Imaging settings

• for phase contrast, binning is usually not needed. Exposure time should be about 10-20ms.

Light source

• green LED for phase contrast
• 465 nm LED  for GFP
• 535 nm LED for Cy3
• 445 nm LED for CFP

Fluorescence excitation light system by CoolLed

Fluorescence filter cubes

GFP-A-Basic

• Excitation transmission band: average transmission > 90% 451.5 - 486.5 nm
• Emission transmission band: average transmission > 90% 505.5 - 544.5 nm

Cy3-4040B (from semrockcatalog2009.pdf)

• Excitation 531 nm, > 93% over 40 nm
• Emission 593 nm, > 93% over 40 nm

CFP-A-Basic

• Excitation transmission band: average transmission > 90% 425.5 - 442.5 nm
• Emission transmission band: average transmission > 90% 459 - 499 nm

You can check fluorophore compatibility on the Semrock website, see the the links above, tab "Fluorophore". For Cy3-4040B you can check the updated set Cy3-4040C.

Objective

Fluorescence Cell-IQ:Nikon Plan Fluor 10x/0.30 Ph1 DL
Phase contrast Cell-IQ:Nikon

Camera

Camera: Qimaging Retiga EXi
Pixel number (W x H): 1392 x 1040
Pixel size: 6.45 x 6.45 um.
11 frames/s with full resolution
Cooled 25 degrees below the ambient temperature
High 63% QE sensitivity with low 8e
ˉ
 read noise and 0.15e-/pix/s dark current.
Objective: Nikon Plan Fluor 10x/0.30 Ph1 DL
Image resolution: (About) 0.700 um/pixel, 995 x 745 um field of view. (Calibrated for each system)

Software

Imaging: Cell-IQ Imagen 4.1.0,Imagen_manual_4.0.0.pdf

Analysis: Cell-IQ Analyser 4.4.0,Analyser_4-4-0_help.pdf