Dave Piston May 18, 2014 Optical Sectioning 2: Confocal Designs and “Pseudo-confocals” Outline 1.PMT detectors 2.Laser Scanning Design Features 3.Pseudo-confocals 4.Signal and Background

Photomultiplier Tube (PMT) High-gain leads to high S/N Can detect single photons PMTs are very fast Drawback is relatively low QE

Newer PMTs (GaAsP) From Hamamatsu

CCD –QE Comparison

Non 90 ° Dichroic Mirror Alignment From Nikon Using laser excitation, it is no longer necessary to use a 90 ° orientation for the dichroic mirror. This has tremendous impact on multiband dichroic design and also for spectral imaging.

2 Laser Synchronized Scanning One laser stimulates, the second laser simultaneously images. Coordination of stimulation and imaging is useful for FRAP, FLIP and photoactivation. From Olympus

Multi-Tracking: Rapid Laser Switching via AOTF FITC 488 nm Cy3 543 nm Overlay Both lasers ONMulti-Tracking: Lasers switching off and on From Zeiss Web Page

Spectral windows instead of filters: Leica AOBS Prism and slit system gives spectrometer type abilities No barrier filters No dichroic filters Up to 4 channels From Leica

AOBS Operation From Leica

1. Order out (= ex in) 0. Order out (= em out) In (= ex in = em out) AOBS Operation From Leica

AOBS Operation From Leica

PMT array with 32 elements ZEISS LSM 510 META Contrast by Fluorescence Spectra

ZEISS LSM 710 Low-angle Spectral recycling

White Light Continuum Generation Laser From Leica

Excitation-Emission Contour Courtesy : Alberto Diaspro, Ph.D. University of Genoa From Leica

Spinning Disk with Microlens Array Only 1% of Nipkow disk is holes, this leads to low excitation rates Lens array directs >50% of the excitation light through the Nipkow Disk pinholes But, faster acquisition means faster photobleaching!

Laser Intensity Confocal Detection Confocal Microscopy

2-D Array Scanning with Microlens Array is scanned and resulting fluorescence is descanned by a galvo Lens array directs many spots to the sample Again, faster acquisition means faster photobleaching!

Sweptfield Confocal (Prairie/Nikon) Prairie Website

Sweptfield Confocal (Prairie/Nikon) Prairie Website

Sweptfield Confocal (Prairie/Nikon) Prairie Website

Fast Confocal Line Scanning Illuminate whole line Detect through confocal slit Optical “trick” instead of a traditional dichroic mirror Uses a CCD-type detector Zeiss 5Live

Spinning Slit Microscope Olympus DSU

Laser Intensity Confocal Detection Confocal Microscopy

Pinhole Effect on Confocal Resolution Theory Experiment Widefield Pinhole 1 AU 0.2 AU Confocal Detection

t = 0.07umt = 0.75umt = 7.5um t = 75 um Confocal Spinning disk Line illumination Conventional NA oil-immersion objective, and 488 nm. Signal-to-Background vs. Sample Thickness