Advice for confocal training

[Jonathan M Krupp]

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Greetings

Just a quick note to ask if anyone would like to give me some advice about
getting started with a confocal training program.

We do EM and LM already, thinking of adding LSCM. Any advice about must
includes,  good practice specimens , techniques etc?

How about insights into instrurments, how basic can we go?, what cost range
should we be thinking.

Thanks

Jon

[phil laissue]

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Hi there,

it really depends on the targeted users - needs differ much depending on
the place/lab. I tailor practical sessions usually to individual needs, but
have general lectures at the start. Basic lectures include: when to use a
confocal, dynamic range, histograms, saturation, pixels, LUTs + real
colour, basic filter settings, bleed-through; z-stack, zoom, Nyquist,
step/pinhole/image sizes, scan speed, laser power vs detector gain, SNR,
averaging/summing, considerations for experimental setup and live cell
imaging, sample quality control; PSF deconvolution, visualisation
(tiling/projections, scalebars, contrast) and preparing figures/movies for
presentation/publication. Basics in practicals include finding your cells
without crashing the lens (for automated XY stages), adjusting channels,
bleaching, taking a z-stack. We don't have too many users wanting advanced
techniques, so I keep information on TIRF/spectral/resonant/FRAP et
al./advanced image analysis options to a 'this is what it can do' level,
and get into details if they want to/need to use it.
For basic use, I hear that the LSM 700 does the job, but myself would
consider a Thorlab kit (although that again depends on the user basis) or
second-hand model (510, SP2, Radiance in good shape). I'm happy with our A1
workhorse.
Samples depend on the users in question, but transiently transfected cells
with varying intensities, strongly autofluorescent samples, diatoms, beads,
bulky whole mounts and poor slides (e.g. with air bubbles or poor signal)
all come in handy to make certain points...
I'm sure I forgot a few things, but hope this helps for starters.

Kind regards

Philippe

_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue <http://privatewww.essex.ac.uk/%7Eplaissue>


On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Greetings
>
> Just a quick note to ask if anyone would like to give me some advice about
> getting started with a confocal training program.
>
> We do EM and LM already, thinking of adding LSCM. Any advice about must
> includes,  good practice specimens , techniques etc?
>
> How about insights into instrurments, how basic can we go?, what cost range
> should we be thinking.
>
> Thanks
>
> Jon
>

[John Oreopoulos]

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Currently, there are two books I recommend as a starting point for new users to confocal microscopy for biological imaging applications:

http://www.amazon.ca/Confocal-Microscopy-Biologists-Alan-Hibbs/dp/0306484684
http://www.amazon.ca/Basic-Confocal-Microscopy-Robert-Price/dp/0387781749

(No commercial interest). Most, if not all questions that a new confocal user might have are answered in these books. I suppose one could develop a short training program or course that mirrors the concepts addressed in these book chapters. They also serve as excellent reference materials.

Also, I find the best way to really understand how to use an instrument like this is to open it up where you can (lasers off or safely controlled) and examine the innards of the instrument. When you understand the optical pathway, you understand how to best optimize the image acquisition for a given situation. I leave you with a quote from Dave Piston that echos this point:

"For many reasons, students today are not taught to be curious about things. They are driven too much by trying to get the results they expect, which makes it easier to get a paper published. Take microscopy as an example of the situation and the need for students to know the equipment they are using. Without question, a top level microscopist needs to know how a microscope works. If you are going to use a microscope to discover new things, you need to know how it works. Complicated (biological) systems and complicated processes require an observer with detailed knowledge of how everything works. If you don’t know how your equipment should work, your scientific understanding can be easily misled by artifacts. You must know if your equipment is working properly. We see it at the microscopy courses (where I co-chair and guest lecture): many students only want to learn how to USE one instrument (of the several available) because it is the one they have at home. Unfortunately, they are not interested in learning the technology and how it works but only the correct buttons to press to obtain results. They only want to learn how to DRIVE the microscope, not how the technology works. This is a shortcoming in the making because when you know how a microscope works, you can immediately see (and feel) it when the instrument is working well or not. Complicated things are very prone to artifacts. We can’t be experts in everything, but researchers should have a base knowledge and also know who the experts are. This is because when you know enough, you question how the data looks, and you will know who the expert is to approach with relevant questions. For example, a lot of researchers are looking at GFP and they don’t know Kohler illumination, or why their DIC looks so poor. Students notice that the images from others in their field look better, but are not curious enough to ask: “Why are my results not of the same quality?” A healthy cynicism of their data/results is lacking."

