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Hi all,
I completely agree with Guy. Resonant scanner confocal can be very useful depending on your application. To me it is always a compromise, every system has it's advantages and disadvantages. You have to think what are the needs of your application, and decide which system serves better your needs. I have been working with a Leica resonant scanner and an SP2-AOBS in parallel and my evaluation of the RS microscope was very positive.
1. Regarding speed, I found that in the same time interval, RS was capable of obtaining 6 to 7 times more images than the SP2. Which meant the RS was 6 to 7 times faster than the SP2 in acquiring an image without averaging.
2. My main concern was that the acquisition speed is fixed in this system, which could be limiting the quality of images with poor fluorescence signal. In this sense I found that when the quality of the image was insufficient, average could be used to improve it. It was determined that 6x averaging in these cases rendered the same image quality that was obtained with the SP2. All these tests were performed using the same objective that was changed from one microscope to the other.
3 In independent experiments, photo bleaching effects of the RS system was tested. The number of scans necessary to bring the signal to a steady state background level was around 100 for the SP2 and 600 for the RS. That is, the SP2 bleaches 6 times more compared to the RS in one scan, which is completely reasonable and expected. This meant that during the scanning previous to the real experiment, during the time spent setting your gain threshold etc..., your sample is bleached to the same extent with the two systems, even though you are scanning the sample many more times with the RS.
My conclusion about the RS system is that it can be used for high speed acquisition when the fluorescence signal is strong enough, or when the quality of the image is not critical (Calcium mobilization experiments for example). However, when the quality of the image matters, acquisition can be done averaging every 6 frames in which case the system would be functioning the same as the conventional SP2 in terms of speed, image quality and bleaching of the sample. This means that we could use this microscope for high speed live cell experiments with the advantages of a confocal microscope (spectral imaging, simultaneous acquisition of different channels, etc...), and with no limitations for performing low speed, long time lapses. For us, it is the perfect complement to the SP2-AOBS microscope that we already have, in order to free this microscope of all the live cell, time lapse experiments. Sorry, I have no experience with a spinning disk confocal that I can share with you.
María C. Montoya Ph. D.
Head of Confocal Microscopy Unit
Biotechnology Program
Centro Nacional de Investigaciones Oncológicas (CNIO)
Melchor Fernandez Almagro, 3
E-28029 Madrid, SPAIN
Phone: 34 91 7328012
Fax: 34 91 2246972
e-mail: [log in to unmask]
www.cnio.es
-----Mensaje original-----
De: Guy Cox [mailto:[log in to unmask]]
Enviado el: martes, 15 de abril de 2003 9:41
Para: [log in to unmask]
Asunto: Re: Leica Resonant Scanner
Search the CONFOCAL archive at
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Nathalie Garin wrote:
> The Leica was great BUT to obtain an image equivalent on the Leica RS as
>on a spinning disk, we had to increase the average number, doing so
>reduces the speed of acquisition to the same speed as a standard confocal.
>In summary, we obtain the same image on the RS as on a standard confocal
>at the same speed. We bought the spinning disk.
With no disrespect intended, this is the result you'd expect. The Leica
RS is no different optically to the slow scan model so you should get the
same image on both with the same accumulation time. High speed spot
scan confocals (whether Leica RS or the now discontinued Bio-Rad/Nikon
RTS 2000) will give you true, fully confocal images at high speed and
if your signal is bright enough this can be really useful.
Sampling 1000 spots at the same time (Yokogawa-based system) will
give you much better s/n for a given time of aaccumulation - the price
is some sacrifice of confocality.
Sampling 1,000,000 points at the same time (ie CCD camera) will
give you even better s/n for a given time of accumulation at the price
of a total sacrifice of confocality!
There's really no way around this tradeoff, and in the end it's the
experiment that will drive the purchase decision.
The advantage of something like an RS is that you can use it at high
speed but at the same time you still have almost all of the features
of a high-performance slow-scan confocal - which you don't have with
a Yokogawa system. So for mixed use and a limited budget it would
seem to have a useful place.
Guy Cox
Assoc. Prof. Guy Cox, ooOOOOOOoo
E.M. Unit, F09 # oOOOO | | OOOOo #
University of Sydney ### OOO| | | | | |OOO ###
NSW 2006, Australia ### OOO | | | | | | OOO ###
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