Basic live cell imaging question...

[Gisele Giorgi]

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For a few dollars you can buy a depression slide! Gisele

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On Nov 3, 2010, at 10:01 PM, CONFOCALMICROSCOPY automatic digest system <[hidden email]> wrote:

> There are 6 messages totalling 703 lines in this issue.
>
> Topics of the day:
>
>  1. Pulsed vs CW laser sources for single-photon fluorescence
>  2. Basic live cell imaging question... (5)
>
> ----------------------------------------------------------------------
>
> Date:    Wed, 3 Nov 2010 09:33:08 +0100
> From:    Christian Schumann <[hidden email]>
> Subject: Re: Pulsed vs CW laser sources for single-photon fluorescence
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> The "molecular memory" of the excitation process is lost after elaxation
> from the Franck-Condon region, which happens within 50-200 fs. After
> that, the molecular environment has the most influence of the reaction
> route, and this is your best guess if you want to influence triplet
> population (see  ie. Vogelsang et al., Angew Chem Int Ed, 47(29):5465).
>
> Of course people have devised clever pulse shaping experiments to
> prepare coherent wavepackets on the S1 surface to influence the course
> of photochemical reactions. But although it's not impossible, I think it
> would be extremly demanding to implement this in a confocal microscope,
> where you have a lot of optics in your beam path as compared to
> spectroscopy experiments. Also, optimization of the pulse shapes mostly
> depends on feedback of the measurement of a reaction product and genetic
> algorithms, which would also have to be implemented in the microscope.
>
> Speaking of the spin of the photon: This is already required to fulfill
> the selection rules for molecular excitation.
>
> But it seems that this is getting really academic now ...
>
> Christian
>
> Am Dienstag, den 02.11.2010, 12:13 -0600 schrieb Craig Brideau:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>> *****
>> =20
>> Here's a question: Would the spin of the incident photons influence
>> intersystem crossing?  As I understand it, crossing requires angular
>> momentum changes.  If the incident photon possesses + or - spin would it
>> make a difference?
>> =20
>> Craig
>> =20
>> =20
>> On Tue, Nov 2, 2010 at 12:06 PM, Christian Schumann <
>> [hidden email]> wrote:
>> =20
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>> *****
>>>
>>> Craig,
>>>
>>> I don't know if there is much data out there on the 2P excitation cross
>>> sections of triplet states, but I'd guess that these are probably not
>>> really high.
>>> Excitation directly to the triplet state is quantum mechanically not
>>> allowed (at least for one-photon transitions) due to the unequal spin
>>> multiplicities, so the cross section is practically non-existent. I'd
>>> intuitively say that it's even lower for a 2P transition.
>>> The only way to get to the triplet state is by intersystem crossing,
>>> which is mostly based on spin-orbit coupling and depends a lot on the
>>> specific molecule and environment. But perhaps some FCS people have
>>> measured triplet population ratios after 2P excitation.
>>>
>>> I might be terribly wrong, my spectroscopy days were quite a while back=
> ,
>>> as was my QM course. Probably anyone else has some more input on this.
>>>
>>> Christian
>>>
>>> Am Dienstag, den 02.11.2010, 11:48 -0600 schrieb Craig Brideau:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>> *****
>>>>
>>>> Thanks for your insight, Christian.  So what it boils down to is that=
> for
>>>> single photon interactions the primary method of damage is intrasyste=
> m
>>>> crossing into triplet states.  So the solution is to allow time for t=
> hese
>>>> states to relax.  Conversely, for two photon the primary damage mecha=
> nism
>>>> are unwanted 3rd order or higher effects so lowering peak energy is t=
> he
>>> way
>>>> to go.  I'm just curious how much variability there is depending on y=
> our
>>>> sample.  Would triplet state excitation also be a factor at all in 2p=
> , or
>>>> are the photon energies really low enough?  After all, couldn't 2p
>>>> interactions excite to triplet states as well?  Or is there just not
>>> enough
>>>> energy to cause crossing?
