PSI SideView™ Imaging Microplate

Image System Optimization

Fluorescence:

Zebrafish exhibit a light avoidance response that can pose additional challenges for fluorescence imaging. Standard fluorescence microscopy delivers excitation light through the objective lens. At magnifications greater than approximately 2.5x this will result in non-uniform illumination of the SideView™ well. The zebrafish will tend to move away from the focal point to the dimmer regions of the well. As the objective power is increased, the intensity of the illumination beam is increased and the animals light avoidance response is further aggravated. Even at low magnification when the well is uniformly illuminated, introduction of light through the side of the well may cause some of the animals to roll onto their sides.

The solution is to flood the SideView™ wells will excitation light from above. PSI has used this approach with great success.

An LED-based illuminator similar to the one depicted below may be ordered on a custom basis.

Optimized Illumination:

Top illumination floods entire well with excitation source.

• 4W 473 nm LED source
  • Long lifetime
  • Little waste heat
• Uniform illumination reduces light avoidance response, especially at higher magnification

LED Excitation Source Design:

• Luxeon 3 Star 4 W LED (Lumileds)
  • Emission peak at 470 nm
• The LED was bonded to an air-cooled heat sink to maintain constant temperature
• Optics
  • Collimating lens (Lumileds
  • 10° diffuser optic provided uniform illumination
  • 450-490 nm bandpass filter (Chroma)
  • Second collimating lens at the end of the lens tube provided a uniform column of light

LED Performance versus Mercury Lamp:

• Test grids placed in SideView wells and imaged using 2.5x objective
• 450-490 nm excitation filter (Chroma)
• 500-530 nm emission filter (Piston GFP Bandpass; Chroma)

Results:

• LED illumination more uniform
• LED contrast (peak to trough ratio) 40% greater than with epiillumination
  • LED contrast = 1.7
  • Epiilumination contrast = 1.2
• LED gave overall superior results with both test targets and zebrafish larvae