Plant preparation for confocal microscopy

[Rosemary.White]

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*****

Hi Christian,

There are a few papers out there with FPs in the vacuole lumen. Some have
said the vacuole is too acidic for good FP fluorescence, but we've seen it
reasonably often - GFP, YFP and CFP but not RFP (haven't looked). The
vacuolar location in each case above was unwanted! - thought to be
incorrect targeting.

A couple of published examples:
Di Sansebastiano, G.-P., Paris, N., Marc-Martin, S., and Neuhaus, J.-M.
(2001). Regeneration of a lytic central vacuole and of neutral peripheral
vacuoles can be visualised by GFP targeted to either type of vacuoles.
Plant Physiol, 126, 78-86
Flückiger, R., De Caroli, M., Piro, G., Dalessandro, G., Neuhaus, J. M.,
and Di Sansebastiano, G. P. (2003). Vacuolar system distribution in
Arabidopsis tissues, visualized using GFP fusion proteins. Journal of
Experimental Botany 54, 1577-1584


These guys have an explanation, but the problem with this and similar
papers is assuming all plant vacuoles are the same - they are not, and
different cell types will also accumulate vacuolar FPs differently:
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi,
S.-i., Watanabe, M., Nishimura, M. and Hara-Nishimura, I. (2003), Why
green fluorescent fusion proteins have not been observed in the vacuoles
of higher plants. The Plant Journal, 35: 545­555


cheers,
Rosemary

On 27/01/12 4:32 AM, "Christian" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>As we're on plants...  has anyone imaged GFP or RFP in the vacuole of
>plants?  Tonoplast is quite routine, but what about the lumen of the
>vacuole?  I have a user who believes there is a cleavage event which
>should lead to the build up of GFP inside the vacuole, my concern was
>degradation of the signal due to pH issues.
>
>Does anyone have any experience with imaging inside the vacuole of
>plants?  This will be in tobacco until the stable Arabidopsis lines are
>available.
>
>Thanks
>
>Christian
>

[Rosemary.White]

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

Hi Monique et al.,

Chlorophyll is easily imaged with 633 excitation and you can detect very
small pre-chloroplasts this way - e.g. Spencer et al. 2005 Protoplasma
225:185-190 (yes, I'm a co-author...). The reason 633 is used is because
you need very little excitation to get a signal, the chlorophyll doesn't
bleach at this wavelength, and there is little overlap with FP emission -
in my experience, at least. You can even separate this from propidium
iodide emission if you want to image cell walls as well.

488 nm EX works, of course, but can bleach the chlorophyll, producing
damaged chloroplasts.  My interpretation, which could be wrong, is that
when the chlorophyll is bleached or overloaded, the energy isn't
transferred properly to the chlorophyll at the centre of the photosystem
so instead of mostly far-red chlorophyll autofluorescence you also get
fluorescence from the accessory pigments (carotenoids, etc), which shows
up as green-yellow emission.

Sequential scanning is great in many cases, except if your fluorescent
targets of interest are moving with streaming cytoplasm! Have separated
CFP, YFP, GFP and chlorophyll in the one image by collecting CFP plus GFP
in one pass then YFP plus chl in the second pass in non-streaming
meristematic cells.