>>>>
>>>> Craig
>>>>
>>>>
>>>> On Tue, Nov 2, 2010 at 2:15 AM, Christian Schumann <
>>>> [hidden email]> wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>>> *****
>>>>>
>>>>> Hi,
>>>>>
>>>>> as I understand it, in Donnert's paper the assumption is that bleac=
> hing
>>>>> (especially due to action of the STED laser) is due to excitation o=
> f
>>>>> triplet states of the fluorophores, which have a lifetime of severa=
> l =C2=B5s
>>>>> and are populated by intersystem crossing from the fluorescent S1
>>> state.
>>>>> So the idea is to let the triplet states relax back to the S0 state=
> ,
>>>>> either by reducing laser rep rate or faster scanning.
>>>>> I haven't read Ji's paper, but in 2P work you should have photon
>>>>> energies low enough that excitation from the S1 or T1 states is not=
> of
>>>>> major concern. On the other hand side, the pulse lengths in 2P are =
> much
>>>>> shorter (~200 fs) than in pulsed STED (~200-300 ps), so you could
>>> direct
>>>>> excitation S0->Sn via 3P absorption or other higher-order effects.
>>>>> Reducing exciation power and incresing pulse rate should give you t=
> he
>>>>> same number of photons (ie SNR) with lower probability of higher-or=
> der
>>>>> effects. Increasing pulse length on the other hand side would reduc=
> e
>>> the
>>>>> excitation probability for 2P as well.
>>>>> As stated, I haven't read Ji's paper, but that's what would make se=
> nse
>>>>> to me.
>>>>>
>>>>> Christian
>>>>>
>>>>> Am Montag, den 01.11.2010, 16:20 -0600 schrieb Craig Brideau:
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>>>> *****
>>>>>>
>>>>>> The papers are very interesting, but seem to be saying different
>>> things.
>>>>>> Donnert's paper basically says that giving the flurophore time to
>>> recover
>>>>>> helps increase signal and reduce bleaching.  Ji's paper, on the o=
> ther
>>>>> hand
>>>>>> (note it is for 2p rather than confocal) states that increasing t=
> he
>>>>> number
>>>>>> of pulses per unit time achieves the same effect.  The papers in =
> a
>>> sense
>>>>>> seem to contradict each other.  Any thoughts or comments, anyone?
>>> Have
>>>>>> other groups verified these results?
>>>>>>
>>>>>> Craig
>>>>>>
>>>>>>
>>>>>> On Sun, Oct 31, 2010 at 9:25 PM, Peng Xi <[hidden email]> wrot=
> e:
>>>>>>
>>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go t=
> o:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>>>>> *****
>>>>>>>
>>>>>>> Dear Craig,
>>>>>>>     These two articles are very useful:
>>>>>>>
>>>>>>> Gerald Donnert, Christian Eggeling & Stefan W Hell, "Major sign=
> al
>>>>>>> increase in fluorescence microscopy through dark-state relaxati=
> on",
>>>>>>> Nature Methods - 4, 81 - 86 (2007)  doi:10.1038/nmeth986
>>>>>>> In this paper, the photobleaching is minimized with dark state
>>>>>>> relaxation (single photon excitation), which needs a low repeti=
> tion
>>>>>>> rate (<=3D1MHz).
>>>>>>>
>>>>>>>
>>>>>>> Na Ji, Jeffrey C Magee & Eric Betzig, "High-speed, low-photodam=
> age
>>>>>>> nonlinear imaging using passive pulse splitters",
>>>>>>> Nature Methods 5, 197 - 202 (2008)
>>>>>>> In this paper, by increasing the repetition rate in two-photon
>>>>>>> excitation, the effective excitation power is decreased, theref=
> ore
>>> a
>>>>>>> lower photobleaching is obtained.
>>>>>>>    Thank you.
>>>>>>>
>>>>>>>
>>>>>>> Sincerely,
>>>>>>> Peng Xi
>>>>>>> Ph. D.    Associate Professor
>>>>>>> Dept. of Biomedical Engineering, College of Engineering
>>>>>>> Peking University, Beijing, China
>>>>>>> Tel: [phone]
>>>>>>> Email: [hidden email]
>>>>>>> http://bme.pku.edu.cn/~xipeng <http://bme.pku.edu.cn/%7Exipeng>=
> <
>>> http://bme.pku.edu.cn/%7Exipeng> <
>>>>> http://bme.pku.edu.cn/%7Exipeng>
>>>>>>>
>>>>>>> On Sat, Oct 30, 2010 at 2:30 AM, Craig Brideau <
>>>>> [hidden email]>
>>>>>>> wrote:
>>>>>>>> *****
>>>>>>>> To join, leave or search the confocal microscopy listserv, go=
> to:
>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>>>>>> *****
>>>>>>>>
>>>>>>>> Hi folks.  I've been noticing a number of microscope companie=
> s
>>> have
>>>>> been
>>>>>>>> offering 'white' tunable lasers with their confocals.  Most o=
> f
>>> these
>>>>>>> systems
>>>>>>>> appear to be pulse-laser driven supercontiuum-based light
>>> sources.
