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Hi Doug
Advantages of an HCS system:
- integration of acquisition, analysis and data management (not the case for all HCS though) with only 1 company to talk to
- possibility to load plates (at extra cost)
- possibility to have a dispenser for one or several wells (at extra cost)
- you can image a whole 96 well plate including the outer wells (which you might want to exclude anyway to avoid edge effects)
Disadvantages:
- amazingly expensive compared to buying a microscope of equivalent specs
- no flexibility (eg: no space for a different camera, for a Yokagawa spinning disk...)
- some of them are not laser based but lamp based, sometimes with large power fluctuation over time
For the price of an HCS system you may be able to buy a microscope based laser scanning confocal with a resonant scanner. You get the speed and the flexibility. You probably need to give up on the plate loader and the dispenser though and you will probably need to deal with acquisition, data analysis and data management as three separate aspects of your experiment.
In any case make sure you have a hardware/laser based autofocus and make sure it is very fast.
In many case it is not possible/difficult to image dishes or slides on a HCS system (no stage inserts for example).
Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14183 Huddinge
Sweden
office: +46 (0) 8 5248 1107
LCI room: +46 (0) 8 5248 1172
mobile: +46 (0) 73 733 5008
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Gustin,
> Emmanuel [JRDBE]
> Sent: 05 June 2012 11:08
> To: [log in to unmask]
> Subject: Re: confocal HCS instruments - are they ready to occasionally replace a
> confocal microscope?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Doug,
>
> Because of the additional cost of automation, not just for the hardware, but also
> the software, you would have a good confocal microscope (and possibly a two-
> photon system) for the price of a confocal HCS reader. I guess that from an
> imaging facility's point of view, an advantage of a modern HCS reader may be that
> it comes as a closed box with software settings only -- no fiddling with filters or
> scratching of objectives; or at least less of it. And it is generally true that there are
> also fewer imaging settings to adjust than on a confocal microscope. However,
> there are the additional settings for automated acquisition that have to be done,
> and a fair amount of calibration work if you want quantitatively reproducible data.
> The software can be fairly complicated, although that depends on user expectation
> and usage patterns.
>
> HCS readers are designed to scan a lot of fields automatically and do statistical
> sampling, while the user of a confocal microscope usually wants to identify
> specific areas of interest and scan them. The HCS system will do "6 fields of view
> in every well of a plate, with 3 fluorescence channels" perfectly well, because that
> is what it is designed for, but on some instruments there is no practical way to get
> "an image of THIS spot", and finding the area of interest in a slide can be a pain.
> The gap can be bridged: Some HCS readers have "manual imaging modes" and
> even oculars. From the other end, there is the Leica "Matrix" option, which takes
> the reverse approach: Taking a standard CLSM and providing it with a software
> package and immersion system that allow automated HCS. But I don't expect there
> will be a system that is equally well suited for both operating modes any time
> soon.
>
> For live-cell imaging, some HCS readers may be a better and more user-friendly
> choice than equipping a confocal microscope with an external incubator (and
> hoping that the software will be stable enough for a long run). And you have the
> option of linking the instrument to an automated incubator and a plate handling
> robot.
>
> Confocal HCS readers come in a variety of optical configurations: There are simple
> Nipkow spinning disk configurations (from BD and PerkinElmer), with the option to
> remove the Nipkow disk from the beam path. There is the more sophisticated
> Yokogawa spinning disk configuration (from PerkinElmer and Yokogawa) with an
> additional microlens disk. There is a slit-scanning confocal system (from GE) and
> for several years there has been a point scanning system (from Molecular
> Devices). Several readers can be operated with oil immersion lenses, for scanning
> small areas. There is no automated application of the oil, so this requires some
> care. A few have automated water immersion systems.
>
> In recent times, most HCS readers have been developed with dual autofocus
> options, i.e. both reflection-based using a small NIR laser, and an image-based
> autofocus that optimizes for contrast. Systems of an older design often have only
> one option. Autofocus remains very sensitive to the quality of your labware and
> the correct definition of the objective.
>
> I think that today, most users of confocal HCS readers use them to enhance
> contrast and suppress fluorescent backgrounds. Fluorescent backgrounds are big
> a nuisance in drug discovery applications because a few % of compounds in
> chemical libraries are significantly fluorescent (up to 11% under UV excitation), and
> besides, screening teams want to have as few reagent washing steps as possible.
> Some people use confocal HCS for 3D imaging (we do), but this is a minority,
> because this is relatively slow, the data volumes are huge, and there are few
> automated analysis options for this type of data.
>
> Best Regards,
>
> Emmanuel
>
> --
> Emmanuel Gustin, Tel. (+32) 14 64 1586, e-mail: [log in to unmask]
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Cromey,
> Douglas W - (dcromey)
> Sent: 04 June 2012 19:25
> To: [log in to unmask]
> Subject: confocal HCS instruments - are they ready to occasionally replace a
> confocal microscope?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Colleagues,
>
> My supervisor has been contacted by one of the high-content screening device
> companies. They are pitching an HCS device with confocal scanning abilities.
> Since the HCS instrument can scan multi-well plates and microscope slides, as well
> as provide environmental conditions for live cells, he was wondering if it would be
> suitable as an entry-level confocal for some of our users with non-demanding
> and/or functional live-cell assay kind of needs?
>
> I recognize that every optical instrument has compromises, are there compromises
> that we don't want to make? Seems like most of the HCS systems favor dry
> lenses, with corresponding lower NAs at any given mag.
>
> The one concern I have is that the device might allow people to collect bad data
> faster. See Dave Piston's recent comment in Nature entitled "Research tools:
> Understand how it works", Nature 484, 440-441 (26 April 2012) doi:10.1038/484440a
>
> I am particularly interested in hearing from people who might have experience with
> both point-scanning confocals and HCS instruments.
>
> Thanks.
> Doug
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