Words to live by in the microscopy field.

Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-03-18, at 8:39 PM, phil laissue wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi there,
>
> it really depends on the targeted users - needs differ much depending on
> the place/lab. I tailor practical sessions usually to individual needs, but
> have general lectures at the start. Basic lectures include: when to use a
> confocal, dynamic range, histograms, saturation, pixels, LUTs + real
> colour, basic filter settings, bleed-through; z-stack, zoom, Nyquist,
> step/pinhole/image sizes, scan speed, laser power vs detector gain, SNR,
> averaging/summing, considerations for experimental setup and live cell
> imaging, sample quality control; PSF deconvolution, visualisation
> (tiling/projections, scalebars, contrast) and preparing figures/movies for
> presentation/publication. Basics in practicals include finding your cells
> without crashing the lens (for automated XY stages), adjusting channels,
> bleaching, taking a z-stack. We don't have too many users wanting advanced
> techniques, so I keep information on TIRF/spectral/resonant/FRAP et
> al./advanced image analysis options to a 'this is what it can do' level,
> and get into details if they want to/need to use it.
> For basic use, I hear that the LSM 700 does the job, but myself would
> consider a Thorlab kit (although that again depends on the user basis) or
> second-hand model (510, SP2, Radiance in good shape). I'm happy with our A1
> workhorse.
> Samples depend on the users in question, but transiently transfected cells
> with varying intensities, strongly autofluorescent samples, diatoms, beads,
> bulky whole mounts and poor slides (e.g. with air bubbles or poor signal)
> all come in handy to make certain points...
> I'm sure I forgot a few things, but hope this helps for starters.
>
> Kind regards
>
> Philippe
>
> _____________________________________
> Philippe Laissue, PhD, Bioimaging Manager
> School of Biological Sciences, Room 4.17
> University of Essex, Colchester CO4 3SQ, UK
> (0044) 01206 872246 / (0044) 07842 676 456
> [hidden email]
> privatewww.essex.ac.uk/~plaissue <http://privatewww.essex.ac.uk/%7Eplaissue>
>
>
> On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Greetings
>>
>> Just a quick note to ask if anyone would like to give me some advice about
>> getting started with a confocal training program.
>>
>> We do EM and LM already, thinking of adding LSCM. Any advice about must
>> includes,  good practice specimens , techniques etc?
>>
>> How about insights into instrurments, how basic can we go?, what cost range
>> should we be thinking.
>>
>> Thanks
>>
>> Jon
>>

[Arvydas Matiukas]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Philippe,

I am impressed by the content and scope of your general lectures.
I wonder how many hours they take. I wish I  could implement
something similar, however, I am 99% positive that biomedical grad
students that are typical users will get bored to death. On the
other side, without a  proper prerequisite of math/physics/optics
users woudn't be able to in depth grasp the basic microscopy
elements.

Regarding Jon's question I would like to suggest to use Zeiss or
Leica's confocal training presentations (just google them).
They contain a well balanced mixture of theory/practise/applications, and
take about 3-4  hrs to cover.
Regarding training cost, basic trainingin my Core  is below $100 ( the policy is
to atract a new customer without scaring him/her with high price).

Best regards,
Arvydas
---------------------------------



Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Imaging Core Facility
Department of Pharmacology
SUNY Upstate Medical University
766 Irving Ave., WH 3159
Syracuse, NY 13210
tel.: [phone]
fax: [phone]
email: [hidden email]

>>> phil laissue  03/18/13 8:40 PM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi there,

it really depends on the targeted users - needs differ much depending on
the place/lab. I tailor practical sessions usually to individual needs, but
have general lectures at the start. Basic lectures include: when to use a
confocal, dynamic range, histograms, saturation, pixels, LUTs + real
colour, basic filter settings, bleed-through; z-stack, zoom, Nyquist,
step/pinhole/image sizes, scan speed, laser power vs detector gain, SNR,
averaging/summing, considerations for experimental setup and live cell
imaging, sample quality control; PSF deconvolution, visualisation
(tiling/projections, scalebars, contrast) and preparing figures/movies for
presentation/publication. Basics in practicals include finding your cells
without crashing the lens (for automated XY stages), adjusting channels,
bleaching, taking a z-stack. We don't have too many users wanting advanced
techniques, so I keep information on TIRF/spectral/resonant/FRAP et
al./advanced image analysis options to a 'this is what it can do' level,
and get into details if they want to/need to use it.
For basic use, I hear that the LSM 700 does the job, but myself would
consider a Thorlab kit (although that again depends on the user basis) or
second-hand model (510, SP2, Radiance in good shape). I'm happy with our A1
workhorse.
Samples depend on the users in question, but transiently transfected cells
with varying intensities, strongly autofluorescent samples, diatoms, beads,
bulky whole mounts and poor slides (e.g. with air bubbles or poor signal)
all come in handy to make certain points...
I'm sure I forgot a few things, but hope this helps for starters.

