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confocal HCS instruments - are they ready to occasionally replace a confocal microscope?

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Cromey, Douglas W - (dcromey)

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

Arne Seitz

Re: confocal HCS instruments - are they ready to occasionally replace a confocal microscope?

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Hi Doug,

first of all it would be extremely helpful if you could specify/name the confocal principle the HCS instrument(s) is/are based on.
I assume that they are spinning disk based (thus all following comments refer to this technology):

The use of lenses without immersion media is recommended if you want to scan an entire 96/384 well plate. Unless you have a dispenser which is "refilling" the immersion media. Without such a device you risk that the objective is running out of immersion media. But if you are not planning to do multi position experiments you should be able to use oil/water immersion lenses with these systems as well.
Then it is just the well-known trade-off between a point scanning confocal microscope and a spinning disk microscope (you should probably find dozens of posting concerning this topics here, otherwise it is nicely summarized in the "Handbook of Confocal microscopy" J. Pawley, 3rd edition).

In short:
Spinning disk: + faster, wide-field detection scheme, less photobleaching. - less good optical sectioning capabilities, fixed pinhole size

Thus spinning disk microscope are well suited for live cell imaging, less ideally suited for fixed specimens. So does the HCS instrument if is based on this technology.

Best regards
Arne

---------------------------------------------------------------
Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne

Phone: +41 21 693 9618
Fax: +41 21 693 9585
http://biop.epfl.ch/
---------------------------------------------------------------

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Cromey,
> Douglas W - (dcromey)
> Sent: lundi 4 juin 2012 19:25
> To: [hidden email]
> 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

Cromey, Douglas W - (dcromey)

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
*****

Arne,

My boss specifically was asking about the GE InCell 6000, which is a slit scanning confocal. But I expect that this issue will arise for a lot of us in the near future, and I was hoping for a broad discussion of the merits and short-comings of HCS devices.

Thanks for your feedback.
Doug

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Seitz Arne [[hidden email]]
Sent: Monday, June 04, 2012 12:44 PM
To: [hidden email]
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,

first of all it would be extremely helpful if you could specify/name the confocal principle the HCS instrument(s) is/are based on.
I assume that they are spinning disk based (thus all following comments refer to this technology):

The use of lenses without immersion media is recommended if you want to scan an entire 96/384 well plate. Unless you have a dispenser which is "refilling" the immersion media. Without such a device you risk that the objective is running out of immersion media. But if you are not planning to do multi position experiments you should be able to use oil/water immersion lenses with these systems as well.
Then it is just the well-known trade-off between a point scanning confocal microscope and a spinning disk microscope (you should probably find dozens of posting concerning this topics here, otherwise it is nicely summarized in the "Handbook of Confocal microscopy" J. Pawley, 3rd edition).

In short:
Spinning disk: + faster, wide-field detection scheme, less photobleaching. - less good optical sectioning capabilities, fixed pinhole size

Thus spinning disk microscope are well suited for live cell imaging, less ideally suited for fixed specimens. So does the HCS instrument if is based on this technology.

Best regards
Arne

---------------------------------------------------------------
Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne

Phone: +41 21 693 9618
Fax: +41 21 693 9585
http://biop.epfl.ch/
---------------------------------------------------------------

Casey Laris-2

Re: confocal HCS instruments - are they ready to occasionally replace a confocal microscope?

In reply to this post by Arne Seitz

*****
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*****

Hi Doug,

An HCS machine can definitely be used for less demanding microscopy tasks but do be mindful about what you are trying to accomplish. The big advantage of HCS is the throughput of getting numbers from images of your samples. That may sound obvious but the compromises to accomplish this goal are generally significant.

The lower N.A./dry objectives are often favored because of autofocus issues. The reflective positioning used by most vendors keeps you close to a surface of the plate or slide but generally doesn't account for sample variation. In thicker - but not too thick - samples, this is less of an issue. For monolayers you'll have a significant percentage of out of focus images. Without fast, image sample based autofocus, confocal imaging on a 0.6 N.A dry objective is a bit of an optical trick to enable crisper images for slightly out of position optics.

HCS is a little strange in that the acquired images could be thought of as just intermediate data waiting to be processed into final measurements. On something like a plate reader, you might not normally care what that looks like and focus more on accuracy and precision of the final well to well numbers. Because those intermediate data sets are images, it's very natural to dive in and see what's there.

The other big thing I'd look at is the software. Many of the systems can technically acquire image data from slides, plates etc but they really aren't designed to do so as you might expect. This varies greatly by manufacturer, but because of this focus on high-speed automated image acquisition/throughput, they might not have very usable joysticks and basic flexible controls you'd think would be easy for quick snap by snap users.

There's nothing wrong with any of this and you can get very nice images from these systems. It's more about being aware of the tradeoffs and the intended use.