cheers,
Rosemary

On 27/01/12 4:13 AM, "Christian" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Monique,
>
>Yes, I have worked in variegated leaves.
>
>As for imaging techniques, nearly all of my imaging is done with only a
>488 laser and a "GFP" and "CY5" emission.  The 488 excites the pigments
>in the chloroplasts just fine, and allows for simultaneous acquisition,
>which matters greatly in living cells which have cytoplasmic streaming.
>Using sequential imaging with the two laser lines maybe too slow.
>
>In the variegated section of the leaves, the proplastids, plastids, and
>chloroplasts can be maddening to image.  The reason again comes down to
>what pigments are there, and where in development those organelles are.
>Furthermore, how successful you are is often tied to how strong a
>promoter is being used.  Identifying YFP or GFP which is highly expressed
>is quite easy, but at low levels, autofluorescence becomes a real
>problem.  I have actually been urging labs to switch to constructs using
>RFP or mCherry when possible.  It is a very "clean" channel to image in
>for plants.
>
>Please contact me off-list if you'd like more details, or would like to
>share images.
>
>Christian
>
>
>
>--- On Thu, 1/26/12, Vasseur Monique <[hidden email]> wrote:
>
>From: Vasseur Monique <[hidden email]>
>Subject: Re: Plant preparation for confocal microscopy
>To: [hidden email]
>Date: Thursday, January 26, 2012, 10:28 AM
>
>Christian,
>
>Do you have experience with variegated leaves?
>Also, as control of the chlorophylls, some people use 458 nm as
>excitation, others 633 nm. What would you suggest the best?  Thanks again.
>
>Monique Vasseur
>tél. [phone] poste 5148
>
>-----Message d'origine-----
>De : Confocal Microscopy List [mailto:[hidden email]]
>De la part de Christian
>Envoyé : 26 janvier 2012 11:06
>À : [hidden email]
>Objet : Re: Plant preparation for confocal microscopy
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>*****
>
>Guy,
>
>I will have to disagree with you on the etiolated plant material.  Large,
>healthy chloroplasts have very strong autofluorescence in the "far red"
>channel, and much smaller peak in the "GFP" range.  When plants are
>stressed, not only do they not produce normal chlorophylls, but they can
>also produce an entire gambit of other pigments.  These pigments may
>throw the autofluorescence curves all over the place making
>identification of GFP or YFP much more difficult.  The same thing occurs
>in leaves which are stressed or experiencing hypersensitive responses to
>transformation.
>
>The best bet is to keep the plants healthy as possible.
>
>Christian
>
>
>--- On Wed, 1/25/12, Guy Cox <[hidden email]> wrote:
>
>From: Guy Cox <[hidden email]>
>Subject: Re: Plant preparation for confocal microscopy
>To: [hidden email]
>Date: Wednesday, January 25, 2012, 10:50 PM
>
>What I do with leaves is to peel off one or other epidermis.  This
>enables you to flood the leaf and you can put both the peeled epidermis
>and the remaining leaf on a slide and image without air problems.
>Chloroplast fluorescence is going to be a problem but if you can work
>with etiolated leaves that might help.
>
>                                  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 [phone]     Fax [phone]
>             Mobile [phone]
>______________________________________________
>      http://www.guycox.net
>
>
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Christian
>Sent: Thursday, 26 January 2012 3:18 AM
>To: [hidden email]
>Subject: Re: Plant preparation for confocal microscopy
>
>Localization to chloroplasts at low expression levels can be challe
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>
>*****
>
>
>
>Localization to chloroplasts at low expression levels can be challenging
>depending on your system and familiarity with dealing with sample with
>autofluorescence.  If you find you're having some difficulty, get back to
>us, and as a group, I'm quite sure we can talk you through that part.
>
>
>
>As for mounting samples, it depends on what plant you're dealing with.
>Zea mays, you would probably want to hand cut cross sections, mount in
>water under a coverslip.  Arabidopsis, you will probably mount with the
>"bottom" of the leaf towards the lens (are you upright or
>inverted?)  This will help avoid not only most of the trichomes which
>cause more air bubble issues and refraction, but the epidermal cells
>under them require deep z-series to see.  Of course that is if you're
>working stable lines, bombardment samples I usually view from the side
>which was bombarded.  Lastly, tobacco, if transiently expressing from
>Agrobacterium, is often infiltrated from the bottom of the leaf, so
>that¹s where I look, on the bottom.
>
>As for air bubblesŠ I do not fret the air bubbles at all, but this is
>mostly because I¹m NOT trying to collect the transmitted light image.  If
>you need it, then you might worry about them, and I highly recommend
>checking out perfluorodecalin.  I personally have not had luck with
>pulling a weak vacuum , actually, what I do is even more simplistic.
>
>I place the leaf sample (disks are too small to be worth the effort in my
>hands) with the side I¹d like to image up on a slide.  Add water, and
>place a 22x60mm cover slip over the sample, which is usually about
>22x22mm.  I then tap out the air bubbles I can see, and place the entire
>thing on a ³steel slide² and then place two small magnets on top of the
>cover slip to hold the works flat. If I avoid taking samples from area
>with large veins, I can do z-series as long as I¹d like without lateral
>shifting. The only major problem I will warn you about is that you¹re
>making your ³slide²
>much thicker and if you use an automated stage, this could be a real
>problem.  Secondly, and obviously, you will not get a transmitted light
>image. Of course this only works on an upright, but it has worked for at
>least four years.
>
>Good luck.
>
>Christian
>
>
>
>
>
>>Dear all,
>
>>
>
>>Do some of you could suggest how to prepare live sample of plant leaf
>>for
>
>>confocal microscopy.  We don't have much idea how to
>"sandwich" a leaf
>
>>between slide and coverslip so that it is flat et not too thick for
>
>>microscopy
>
>>(and without destroying it)? What should we use as mounting media? What
>
>>should we be aware of?  Our plant will have YFP in chloroplastides.
>
>>
>
>>Thanks a lot in advance to all of you.
>
>>