Kind regards

Philippe

_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue


On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp  wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Greetings
>
> Just a quick note to ask if anyone would like to give me some advice about
> getting started with a confocal training program.
>
> We do EM and LM already, thinking of adding LSCM. Any advice about must
> includes,  good practice specimens , techniques etc?
>
> How about insights into instrurments, how basic can we go?, what cost range
> should we be thinking.
>
> Thanks
>
> Jon
>

[Stanislav Vitha-2]

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To join, leave or search the confocal microscopy listserv, go to:
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*****

As far as what confocal to get, it is hard to give you any specific advice. It is
like buying a car. It depends what your users need.
I like them all, each of the big four has their strong points and offers a range
of models. Some of the lower-end models are upgradeable, some are not so
easily. And, as was mentioned in this thread, there are other options, like the
Thor Labs kit, and few others if you are looking for a single-point scanning
confocal system. Spinning disk/micromirror instruments are yet another group.

 
We have a dual approach for training.

There is a graduate - level course where students learn the theory of image
formation and a range of light microscopy techniques, and receive practical
training on a standard light microscope (BF, DIC, phase contrast,
fluorescence) as well as on a confocal microscope.

For users that just want to learn how to use the confocal microscope I do the
training (2 people max) in two sessions, ~2 hours each.
We start with general and lab safety, biosafety rules (we are BL-1), each new
user is required by our university to have passed an online laser safety
training.

I have a short guide for users to download, it also includes startup/shutdown
procedures.

In the first session we cover the operation of the microscope, what do those
buttons do, setting up Kohler illumination, using standard epifluorescence to
check the sample, and basic functions in the software: scan size, speed,
setting the detector voltage and offset for maximum contrast and to avoid
saturation, choosing fluorescent dyes from the dye database, confocal zoom
and setting the pixel size for Nyquist sampling, sequential scanning to minimize
spectral cross-talk in multi-labeled samples. We also cover how to deal with
weak signals and noise by changing scanning speed, Kalman filtering, opening
the pinhole.
Usually in the first session we only use the 10x and 20x dry objectives and a
standard teaching sample (I use pollen grains).
We also cover the file formats, the importance of saving the original data files,
adding a scale bar, exporting the images for a Powerpoint resentation.

I like to demonstrate the effect of the DIC prism on the confocal image
resolution and signal brightness.


In the second session we cover the use of objectives with coverglass
thickness correction collar (dry 40x and water immersion 60x), using oil
immersion objectives, and general rules how to clean up after using the
immersion optics. Z-stacks - setting the z-step size to Nyquist, adjusting the
laser power or detector voltage to compensate for loss of signal  with depth.
Time lapse imaging.
Towards the end of the second session user get to look at their own
specimen, if they have one, and get help with optimizing the imaging
parameters for their sample.

What exactly is covered depends on each user's need. Some people will never
use a water immersion objective, some people only look at thick live samples  
and will not use the oil immersion lenses.

I inform the users about the more advanced features, like the photon counting
mode, bi-directional scanning, line-scan and point-scan, FRAP, FRET and
acceptor photobleaching, using a SIM scanner (it is an Olympus FV1000) for
photobleaching/photoactivavtion, Multi-area Time lapse (uses the motorized
stage), assembling complex imaging sequences ("macro"), Raster Image
Correlation Spectroscopy, ... but for 99% of the users this would be more than
they need at the moment and it does not make sense to train them for this.
     
After the two training sessions the users should be able to use the confocal
microscope more or less independently, with some occasional help. They can
use the instrument during normal operating hours (~9am-5PM).  After one or
more sessions, when they are comfortable in using the microscope and do not
need any more help, they let me know and we go through a short practical
test, where they show me they can indeed do the imaging without
endangering themselves or the microscope.  After the successful practical test
they become fully qualified users, they get access to the online scheduler and
get a key to the microscope, so they can use it any time.

