector Valence Band Conduction Band h e-e- EmissionAbsorption 10057p1-2 here

6. May 02002 Chuck DiMarzio, Northeastern University 10100-6-6 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 20-100  m Channel: 2 to 500  m into page NMOS Metal-Oxide-Semiconductor

7. May 02002 Chuck DiMarzio, Northeastern University 10100-6-7 Charge-Coupled Device (CCD) Channel Length 1 to 10  m B S G D S D B ~10  m X n Rows

8. May 02002 Chuck DiMarzio, Northeastern University 10100-6-8 CCD Charge Transfer Clock Signals time One Line V V

9. May 02002 Chuck DiMarzio, Northeastern University 10100-6-9 Formats Collection FrameTransfer Frame Frame Transfer Line Transfer

10. May 02002 Chuck DiMarzio, Northeastern University 10100-6-10 Signals and Noise 10057p1-3 and 4 on this page (Gaussian)

11. May 02002 Chuck DiMarzio, Northeastern University 10100-6-11 Linearity and AGC 00.20.40.60.81 0 0.2 0.4 0.6 0.8 1 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

12. May 02002 Chuck DiMarzio, Northeastern University 10100-6-12 Pixelation and Digitization “Brightness” Count 0 255

13. May 02002 Chuck DiMarzio, Northeastern University 10100-6-13 Digitization and Dynamic Range Signal Voltage 2 N -1 0 Pedestal Saturation Step Size Dark Minimum Signal Maximum Signal

14. May 02002 Chuck DiMarzio, Northeastern University 10100-6-14 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

15. May 02002 Chuck DiMarzio, Northeastern University 10100-6-15 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.

16. May 02002 Chuck DiMarzio, Northeastern University 10100-6-16 Camera Port and Eyepiece F’ F A’ A F’ F Options: Beamsplitter, Movable Mirror, Movable Beamsplitter/Mirror Lens in Camera Port (relay or magnifier)

17. May 02002 Chuck DiMarzio, Northeastern University 10100-6-17 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

18. May 02002 Chuck DiMarzio, Northeastern University 10100-6-18 Confocal Microscopy Confocal Concept Reflectance Imaging Fluorescence Imaging Details of the Optics

19. May 02002 Chuck DiMarzio, Northeastern University 10100-6-19 Basic Concept

20. May 02002 Chuck DiMarzio, Northeastern University 10100-6-20 Confocal Sensing

21. May 02002 Chuck DiMarzio, Northeastern University 10100-6-21 Confocal Microscopy Polygonal Mirror Scanner Galvo Scanner Laser Sample Detector

22. May 02002 Chuck DiMarzio, Northeastern University 10100-6-22 Hints on Designing A Scanner Place the mirrors at pupils Put Mirrors Here

23. May 02002 Chuck DiMarzio, Northeastern University 10100-6-23 Research Confocal Microscope

24. May 02002 Chuck DiMarzio, Northeastern University 10100-6-24 Confocal Reflectance Images of Oocytes 100200300400500600 100 200 300 400 500 100 200 300 400 500 100200300400500600 pxl

25. May 02002 Chuck DiMarzio, Northeastern University 10100-6-25 VivaScope 2000 - imaging ex vivoVivaScope 1000 - imaging in vivo Commercial Confocal Microscopes

26. May 02002 Chuck DiMarzio, Northeastern University 10100-6-26 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

27. May 02002 Chuck DiMarzio, Northeastern University 10100-6-27 Fluorescence & Confocal Imaging: A Great Combination! Alexa Dye Injected Neuron Image Dimensions: 512x480x301 this slide borrowed from Prof. Badri Roysam, RPI