[Christian-103]

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

Rosemary,

I can believe this, but have not had the experience.  Of course most of my imaging is in Nicotiana epidermal cells.  I suspect working on mesophyll cells or using much longer dwell times might result in green shifting. 

I am actually going to give it a try next time I have a few moments.

Christian


488 nm EX works, of course, but can bleach the chlorophyll, producing
damaged chloroplasts.  My interpretation, which could be wrong, is that
when the chlorophyll is bleached or overloaded, the energy isn't
transferred properly to the chlorophyll at the centre of the photosystem
so instead of mostly far-red chlorophyll autofluorescence you also get
fluorescence from the accessory pigments (carotenoids, etc), which shows
up as green-yellow emission.

[Christian-103]

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

Rosemary,

Thank you for the references.  If I have any success with the RFP I will let you know.  I am hoping whichever flavor of vacuole is being targeted does not degrade the fluors.

Christian


--- On Thu, 1/26/12, Rosemary White <[hidden email]> wrote:

From: Rosemary White <[hidden email]>
Subject: Re: Plant vacuoles
To: [hidden email]
Date: Thursday, January 26, 2012, 4:02 PM

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

Hi Christian,

There are a few papers out there with FPs in the vacuole lumen. Some have
said the vacuole is too acidic for good FP fluorescence, but we've seen it
reasonably often - GFP, YFP and CFP but not RFP (haven't looked). The
vacuolar location in each case above was unwanted! - thought to be
incorrect targeting.

A couple of published examples:
Di Sansebastiano, G.-P., Paris, N., Marc-Martin, S., and Neuhaus, J.-M.
(2001). Regeneration of a lytic central vacuole and of neutral peripheral
vacuoles can be visualised by GFP targeted to either type of vacuoles.
Plant Physiol, 126, 78-86
Flückiger, R., De Caroli, M., Piro, G., Dalessandro, G., Neuhaus, J. M.,
and Di Sansebastiano, G. P. (2003). Vacuolar system distribution in
Arabidopsis tissues, visualized using GFP fusion proteins. Journal of
Experimental Botany 54, 1577-1584


These guys have an explanation, but the problem with this and similar
papers is assuming all plant vacuoles are the same - they are not, and
different cell types will also accumulate vacuolar FPs differently:
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi,
S.-i., Watanabe, M., Nishimura, M. and Hara-Nishimura, I. (2003), Why
green fluorescent fusion proteins have not been observed in the vacuoles
of higher plants. The Plant Journal, 35: 545­555


cheers,
Rosemary

On 27/01/12 4:32 AM, "Christian" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>As we're on plants...  has anyone imaged GFP or RFP in the vacuole of
>plants?  Tonoplast is quite routine, but what about the lumen of the
>vacuole?  I have a user who believes there is a cleavage event which
>should lead to the build up of GFP inside the vacuole, my concern was
>degradation of the signal due to pH issues.
>
>Does anyone have any experience with imaging inside the vacuole of
>plants?  This will be in tobacco until the stable Arabidopsis lines are
>available.
>
>Thanks
>
>Christian
>

