Fluorescence and Confocal Microscopy

More documents

Recommendations

Info

Ways to increase the signal that the camera sees •Brighter sample •Align illumination optics (Koehler illumination) & bulb •Brighter objective lens (B = NA 4 / M 2 ) •Higher transmission objective lens (less aberration correction) •Decrease specimen noise (mounting medium, BG fluorescence) •Decrease other optical noise (minimize reflective surfaces, use field diaphragm, work in dark, no dirt) Detectors: Photomultiplier Tube (PMT) Strengths: very low noise and allow rapid data collection Weaknesses: old designs don’t count every photon (GaAs PMTs, 10% efficient), but new PMTs GaAsP) are about 40% efficient at their spectral optimum. Nikon (www.microscopyu.com) A metaphore for CCD camera readout Gain: • The amount that an analog signal has been amplified. For video, gain is used to increase or decrease the dynamic range of a video camera by selecting the voltage levels that the digitizer will accept. • For example, when viewing a faint image, the gain is often raised to increase the minute changes into those that are more readily detectable. Increasing gain reduces the number of gray levels Increasing gain, Same exposure time Offset (Black Level), can be used to re-zero the gray scale Detectors: Charged Coupled Device (CCD) Images collected by JWS in the Nikon Imaging Center at Harvard Medical School The sensitivities of various electronic cameras: Video - CCD Photodiodes • A light-sensitive semi-conductor set up so incident light can knock electrons loose. • A “loose” electron leaves behind a zone of positive charge or a “hole” • Electrons and holes can move in response to an electric field • Current is then proportional to the number of “loose” electrons, i.e., to number of incident photons 8
Diagram of an individual CCD “Well” or “Pixel” full well capacity on the order of ~1000 electrons/µm 2 Spectral Sensitivity & QE Detector: Electron Multiplier CCD(EM-CCD) Hazelwood et al., from Shorte and Frischknecht 2007 Graph from microscopyu.com Hamamatsu Improvements in Interline CCDs Single microlens added No microlens Detector: Charged Coupled Device (CCD) ORCA- ER 500ms, High Gain Input light EM-CCD Performance EM-CCD 200ms, Low Multiplication Open window EM-CCD 200ms, Med Multiplication Microlens Image from Butch Moomaw, Hamamatsu Hamamatsu EM-CCD 50ms, High Multiplication Images collected by JWS in the Nikon Imaging Center at Harvard Medical School 9
Page 1 and 2: Fluorescence and Confocal Microscop
Page 3 and 4: • Physical Process - Excitation:
Page 5 and 6: Nikon (www.microscopyu.com) Nikon (
Page 7: Controlling Image Quality: • Spin
Page 11: Starting Images Ending Images 16 Bi

Fluorescence and Confocal Microscopy

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?