>>>>> My
>>>>>>>> question for the list is will the pulsed excitation be more
>>> effective
>>>>> for
>>>>>>>> even single photon fluorescence compared to conventional CW
>>>>> excitation?
>>>>>>> I'm
>>>>>>>> thinking the relaxation time between pulses may help with
>>>>> photobleaching.
>>>>>>>> Does anyone have any thoughts or experiences to share on the
>>> matter?
>>>>>>>>
>>>>>>>> Thanks,
>>>>>>>>
>>>>>>>> Craig
>>>>>>>>
>>>>>>>
>>>>> --
>>>>> Dr. Christian Schumann
>>>>> INM
>>>>> Leibniz-Institut f=C3=BCr Neue Materialien gGmbH
>>>>> Campus D2 2
>>>>> 66123 Saarbr=C3=BCcken
>>>>>
>>>>> Telefon: [phone]
>>>>> Telefax: [phone]
>>>>> E-Mail: [hidden email]
>>>>> Homepage: www.inm-gmbh.de
>>>>>
>>>>>
>>> -----------------------------------------------------------------------=
> -
>>>>> Sitz der Gesellschaft: Saarbr=C3=BCcken
>>>>> Rechtsform: gGmbH
>>>>> Amtsgericht Saarbr=C3=BCcken, HRB 8525
>>>>> Gesch=C3=A4ftsf=C3=BChrer: Prof. Dr. Eduard Arzt (Vorsitz),
>>>>> Prof. Dr. Michael Veith, Dr. Roland Rolles
>>>>> Kuratoriumsvorsitzender: StS Peter Hauptmann
>>>>> USt.-ID: DE 138167776
>>>>>
>>> -----------------------------------------------------------------------=
> -
>>>>>
>>> --
>>> Dr. Christian Schumann
>>> INM
>>> Leibniz-Institut f=C3=BCr Neue Materialien gGmbH
>>> Campus D2 2
>>> 66123 Saarbr=C3=BCcken
>>>
>>> Telefon: [phone]
>>> Telefax: [phone]
>>> E-Mail: [hidden email]
>>> Homepage: www.inm-gmbh.de
>>>
>>> -----------------------------------------------------------------------=
> -
>>> Sitz der Gesellschaft: Saarbr=C3=BCcken
>>> Rechtsform: gGmbH
>>> Amtsgericht Saarbr=C3=BCcken, HRB 8525
>>> Gesch=C3=A4ftsf=C3=BChrer: Prof. Dr. Eduard Arzt (Vorsitz),
>>> Prof. Dr. Michael Veith, Dr. Roland Rolles
>>> Kuratoriumsvorsitzender: StS Peter Hauptmann
>>> USt.-ID: DE 138167776
>>> -----------------------------------------------------------------------=
> -
>>>
> --=20
> Dr. Christian Schumann
> INM
> Leibniz-Institut f=C3=BCr Neue Materialien gGmbH
> Campus D2 2
> 66123 Saarbr=C3=BCcken
>
> Telefon: [phone]
> Telefax: [phone]
> E-Mail: [hidden email]
> Homepage: www.inm-gmbh.de
>
> ------------------------------------------------------------------------
> Sitz der Gesellschaft: Saarbr=C3=BCcken
> Rechtsform: gGmbH
> Amtsgericht Saarbr=C3=BCcken, HRB 8525
> Gesch=C3=A4ftsf=C3=BChrer: Prof. Dr. Eduard Arzt (Vorsitz),
> Prof. Dr. Michael Veith, Dr. Roland Rolles
> Kuratoriumsvorsitzender: StS Peter Hauptmann
> USt.-ID: DE 138167776
> ------------------------------------------------------------------------
>
> ------------------------------
>
> Date:    Wed, 3 Nov 2010 21:09:57 +0100
> From:    Gert van Cappellen <[hidden email]>
> Subject: Re: Basic live cell imaging question...