Even after the training is completed I still like to check on my users, ask how it
goes if they have any issues with their images, and dispense some unsolicited
advice, e.g. that using Alexa488 and Cy3 for dual labeling and colocalization
analysis is not the best idea, there are better dye combinations.
 
Overall this training scheme works well, we have had minimal problem with
users doing bad things to our microscope. Sometimes I train the students and
then they realize that they use the microscope so infrequently that they are
better off just letting me do the imaging once or twice a year, rather than
trying to re-learn what they have forgotten since the training.
 
With Regards,


Stan Vitha

Microscopy and Imaging Center
Texas A&M University
http://microscopy.tamu.edu

On Mon, 18 Mar 2013 14:56:50 -0700, jmkrupp jmkrupp <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Greetings
>
>Just a quick note to ask if anyone would like to give me some advice about
>getting started with a confocal training program.
>
>We do EM and LM already, thinking of adding LSCM. Any advice about must
>includes,  good practice specimens , techniques etc?
>
>How about insights into instrurments, how basic can we go?, what cost range
>should we be thinking.
>
>Thanks
>
>Jon

[phil laissue-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Hi Arvydas,

thanks for the feedback. I run four lectures (basics, dynamic range,
colour, 3D & time-lapse) of 1.5 to 2 hours each (depending on questions) at
the start of each term. I also have a three-day course each year with four
1.5h lectures followed by practicals. The third day is used entirely for
samples the users have prepared. A strong point of the lectures is the
practical aspect. I talk about the focal planes, lazy oil, and teach them a
standard procedure to find their cells. This sounds very pedestrian, but
I've seen often how PhDs spend half their time at the 'scope looking for
their cells. We've also had two crashed stages (but luckily no objectives
were hurt) in the first half year of having the new instruments, and the
new way avoids that. It would be good to have parking sensors on stages. I
still have to figure out if it can be done software-wise with NIS-Elements.
I fully agree that many users could possibly care less, but not by a very
large margin. I run the courses with interested students in mind, but also
have the lectures on a training website, so they can be downloaded and
studied in advance of a practical session. Printouts and crib sheets
complement that. What does help with motivation is to make students
understand that bioimaging is a hot area and increasingly desired on CVs.
And so I end up with about one third interested, good, careful users, one
third routine (but responsible) users, and one third of intermittent (and
rather bored) users.
David Piston, and John, are of course right. Rigorously speaking, you can
probably only truly understand (and appreciate) a system if you've built
one yourself. But multi-user facilities with all members having such
insight and skills must be extremely rare. For many, it's just an
instrument they want to use. So to some degree, I have the lectures for
myself. Just showing the users which button to push is not fun. However, in
defense of the students, it seems that the pressure and lack of
understanding for the complexity of the matter (or the time it requires to
do it properly) comes in some cases from the group leader. So another point
I put forward is that it only makes sense to embark on a bioimaging project
if they're willing, ready and able to put in the time; sample optimisation,
proper image acquisition, and image analysis being aspects that can swallow
a lot of time. But there's no 'quick & dirty' way to do it really, unless
all they want is a pretty picture for a publication. So the minimum time
charged for confocal training is 10h. Alison North's article (Seeing is
believing? A beginners' guide to practical pitfalls in image acquisition)
really helps to drive that point home as well. And when a user gets the
imaging bug, all that effort seems worth while...