[Monique Vasseur]

Hi Rosemary,

Is there a reason why you don't do your sequencial scan with the less energy wavelenght first (YFP plus chl)  and then CFP and GFP?  Thanks

Monique Vasseur
tél. [phone] poste 5148

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Rosemary White
Envoyé : 26 janvier 2012 18:26
À : [hidden email]
Objet : Re: Plant preparation for confocal microscopy

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

Hi Monique et al.,

Chlorophyll is easily imaged with 633 excitation and you can detect very small pre-chloroplasts this way - e.g. Spencer et al. 2005 Protoplasma
225:185-190 (yes, I'm a co-author...). The reason 633 is used is because you need very little excitation to get a signal, the chlorophyll doesn't bleach at this wavelength, and there is little overlap with FP emission - in my experience, at least. You can even separate this from propidium iodide emission if you want to image cell walls as well.

488 nm EX works, of course, but can bleach the chlorophyll, producing damaged chloroplasts.  My interpretation, which could be wrong, is that when the chlorophyll is bleached or overloaded, the energy isn't transferred properly to the chlorophyll at the centre of the photosystem so instead of mostly far-red chlorophyll autofluorescence you also get fluorescence from the accessory pigments (carotenoids, etc), which shows up as green-yellow emission.

Sequential scanning is great in many cases, except if your fluorescent targets of interest are moving with streaming cytoplasm! Have separated CFP, YFP, GFP and chlorophyll in the one image by collecting CFP plus GFP in one pass then YFP plus chl in the second pass in non-streaming meristematic cells.