Best,

Casey Laris | DiPath Inc. | Twitter | Mobile: 858-829-9501

On Jun 4, 2012, at 12:44 PM, Seitz Arne wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Doug,
>
> first of all it would be extremely helpful if you could specify/name the confocal principle the HCS instrument(s) is/are based on.
> I assume that they are spinning disk based (thus all following comments refer to this technology):
>
> The use of lenses without immersion media is recommended if you want to scan an entire 96/384 well plate. Unless you have a dispenser which is "refilling" the immersion media. Without such a device you risk that the objective is running out of immersion media. But if you are not planning to do multi position experiments you should be able to use oil/water immersion lenses with these systems as well.
> Then it is just the well-known trade-off between a point scanning confocal microscope and a spinning disk microscope (you should probably find dozens of posting concerning this topics here, otherwise it is nicely summarized in the "Handbook of Confocal microscopy" J. Pawley, 3rd edition).
>
> In short:
> Spinning disk: + faster, wide-field detection scheme, less photobleaching. - less good optical sectioning capabilities, fixed pinhole size
>
> Thus spinning disk microscope are well suited for live cell imaging, less ideally suited for fixed specimens. So does the HCS instrument if is based on this technology.
>
> Best regards
> Arne
>
>
> ---------------------------------------------------------------
> Arne Seitz
> Head of Bioimaging and Optics Platform (PT-BIOP)
> Ecole Polytechnique Fédérale de Lausanne (EPFL)
> Faculty of Life Sciences
> Station 15, AI 0241
> CH-1015 Lausanne
>
> Phone: +41 21 693 9618
> Fax: +41 21 693 9585
> http://biop.epfl.ch/
> ---------------------------------------------------------------
>
>
>
>
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Cromey,
>> Douglas W - (dcromey)
>> Sent: lundi 4 juin 2012 19:25
>> To: [hidden email]
>> 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

George McNamara

Re: confocal HCS instruments - are they ready to occasionally replace a confocal microscope?

In reply to this post by Cromey, Douglas W - (dcromey)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Doug,

If you or anyone else would like to buy an HCS confocal microscope,
please buy our BD Pathway 855 HCS !!! Reference for UM's use of the
Pathway:

*Cabrera* O, Jacques-Silva MC, Berman DM, Fachado A, Echeverri F, Poo R,
Khan A, Kenyon NS, Ricordi C, Berggren PO, *Caicedo* A. Automated,
high-throughput assays for evaluation of human pancreatic islet
function. </pubmed/18351020> Cell Transplant. 2008;16(10):1039-48. PMID:
18351020.

Uses a CARV spinning disk confocal attachment (can slide or out of light
path), has 2 Hg arc lamps, can do Fura-2 350/380 nm excitation ratioing,
ORCA-ER camera - and can look by eyepiece in each of widefield and
confocal modes. Has been off service contract for a year. We can even
add in a somewhat disassembled CARV-1 unit (set up for a Zeiss Axiovert
200M) at no extra charge. For that matter, if the price is right, the
buyer is also welcome to our Zeiss LSM510 scanhead, electronics, lasers,
more (I am turning its ex-LSM's Axiovert 200M into a widefield system,
but could ship that too if the price is right).

GE InCell 6000 - This illuminates one line at a time (presumably
resolution limited by the objective lens?) and detection is user
selectable number of pixels across the line, using the rolling shutter
mode of a 5.5 megapixel sCMOS. They refer to it as acting as a 'virtual
detection slit' (my phrase - you can check GE marketing for the exact
phrase). I suggest that anything other than a physical slit (line
equivalent of a pinhole) means this is not really a confocal microscope.
Could still be quite useful.

With respect to your original question ... if the instrument loading and
unloading is easy, and the software is easy to use and/or you have
brilliant users who can easily learn and deal with any software, sure. I
am going to guess the price of the InCell 6000 is in the $500K-$800K
price range (depending on lasers, service contract, profit margin, etc)
... and its annual service contract price is probably 10% of purchase
price. So, are you really going to be able to make back the service
contract price if you have someone count DAPI+ thingies a couple of
times a day? If you are going to spend the money on an HCS system, best
to have enough expected utilization for HCS screens to pay for at least
the service contract.