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>  Culture your cells on a round coverslip. Take an object glas glue a
> square piece of non-toxic rubber with a round hole on it. Fill this with
> CO2 satured medium somewaht more as the volume of the hole. Put your
> coverslip on it, with the cells to the medium off course. Press it
> gently down and the glass will seal itself to the rubber ring. Now your
> cells will survive for a couple of hours, so you can do the first
> imaging. For real experiments you have to find a way to heat the object
> glass to 37C.
>
> Good luck, Gert
>
> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.
>>
>> Colin
>>
>
> ------------------------------
>
> Date:    Thu, 4 Nov 2010 09:03:17 +0800
> From:    Axel Kurt Preuss <[hidden email]>
> Subject: Re: Basic live cell imaging question...
>
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>
> ------------------------------
>
> Date:    Thu, 4 Nov 2010 09:05:05 +0800
> From:    Axel Kurt Preuss <[hidden email]>
> Subject: Re: Basic live cell imaging question...
>
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>
> ------------------------------
>
> Date:    Wed, 3 Nov 2010 21:39:31 -0400
> From:    "JOEL B. SHEFFIELD" <[hidden email]>
> Subject: Re: Basic live cell imaging question...
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I think Gert's response makes a lot sense.  For a newbie, with limited goal=
> s
> (initially), it is not necessary to have an elaborate setup.  In the (very)
> old days, there was something called a "hanging drop culture" in which
> tissue fragments were placed in a drop of medium on a slide, the slide was
> inverted and examined with a conventional microscope.  Gert is suggesting a
> modern version of the same, assuming that you would visualize the cells
> through the cover slip to which they are attached, Ujnfortunately, the
> original writer did not specify the kind of optics they intended to use. If
> the cells are, indeed, attached to the cover slip, it is possible to use
> properly adjusted phase or Hoffman optics in transmissionto increase the
> contrast.  If fluorescence is needed, then you have to be concerned with
> light toxicity, etc., but the optics should work fine, as long as you don't
> want to see nuclear speckles, or details of mitochondria.
>
> Joel
>
>
> On Wed, Nov 3, 2010 at 9:03 PM, Axel Kurt Preuss <
> [hidden email]> wrote:
>
>> You need a water immersion object or have to build one
>>
>>
>>  Cheers
>>
>> Axel
>> =97=97=97=97=97
>> Axel K Preuss, PhD,
>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,
>> sent from 9271.5622
>>
>>
>> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>> *****
>>>
>>> Culture your cells on a round coverslip. Take an object glas glue a
>>> square piece of non-toxic rubber with a round hole on it. Fill this wit=
> h
>>> CO2 satured medium somewaht more as the volume of the hole. Put your
>>> coverslip on it, with the cells to the medium off course. Press it
>>> gently down and the glass will seal itself to the rubber ring. Now your
>>> cells will survive for a couple of hours, so you can do the first
>>> imaging. For real experiments you have to find a way to heat the object
>>> glass to 37C.
>>>
>>> Good luck, Gert
>>>
>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>> *****
>>>>
>>>> We would like to do live cell imaging - mammalian cell lines - but onl=
> y
>> have direct access to an upright Olympus BX61. We don't really need
>> complicated perfusion chambers, etc just something simple. We're real
>> neophytes so all suggestions gratefully received.
>>>>
>>>> Colin
>>>>
>>
>> Note: This message may contain confidential information. If this Email/Fa=
> x
>> has been sent to you by mistake, please notify the sender and delete it
>> immediately. Thank you.
>>
>
>
>
> --=20
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
>
> ------------------------------
>
> Date:    Thu, 4 Nov 2010 10:51:01 +0800
> From:    Axel Kurt Preuss <[hidden email]>
> Subject: Re: Basic live cell imaging question...
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> Joel, I totally agree.