cheers,
Rosemary

On 27/01/12 4:13 AM, "Christian" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Monique,
>
>Yes, I have worked in variegated leaves.
>
>As for imaging techniques, nearly all of my imaging is done with only a
>488 laser and a "GFP" and "CY5" emission.  The 488 excites the pigments
>in the chloroplasts just fine, and allows for simultaneous acquisition,
>which matters greatly in living cells which have cytoplasmic streaming.
>Using sequential imaging with the two laser lines maybe too slow.
>
>In the variegated section of the leaves, the proplastids, plastids, and
>chloroplasts can be maddening to image.  The reason again comes down to
>what pigments are there, and where in development those organelles are.
>Furthermore, how successful you are is often tied to how strong a
>promoter is being used.  Identifying YFP or GFP which is highly
>expressed is quite easy, but at low levels, autofluorescence becomes a
>real problem.  I have actually been urging labs to switch to constructs
>using RFP or mCherry when possible.  It is a very "clean" channel to
>image in for plants.
>
>Please contact me off-list if you'd like more details, or would like to
>share images.
>
>Christian
>
>
>
>--- On Thu, 1/26/12, Vasseur Monique <[hidden email]> wrote:
>
>From: Vasseur Monique <[hidden email]>
>Subject: Re: Plant preparation for confocal microscopy
>To: [hidden email]
>Date: Thursday, January 26, 2012, 10:28 AM
>
>Christian,
>
>Do you have experience with variegated leaves?
>Also, as control of the chlorophylls, some people use 458 nm as
>excitation, others 633 nm. What would you suggest the best?  Thanks again.
>
>Monique Vasseur
>tél. [phone] poste 5148
>
>-----Message d'origine-----
>De : Confocal Microscopy List [mailto:[hidden email]]
>De la part de Christian
>Envoyé : 26 janvier 2012 11:06
>À : [hidden email]
>Objet : Re: Plant preparation for confocal microscopy
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>*****
>
>Guy,
>
>I will have to disagree with you on the etiolated plant material.  
>Large, healthy chloroplasts have very strong autofluorescence in the "far red"
>channel, and much smaller peak in the "GFP" range.  When plants are
>stressed, not only do they not produce normal chlorophylls, but they
>can also produce an entire gambit of other pigments.  These pigments
>may throw the autofluorescence curves all over the place making
>identification of GFP or YFP much more difficult.  The same thing
>occurs in leaves which are stressed or experiencing hypersensitive
>responses to transformation.
>
>The best bet is to keep the plants healthy as possible.
>
>Christian
>
>
>--- On Wed, 1/25/12, Guy Cox <[hidden email]> wrote:
>
>From: Guy Cox <[hidden email]>
>Subject: Re: Plant preparation for confocal microscopy
>To: [hidden email]
>Date: Wednesday, January 25, 2012, 10:50 PM
>
>What I do with leaves is to peel off one or other epidermis.  This
>enables you to flood the leaf and you can put both the peeled epidermis
>and the remaining leaf on a slide and image without air problems.
>Chloroplast fluorescence is going to be a problem but if you can work
>with etiolated leaves that might help.
>
>                                  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 [phone]     Fax [phone]
>             Mobile [phone]
>______________________________________________
>      http://www.guycox.net
>
>
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]]
>On Behalf Of Christian
>Sent: Thursday, 26 January 2012 3:18 AM
>To: [hidden email]
>Subject: Re: Plant preparation for confocal microscopy
>
>Localization to chloroplasts at low expression levels can be challe
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>
>*****
>
>
>
>Localization to chloroplasts at low expression levels can be
>challenging depending on your system and familiarity with dealing with
>sample with autofluorescence.  If you find you're having some
>difficulty, get back to us, and as a group, I'm quite sure we can talk you through that part.
>
>
>
>As for mounting samples, it depends on what plant you're dealing with.
>Zea mays, you would probably want to hand cut cross sections, mount in
>water under a coverslip.  Arabidopsis, you will probably mount with the
>"bottom" of the leaf towards the lens (are you upright or
>inverted?)  This will help avoid not only most of the trichomes which
>cause more air bubble issues and refraction, but the epidermal cells
>under them require deep z-series to see.  Of course that is if you're
>working stable lines, bombardment samples I usually view from the side
>which was bombarded.  Lastly, tobacco, if transiently expressing from
>Agrobacterium, is often infiltrated from the bottom of the leaf, so
>that¹s where I look, on the bottom.
>
>As for air bubblesŠ I do not fret the air bubbles at all, but this is
>mostly because I¹m NOT trying to collect the transmitted light image.  
>If you need it, then you might worry about them, and I highly recommend
>checking out perfluorodecalin.  I personally have not had luck with
>pulling a weak vacuum , actually, what I do is even more simplistic.
>
>I place the leaf sample (disks are too small to be worth the effort in
>my
>hands) with the side I¹d like to image up on a slide.  Add water, and
>place a 22x60mm cover slip over the sample, which is usually about
>22x22mm.  I then tap out the air bubbles I can see, and place the
>entire thing on a ³steel slide² and then place two small magnets on top
>of the cover slip to hold the works flat. If I avoid taking samples
>from area with large veins, I can do z-series as long as I¹d like
>without lateral shifting. The only major problem I will warn you about
>is that you¹re making your ³slide² much thicker and if you use an
>automated stage, this could be a real problem.  Secondly, and
>obviously, you will not get a transmitted light image. Of course this
>only works on an upright, but it has worked for at least four years.
>
>Good luck.
>
>Christian
>
>
>
>
>
>>Dear all,
>
>>
>
>>Do some of you could suggest how to prepare live sample of plant leaf
>>for
>
>>confocal microscopy.  We don't have much idea how to
>"sandwich" a leaf
>
>>between slide and coverslip so that it is flat et not too thick for
>
>>microscopy
>
>>(and without destroying it)? What should we use as mounting media?
>>What
>
>>should we be aware of?  Our plant will have YFP in chloroplastides.
>
>>
>
>>Thanks a lot in advance to all of you.
>
>>