Lecture5.ConfocalmicroscopyandinstrumentationI.pptVIP

* /primer/java/digitalimaging/avalanche/index.html /primer/flash/photomultiplier/index.html * V^2 = 4kTR Small size Very small cavity, large mode spacing. Very efficient: ~50% efficiency Most band gap is small, so emit IR light (shortest wavelength at ~760nm) Can easily form arrays to increase total power output. Mostly used as pump laser in microscopy applications. DPSS Laser Common Continuous Wave (CW) Lasers for Microscopy Argon UV (cw) 364 nm Argon Vis 488, 514 nm (458, 477 nm) Argon-Krypton 488, 568, 647 nm He-Ne 633 nm (laser pointer) DPSS laser 532 nm, 565 nm Not tunable None appropriate for 2-p absorption, wrong colors, low power Dye lasers are tunable and covers broad spectrum, but very difficult to operate. Wide Field vs Confocal Fluorescence Imaging Confocal Wide-field Greatly reduces Out of focus blur Brighter but No sectioning More examples medulla muscle pollen widefield confocal Epi-illumination widefield is form of Kohler Illumination: Objective is also condenser Lamp or laser detector Detect at 90 degrees Split with dichroic mirror lens Confocal detection with 3 dimensional scanning Image one plane, Move focus Confocal Aperture Decreasing the pinhole size rejects more out of focus light, therefore improving contrast and effective z resolution. Decreasing the pinhole will increase x,y resolution (1.3x widefield) Decreasing pinhole size decreases the amount of the Airy disk that reaches the detector. This results in less light from each point being collected Generally, collecting the diameter of 1 Airy disk is considered optimal. This collects about 85% of light from a sub-resolution point. Limits: Open pinhole: nearly widefield resolution (still some confocality) Closed: no image Signal, S/N (out of focus) opposite trends Closed: better axial sectioning, but no photons for contrast Open: no sectioning, lots of photons Confocal Aperture ALIGNMENT OF APERTURES IS CRITICAL X, Y alignment : Different wavelengths focus at di