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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?

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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