Confocal Microscopy LSCM Advantages Better resolution Optical sectioning

Confocal Microscopy - LSCM Advantages: -Better resolution - Optical sectioning -3 D reconstruction - ROI scan 1

Confocal Microscopy - LSCM focuses a single beam on the specimen plane to sequentially point-scan a region of interest. Limitation: --- Scan speed 2

Laser Scanning Confocal Laser ‘spot’ scanning: - Spot by spot - Relatively slow - 2 -3 frames per second (512 X 512) - requires good dwelling time 3

Resonant Scanner About 30 fps 4

Resonant Scanner Dual scanners 5

LSM 7 Live (Zeiss line scan) 6

Spinning Disk Confocal - single scan - every 30 -degree - 360 -degree rotation 360/30 (= 12 frames/r) - spinning at 2, 000 rpm, ~400 frames per second 7

Spinning Disk confocal Invented by Paul Nipkow in 1884 Archimedean spiral 8

Spinning Disk Confocal Microscopy CCD camera Instead of PMT No laser necessary 9

Spinning Disc confocal Early day design 10

Spinning Disk confocal Yokogawa Spinning Disk Unit (SDU): Microlens - focus light onto perfectly aligned pinholes - more input of illumination 11

Spinning Disc confocal -Yokogawa Nipkow disk 12

Spinning Disk Confocal - 55 -millimeter diameter - 20, 000 pinholes - 250 um spacing (between pinholes) - Pinhole = 50 to 70 um 13

Spinning Disk Confocal - 55 -millimeter diameter - 20, 000 pinholes - 250 um spacing (between pinholes) - Pinhole = 50 to 70 um? 14

Spinning Disk Confocal In Spinning disk, pinholes regulate both illumination and emission (about 90% of light is blocked). 15

Spinning Disk Confocal Larger pinholes bring in more illumination, but with poor axial resolution. 16

Spinning Disk Confocal - 55 -millimeter diameter - 20, 000 pinholes - 250 um spacing (between pinholes)? - Pinhole = 50 and 70 um 17

Spinning Disk Confocal Emission further away from the focal point enters into neighboring pinholes, causing ‘light contamination’ or ‘pinhole crosstalk’.

Spinning Disk Confocal Emission further away from the focal point enters into neighboring pinholes, causing ‘light contamination’ or ‘pinhole crosstalk’.

Spinning Disk Confocal Scanning speed effect (starting 1: 15)

Spinning Disk Confocal 21

Spinning Disk Confocal - When fluorescence is dim, camera exposure time is the limiting factor - Electron-multiplying CCD (EMCCD) requires 100 milliseconds on a weakly fluorescent specimen - Limit the acquisition rate to 10 frames per second. 22

LSCM vs. Spinning Disk Confocal 1. Background rejection and resolution SDCM with 250 um spacing SDCM with 125 um spacing 23

LSCM vs. Spinning Disc Confocal 2. Speed: - Spinning disk is about 100 to 1, 000 times faster - Advanced CCD camera has a higher quantum efficiency (ratio of incident photon converts to electron) 24

LSCM vs. Spinning Disc Confocal 3. Variable Pinhole size 25

LSCM vs. Spinning Disc confocal 4. Simultaneous multi-channel acquisition 5. Optical zooming/ROI scanning Digital camera has a fixed pixel size 6. Cell Viability Less excitation input power per unit area in SD confocal 26

Live cell imaging 1. Maintenance of Living Cells 2. Reducing photodynamic Damages 3. Choose the ‘right’ imaging system 27

Live cell imaging 1. Maintenance of Living Cells 28

Live cell imaging - Environment chamber to keep cells “happy”; - Temperature fluctuation may cause focus drift. - Autofocus mechanism 29

Live cell imaging 2. Reducing photodynamic Damages - Sensitivity of detection, - Speed of acquisition, - The viability of the specimen (less damage to cells) 30

Live Cell Imaging 3. Choose the ‘right’ imaging system - Stability of the system (stage etc). - Speed. Spinning Disc Confocal Microscopy Multi-photon Microscopy - Photobleaching. Spinning Disc Confocal Microscopy Multi-photon Microscopy 31