>
> All what Gert needs is to avoid cell jitter or media perturbations. That s =
> best achieved by having an immersion objective or by placing a coverslid on=
> the cells with some space left for them to "breathe". In other words, not =
> get squished or dry up. Or, to grow them on a coverslip and flip that in on=
> e way or the other upside down
>
> There are three ways to achieve that
> 1) and 2) are leaving cells on their substrate and cover them with a covers=
> lip
> 1) by placing spacers on the slid and place that on the cell culture
> 2) by placing the lid on the objective and glue it (reversibly with some no=
> n damaging glue or wax if it has to be) on the coverslip and make sure the =
> edges of the slid are cemented in a way that they don't let media in
> 3)
> The third way is the upside down approach . In this way, cells are grown on=
> a coverslip and flipped upside down and best placed on some mold with enou=
> gh medium volume. If the molded carrier is glass he may not need Hoffman. I=
> think that s what Gert meant.
>
> You also  can buy a cheap small  perfusion chamber which you can place upsi=
> de down, (cells grown on coverslip are mounted into chamber, chamber gets f=
> lipped upside down, cells face downwards and their coverslip upwards toward=
> s objective, the whole chamber gets perfused and you can put it upside down=
> and with some luck you don't get air bubbles killing your cells).
>
> Colin needed to tell us whether he wants to stimulate the cells or not, and=
> whether the BX has a water objective.
>
> Thanks, Cheers
> Best Regards
> Axel  cell  +65 9271.5622
> ------------------
>  "We focus on your objectives!"  -Axel K Preuss PhD,  Central Imaging @IMC=
> B,  6-19B,  Singapore 138673
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On=
> Behalf Of JOEL B. SHEFFIELD
> Sent: Thursday, November 04, 2010 9:40 AM
> To: [hidden email]
> Subject: Re: Basic live cell imaging question...
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I think Gert's response makes a lot sense.  For a newbie, with limited goal=
> s
> (initially), it is not necessary to have an elaborate setup.  In the (very)
> old days, there was something called a "hanging drop culture" in which
> tissue fragments were placed in a drop of medium on a slide, the slide was
> inverted and examined with a conventional microscope.  Gert is suggesting a
> modern version of the same, assuming that you would visualize the cells
> through the cover slip to which they are attached, Ujnfortunately, the
> original writer did not specify the kind of optics they intended to use. If
> the cells are, indeed, attached to the cover slip, it is possible to use
> properly adjusted phase or Hoffman optics in transmissionto increase the
> contrast.  If fluorescence is needed, then you have to be concerned with
> light toxicity, etc., but the optics should work fine, as long as you don't
> want to see nuclear speckles, or details of mitochondria.
>
> Joel
>
>
> On Wed, Nov 3, 2010 at 9:03 PM, Axel Kurt Preuss <
> [hidden email]> wrote:
>
>> You need a water immersion object or have to build one
>>
>>
>>  Cheers
>>
>> Axel
>> -----
>> Axel K Preuss, PhD,
>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,
>> sent from 9271.5622
>>
>>
>> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>> *****
>>>
>>> Culture your cells on a round coverslip. Take an object glas glue a
>>> square piece of non-toxic rubber with a round hole on it. Fill this wit=
> h
>>> CO2 satured medium somewaht more as the volume of the hole. Put your
>>> coverslip on it, with the cells to the medium off course. Press it
>>> gently down and the glass will seal itself to the rubber ring. Now your
>>> cells will survive for a couple of hours, so you can do the first
>>> imaging. For real experiments you have to find a way to heat the object
>>> glass to 37C.
>>>
>>> Good luck, Gert
>>>
>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>> *****
>>>>
>>>> We would like to do live cell imaging - mammalian cell lines - but onl=
> y
>> have direct access to an upright Olympus BX61. We don't really need
>> complicated perfusion chambers, etc just something simple. We're real
>> neophytes so all suggestions gratefully received.
>>>>
>>>> Colin
>>>>
>>
>> Note: This message may contain confidential information. If this Email/Fa=
> x
>> has been sent to you by mistake, please notify the sender and delete it
>> immediately. Thank you.
>>
>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
>
> Note: This message may contain confidential information. If this Email/Fax =
> has been sent to you by mistake, please notify the sender and delete it imm=
> ediately. Thank you.
>
> ------------------------------
>
> End of CONFOCALMICROSCOPY Digest - 2 Nov 2010 to 3 Nov 2010 (#2010-61)
> **********************************************************************