Best regards

Philippe


_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue


On Tue, Mar 19, 2013 at 1:23 AM, Arvydas Matiukas <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Philippe,
>
> I am impressed by the content and scope of your general lectures.
> I wonder how many hours they take. I wish I  could implement
> something similar, however, I am 99% positive that biomedical grad
> students that are typical users will get bored to death. On the
> other side, without a  proper prerequisite of math/physics/optics
> users woudn't be able to in depth grasp the basic microscopy
> elements.
>
> Regarding Jon's question I would like to suggest to use Zeiss or
> Leica's confocal training presentations (just google them).
> They contain a well balanced mixture of theory/practise/applications, and
> take about 3-4  hrs to cover.
> Regarding training cost, basic trainingin my Core  is below $100 ( the
> policy is
> to atract a new customer without scaring him/her with high price).
>
> Best regards,
> Arvydas
> ---------------------------------
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Imaging Core Facility
> Department of Pharmacology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3159
> Syracuse, NY 13210
> tel.: [phone]
> fax: [phone]
> email: [hidden email]
>
> >>> phil laissue  03/18/13 8:40 PM >>>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi there,
>
> it really depends on the targeted users - needs differ much depending on
> the place/lab. I tailor practical sessions usually to individual needs, but
> have general lectures at the start. Basic lectures include: when to use a
> confocal, dynamic range, histograms, saturation, pixels, LUTs + real
> colour, basic filter settings, bleed-through; z-stack, zoom, Nyquist,
> step/pinhole/image sizes, scan speed, laser power vs detector gain, SNR,
> averaging/summing, considerations for experimental setup and live cell
> imaging, sample quality control; PSF deconvolution, visualisation
> (tiling/projections, scalebars, contrast) and preparing figures/movies for
> presentation/publication. Basics in practicals include finding your cells
> without crashing the lens (for automated XY stages), adjusting channels,
> bleaching, taking a z-stack. We don't have too many users wanting advanced
> techniques, so I keep information on TIRF/spectral/resonant/FRAP et
> al./advanced image analysis options to a 'this is what it can do' level,
> and get into details if they want to/need to use it.
> For basic use, I hear that the LSM 700 does the job, but myself would
> consider a Thorlab kit (although that again depends on the user basis) or
> second-hand model (510, SP2, Radiance in good shape). I'm happy with our A1
> workhorse.
> Samples depend on the users in question, but transiently transfected cells
> with varying intensities, strongly autofluorescent samples, diatoms, beads,
> bulky whole mounts and poor slides (e.g. with air bubbles or poor signal)
> all come in handy to make certain points...
> I'm sure I forgot a few things, but hope this helps for starters.
>
> Kind regards
>
> Philippe
>
> _____________________________________
> Philippe Laissue, PhD, Bioimaging Manager
> School of Biological Sciences, Room 4.17
> University of Essex, Colchester CO4 3SQ, UK
> (0044) 01206 872246 / (0044) 07842 676 456
> [hidden email]
> privatewww.essex.ac.uk/~plaissue
>
>
> On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp  wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Greetings
> >
> > Just a quick note to ask if anyone would like to give me some advice
> about
> > getting started with a confocal training program.
> >
> > We do EM and LM already, thinking of adding LSCM. Any advice about must
> > includes,  good practice specimens , techniques etc?
> >
> > How about insights into instrurments, how basic can we go?, what cost
> range
> > should we be thinking.
> >
> > Thanks
> >
> > Jon
> >
>

[Arvydas Matiukas]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Philippe,
 
thanks for the details how you train your users. My experience
is very similar to yours  that a new user requires about 10hrs of
training/work with confocal before he/she can start productively
work on their imaging project independently. On the other
hand about 3hrs of  practical training (i.e showing which buttons
to push and which icons to click) is fairly enough to prevent
users from breaking the equipment. Most of my users assume
that this is enough of me wasting their time and typically
prefer as a first choice to ask their previously trained coworkers which buttons
to push. I kind of understand their quick pace which (as you wrote) often is imposed
by a lab leader requesting quick results. Moreover, usually sample preparation
takes much longertime than imaging and still does not guarantee good picture.
 
 
Unfortunately, until now our campus did not have theoretical
course on bioimaging/microscopy but I am working hard to change
this. It that respect your experience is very useful.
 
Finally, I wonder how it is possible to crash a stage (never saw or heard
anything of such kind). Our inverted Axiovert 200M has a button to set the top
limit of objective advancement which protects from crashing lens. However,
the caveat is if somebody pushes that button at wrong z-position (while objective
is outside working distance) then it is not possible to focus without restarting
the microscope power.
 
Best,
Arvydas


>>> phil laissue <[hidden email]> 3/19/2013 12:14 PM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Arvydas,