Sincerely,

George
University of Miami

On 6/4/2012 4:27 PM, Cromey, Douglas W - (dcromey) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Arne,
>
> My boss specifically was asking about the GE InCell 6000, which is a slit scanning confocal. But I expect that this issue will arise for a lot of us in the near future, and I was hoping for a broad discussion of the merits and short-comings of HCS devices.
>
> Thanks for your feedback.
> Doug
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on behalf of Seitz Arne [[hidden email]]
> Sent: Monday, June 04, 2012 12:44 PM
> To: [hidden email]
> 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,
>
> first of all it would be extremely helpful if you could specify/name the confocal principle the HCS instrument(s) is/are based on.
> I assume that they are spinning disk based (thus all following comments refer to this technology):
>
> The use of lenses without immersion media is recommended if you want to scan an entire 96/384 well plate. Unless you have a dispenser which is "refilling" the immersion media. Without such a device you risk that the objective is running out of immersion media. But if you are not planning to do multi position experiments you should be able to use oil/water immersion lenses with these systems as well.
> Then it is just the well-known trade-off between a point scanning confocal microscope and a spinning disk microscope (you should probably find dozens of posting concerning this topics here, otherwise it is nicely summarized in the "Handbook of Confocal microscopy" J. Pawley, 3rd edition).
>
> In short:
> Spinning disk: + faster, wide-field detection scheme, less photobleaching. - less good optical sectioning capabilities, fixed pinhole size
>
> Thus spinning disk microscope are well suited for live cell imaging, less ideally suited for fixed specimens. So does the HCS instrument if is based on this technology.
>
> Best regards
> Arne
>
>
> ---------------------------------------------------------------
> Arne Seitz
> Head of Bioimaging and Optics Platform (PT-BIOP)
> Ecole Polytechnique Fédérale de Lausanne (EPFL)
> Faculty of Life Sciences
> Station 15, AI 0241
> CH-1015 Lausanne
>
> Phone: +41 21 693 9618
> Fax: +41 21 693 9585
> http://biop.epfl.ch/
> ---------------------------------------------------------------
>
>
>
>
>
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Cromey,
>> Douglas W - (dcromey)
>> Sent: lundi 4 juin 2012 19:25
>> To: [hidden email]
>> 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
>>

Guy Cox-2

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
*****

I don't understand why a single line of the detector is anything other than a genuine slit, assuming that you scan one line and only read one line (ie ignoring any signal on lines above and below). If the detector is capable of Nyquist sampling then you'll have a narrower slit than any physical slit-scanner will give you. It does put a bit of a premium on alignment but for $800K you might expect them to manage this! I'd suggest this is way more confocal than a BD CARV (no slight intended to the latter product).

Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Tuesday, 5 June 2012 11:02 AM
To: [hidden email]
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,

If you or anyone else would like to buy an HCS confocal microscope,
please buy our BD Pathway 855 HCS !!! Reference for UM's use of the
Pathway:

*Cabrera* O, Jacques-Silva MC, Berman DM, Fachado A, Echeverri F, Poo R,
Khan A, Kenyon NS, Ricordi C, Berggren PO, *Caicedo* A. Automated,
high-throughput assays for evaluation of human pancreatic islet
function. </pubmed/18351020> Cell Transplant. 2008;16(10):1039-48. PMID:
18351020.

Uses a CARV spinning disk confocal attachment (can slide or out of light
path), has 2 Hg arc lamps, can do Fura-2 350/380 nm excitation ratioing,
ORCA-ER camera - and can look by eyepiece in each of widefield and
confocal modes. Has been off service contract for a year. We can even
add in a somewhat disassembled CARV-1 unit (set up for a Zeiss Axiovert
200M) at no extra charge. For that matter, if the price is right, the
buyer is also welcome to our Zeiss LSM510 scanhead, electronics, lasers,
more (I am turning its ex-LSM's Axiovert 200M into a widefield system,
but could ship that too if the price is right).

GE InCell 6000 - This illuminates one line at a time (presumably
resolution limited by the objective lens?) and detection is user
selectable number of pixels across the line, using the rolling shutter
mode of a 5.5 megapixel sCMOS. They refer to it as acting as a 'virtual
detection slit' (my phrase - you can check GE marketing for the exact
phrase). I suggest that anything other than a physical slit (line
equivalent of a pinhole) means this is not really a confocal microscope.
Could still be quite useful.

With respect to your original question ... if the instrument loading and
unloading is easy, and the software is easy to use and/or you have
brilliant users who can easily learn and deal with any software, sure. I
am going to guess the price of the InCell 6000 is in the $500K-$800K
price range (depending on lasers, service contract, profit margin, etc)
... and its annual service contract price is probably 10% of purchase
price. So, are you really going to be able to make back the service
contract price if you have someone count DAPI+ thingies a couple of
times a day? If you are going to spend the money on an HCS system, best
to have enough expected utilization for HCS screens to pay for at least
the service contract.

Sincerely,

George
University of Miami

On 6/4/2012 4:27 PM, Cromey, Douglas W - (dcromey) wrote:

Emmanuel Gustin

Re: confocal HCS instruments - are they ready to occasionally replace a confocal microscope?

In reply to this post by Cromey, Douglas W - (dcromey)

*****
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: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: 04 June 2012 19:25
To: [hidden email]
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

Sylvie Le Guyader

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

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

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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:[hidden email]] On Behalf Of Gustin,
> Emmanuel [JRDBE]
> Sent: 05 June 2012 11:08
> To: [hidden email]
> 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:
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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: [hidden email]
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Cromey,
> Douglas W - (dcromey)
> Sent: 04 June 2012 19:25
> To: [hidden email]
> 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