Fluorescence and Confocal Microscopy

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