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From 2014.igem.org

Protocols / Confocal Microscopy

Material:
Prepared MS Samples
Confocal Microscope	(here: Eclipse TE2000-E, Nikon, Japan)
	Settings 100 x magnification For penetration of fluorescence the laser wavelength were adjusted to λ= 408 nm and λ= 488 nm Transmission microscopy: halogen lamp vs. Detection of GFP signals: mercury lamp

Protocol

Before samples are screened by powerful lasers, which could bleach the GFP signal, the halogen transmission light was used for browsing the sample. After the right sample layer was focused, the source of lightening was switched to the lasers (mercury) and the pinhole-value needed to be adjusted. Three pictures were taken: one in the green channel, one in the red channel and one of in pseudo-transmission light. GFP molecules gave a green signal, while the chlorophyll visualized the chloroplasts as red dots. In order to compare the pictures, a scale bar was added in the pictures.