Comparison between CSU and conventional LSM in 4D movies

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Comparison between CSU and conventional LSM in 4D movies

MDCK cells stably expressing GFP-Rab25 were imaged at 2/s for 100s.
XYT-volume movies show apparent difference in the endosome fluorescence decay between the two systems.  

CSU

CSU: Minimal bleaching over 100s

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Conventional LSM:

Conventional LSM: Photo bleaching of each endosome is apparent

(Full size movie Play)

The CSU system is capable of reproducing the highest SNR (signal to noise ratio) measured for an SPSC system(single-beam confocal) at approximately 1/15th of the rate of the photobleaching.

It has been widely recognized among CSU system users, most typically cell biology investigators that extended imaging studies using the CSU10 system are remarkably free of photobleaching. Suggestions were made that its superior performance results from the high efficiency of its CCD detector or from the low illumination dosage of a spinning disc system.

K.W.Dunn et al.,(Department of Medicine, Division of Nephrology, Indiana University Medical Center) thoroughly investigated photon economy of CSU system in comparison with that of conventional point scanning confocal systems.
(Journal of Microscopy, Vol. 218, Pt 2 May 2005, pp. 148 -159ng: Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems)

High-speed Imaging of endosomes in living MDCK cells.

living MDCK cells.

Cells expressing GFP-Rab25, a vesicle-associated protein, imaged at 20 f/s for 30s.

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living MDCK cells.

Cells incubated in TexasRed-labelled transferrin which is internalized into endosomes.
Imaged at 11 f/s for 100s, 3X actual speed. Each endosome can be imaged with high S/N.

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XYT volume

XYT volume movie of endosomes: Vertical axis represents time. Lack of photobleaching in each endosome over time is apparent.

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They quantitatively compared optical efficiency between the CSU system against several single point scanning confocal (SPSC) microscope systems, by measuring the photon economy by the signal to noise ratio(SNR) of images returned for a given level of photobleaching.

Their conclusions are:
*At moderately high imaging rates, the CSU10 system is capable of reproducing the highest SNR measured for an SPSC system at approximately 1/15th of the rate of the photobleaching.

*At higher levels of illumination, CSU system is capable of collecting images with SNRs four (4)-fold higher than the highest observed with the SPSC systems.

*Significant fluorescence saturation was found in the SPSC system, but not in the CSU10 system.

Not only is the CSU10 capable of collecting images much more rapidly than SPSC systems, it does so with much higher efficiency. The performance advantage of the CSU10 system derives not only from its more efficient collection, but also from more efficient excitation made possible by an illumination system that maximizes the population of fluorophores in the ground state, effectively optimizing the amount of fluorescence stimulated from a given number of fluorophore molecules.

Together, these characteristics support extended 4D imaging of living cells at rates sufficient to capture the 3D motion of intracellular vesicles moving up to several micrometers per second.( K.W.Dunn et al. 2005) : Copyright 2005 The Royal Microscopical Society

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