Dear Charu,
All types of spinning disk confocals you refer to use fluorescence (i.e. it is not laser versus fluorescence, but rather laser versus arc lamp). To oversimplify, the main differences between systems is (a) the illumination source, and (b) the type of scanner .
For the illumination source you can use a laser control unit with a set of individual lasers (each laser providing one or a few wavelengths for fluorescence excitation). Alternatively, you can use a mercury lamp or similar broad band excitation light, and use a filter wheel to select specific bands (range of wavelengths) to excite fluorescence. Some big differences between these options are cost (lasers may cost US $ 10,000-20,000 each), plus the cost of all the electronics controlling them. Old style gas lasers may last a few years and need to be replaced, which may add substantial cost down the road. New solid state lasers will last longer, allegedly, but tend to be still quite expensive to purchase. Typically, with a laser system, you will probably want four lasers (405 and/or 440, 488, 560 or so, 633 or so, maybe 514 for YFP), especially for a multi-user facility. Depending on your anticipated applications, you need to chose your lasers
carefully. 440 for instance will be better for CFP, but will be very poor or unusable for nuclear dyes such as DAPI. 405 will work with DAPI and CFP (although less good for CFP than the 440), but will be a bit more toxic if doing CFP, so not as good for live imaging. Lamp based systems will be tens of thousands of dollars cheaper to purchase (but you will have to purchase lamps regularly), and will be more flexible since you can add extra excitation filters at pretty low cost. Most high performance systems tend to use lasers. I don't think it's a matter of power or light intensity, but lasers may have some advantages in terms of speed of switching, beam focusing, synchronization, etc...
A second difference is the type of confocal scanner. The gold standard seems to be Yokogawa, which many vendors of spinning disk systems use. In their latest implementation, these are very light efficient (because of the use of microlenses to gather as much light as possible), and may well be the most high performance systems available. They have one single aperture size, which can be a limitation sometimes. Systems such as the CARV use less sophisticated spinning disk technology, and are not quite as high performing, I think. Olympus DSU uses a set of interchangeable apertures, so that users can choose various slit sizes. Combined with an arc lamp illumination, the DSU is a quite affordable system, and does work pretty well for many samples and applications, but I suspect it may not offer the same level of performance as a high end Yokogawa system. The only way to know if this will work for you is probably to run tests on such a system with actual
samples and see what happens (or to get feed-back from someone who has both types of systems and has thoroughly compared them). An Olympus DSU with arc lamp illumination may easily cost US $ 100,000-150,000 less than a four laser Yokogawa system, because of the lower cost of the lamp versus lasers, and the lower cost of the DSU scanner versus the Yokogawa (but note that Yokogawa may have several different models).
Finally, there are other types of systems that may be somewhere in between these two extremes in terms of cost, performance and functionality, such as the Nikon Live Scan (Prairie technologies swept field), and Zeiss LSM 5 live. The Nikon LiveScan/Prairie swept field allows users to select between pinholes or slits of different sizes and can be tailored to be more sensitive (faster) or more confocal, and is a nice instrument, although possibly requiring more maintenance and calibration than a spinning disk. The Zeiss 5 Live is a slit scanning system. Therefore, these are not strictly speaking spinning disk systems, although they are functionally similar (faster imaging than conventional confocals through simultaneous illumination of multiple points or lines, but with reduced confocality)
This being said, I would not necessarily exclude conventional laser point scanning confocals. The way they work is by imaging (reading intensities) one point at a time and building the image by scanning a certain area line by line. Each point is read in the order of milliseconds, and therefore you need much higher illumination intensities. On a spinning disk, you image hundreds of points simultaneously, with illumination intensities that are much lower (and integration times much longer). This is considered better for live cell imaging, but you can do live cell imaging with a point scanning confocal as long as you keep illumination intensities below a critical threshold. Regarding speed, in theory spinning disk can run much faster in terms of frames per second, but you can go pretty fast with a conventional confocal, if you are able to keep the imaged area small (such as 128 x 128 pixels or so), or you can do a line scanning (and obtain an intensity
profile over time rather than an image). This may still answer your biological question just fine.
If I had one piece of advice to give you, rather than immediately jumping into buying a new instrument, I would try your calcium experiments on the instruments you already have (assuming they have the required lasers, etc...). This will show you whether or not they can be done, will give you some experience about the particular experiment, and you will get a better sense what to look for if you later on can have a demo for a new instrument, because you will know exactly what you are looking for. If you have a demo where people bring samples or try techniques they had never used, and the demo doesn't work, you will never know if it is because of the instrument, or because of the samples... Some vendors may let you have an extended demo, i.e. 1-2 weeks, which will give you a good sense about an instrument, but even so, demos are rarely perfect....
I can not tell you which specific instrument is better for your situation. This will depend on your financial situation (how much money you have for purchase, as well as for maintenance/replacement parts down the road). In addition, you will have to take into account the needs of your customers, such as which dyes/fluorescent proteins they anticipate using, applications, speed requirements, confocality requirements, and such. Maybe you need to consider getting an instrument that satisfies your immediate needs, but than can be upgraded later if needed. If you can have a demo that you can run on actual samples (that is, run an actual experiment and see if it works; ideally, use samples you are familiar with and know they work as I mentioned above), then, that's probably the best. Second best may be to look at your anticipated applications, find publications about similar work, and do a little survey about what these authors used (maybe contact them
directly and ask about their experience).
This being said, if you have the money (for purchase and long term maintenance), you probably won't go wrong by buying a good laser/yokogawa scanner based spinning disk system. Just make sure you get the right lasers, dichoics, emission filters and objectives for your anticipated applications. Your local vendor should be able to guide you. If funds are limited, you should consider some of the more affordable options, but make sure you test them for your specific applications before you purchase them. They may happen to work just fine...
Good luck.
Julio.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA
==
On Dec 30, 2009, at 3:49 AM, charu tanwar wrote:
No, i have not yet seen any of the spinning disk systems. Yokogawa is very famous but i really want to know that how reasonable is to buy a laser based multipoint scanning system than buying fluorescence based spinning disk. (we already have single point scanning system, one only confocal and another confocal with live cell, both laser based.)
--- On Wed, 30/12/09, Vergara, Leoncio A. <lvergara@...> wrote:
From: Vergara, Leoncio A. <lvergara@...>
Subject: Re: Spinning disk requirement
To: CONFOCALMICROSCOPY@...
Date: Wednesday, 30 December, 2009, 4:12 PM
have you tested the Olympus DSU? I am wondering how it would compare with the yokowaga scanhead based systems. ________________________________________ From: Confocal Microscopy List [ CONFOCALMICROSCOPY@...] On Behalf Of Charu Tanwar [ tanwar_charu@...] Sent: Wednesday, December 30, 2009 1:36 AM To: CONFOCALMICROSCOPY@...Subject: Spinning disk requirement Dear All I request all the members to please help me to decide for a spinning disk confocal microscope for our facility which is a multiuser facility for the whole university. We
already have a point scanning confocal system from Olympus. We will have another point scanning confocal soon with all the live cell imaging accessories (laser based). For very fast phenomenon like measuring Calcium Flux in the cells, we want to have a multi point scanning system. But i am unable to decide whether i should take a fluorescence based spinning disk or laser based spinning disk confocal. Also, please give me some feedback for CARV II (multi point confocal, fluorescence based). Please help. Thanks in advance. Charu Tanwar Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi India.
|
The INTERNET now has a personality. YOURS! See your Yahoo! Homepage.