thanks for the feedback. I run four lectures (basics, dynamic range,
colour, 3D & time-lapse) of 1.5 to 2 hours each (depending on questions) at
the start of each term. I also have a three-day course each year with four
1.5h lectures followed by practicals. The third day is used entirely for
samples the users have prepared. A strong point of the lectures is the
practical aspect. I talk about the focal planes, lazy oil, and teach them a
standard procedure to find their cells. This sounds very pedestrian, but
I've seen often how PhDs spend half their time at the 'scope looking for
their cells. We've also had two crashed stages (but luckily no objectives
were hurt) in the first half year of having the new instruments, and the
new way avoids that. It would be good to have parking sensors on stages. I
still have to figure out if it can be done software-wise with NIS-Elements.
I fully agree that many users could possibly care less, but not by a very
large margin. I run the courses with interested students in mind, but also
have the lectures on a training website, so they can be downloaded and
studied in advance of a practical session. Printouts and crib sheets
complement that. What does help with motivation is to make students
understand that bioimaging is a hot area and increasingly desired on CVs.
And so I end up with about one third interested, good, careful users, one
third routine (but responsible) users, and one third of intermittent (and
rather bored) users.
David Piston, and John, are of course right. Rigorously speaking, you can
probably only truly understand (and appreciate) a system if you've built
one yourself. But multi-user facilities with all members having such
insight and skills must be extremely rare. For many, it's just an
instrument they want to use. So to some degree, I have the lectures for
myself. Just showing the users which button to push is not fun. However, in
defense of the students, it seems that the pressure and lack of
understanding for the complexity of the matter (or the time it requires to
do it properly) comes in some cases from the group leader. So another point
I put forward is that it only makes sense to embark on a bioimaging project
if they're willing, ready and able to put in the time; sample optimisation,
proper image acquisition, and image analysis being aspects that can swallow
a lot of time. But there's no 'quick & dirty' way to do it really, unless
all they want is a pretty picture for a publication. So the minimum time
charged for confocal training is 10h. Alison North's article (Seeing is
believing? A beginners' guide to practical pitfalls in image acquisition)
really helps to drive that point home as well. And when a user gets the
imaging bug, all that effort seems worth while...

Best regards

Philippe


_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue


On Tue, Mar 19, 2013 at 1:23 AM, Arvydas Matiukas <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Philippe,
>
> I am impressed by the content and scope of your general lectures.
> I wonder how many hours they take. I wish I  could implement
> something similar, however, I am 99% positive that biomedical grad
> students that are typical users will get bored to death. On the
> other side, without a  proper prerequisite of math/physics/optics
> users woudn't be able to in depth grasp the basic microscopy
> elements.
>
> Regarding Jon's question I would like to suggest to use Zeiss or
> Leica's confocal training presentations (just google them).
> They contain a well balanced mixture of theory/practise/applications, and
> take about 3-4  hrs to cover.
> Regarding training cost, basic trainingin my Core  is below $100 ( the
> policy is
> to atract a new customer without scaring him/her with high price).
>
> Best regards,
> Arvydas
> ---------------------------------
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Imaging Core Facility
> Department of Pharmacology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3159
> Syracuse, NY 13210
> tel.: [phone]
> fax: [phone]
> email: [hidden email]
>
> >>> phil laissue  03/18/13 8:40 PM >>>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi there,
>
> it really depends on the targeted users - needs differ much depending on
> the place/lab. I tailor practical sessions usually to individual needs, but
> have general lectures at the start. Basic lectures include: when to use a
> confocal, dynamic range, histograms, saturation, pixels, LUTs + real
> colour, basic filter settings, bleed-through; z-stack, zoom, Nyquist,
> step/pinhole/image sizes, scan speed, laser power vs detector gain, SNR,
> averaging/summing, considerations for experimental setup and live cell
> imaging, sample quality control; PSF deconvolution, visualisation
> (tiling/projections, scalebars, contrast) and preparing figures/movies for
> presentation/publication. Basics in practicals include finding your cells
> without crashing the lens (for automated XY stages), adjusting channels,
> bleaching, taking a z-stack. We don't have too many users wanting advanced
> techniques, so I keep information on TIRF/spectral/resonant/FRAP et
> al./advanced image analysis options to a 'this is what it can do' level,
> and get into details if they want to/need to use it.
> For basic use, I hear that the LSM 700 does the job, but myself would
> consider a Thorlab kit (although that again depends on the user basis) or
> second-hand model (510, SP2, Radiance in good shape). I'm happy with our A1
> workhorse.
> Samples depend on the users in question, but transiently transfected cells
> with varying intensities, strongly autofluorescent samples, diatoms, beads,
> bulky whole mounts and poor slides (e.g. with air bubbles or poor signal)
> all come in handy to make certain points...
> I'm sure I forgot a few things, but hope this helps for starters.
>
> Kind regards
>
> Philippe
>
> _____________________________________
> Philippe Laissue, PhD, Bioimaging Manager
> School of Biological Sciences, Room 4.17
> University of Essex, Colchester CO4 3SQ, UK
> (0044) 01206 872246 / (0044) 07842 676 456
> [hidden email]
> privatewww.essex.ac.uk/~plaissue
>
>
> On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp  wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Greetings
> >
> > Just a quick note to ask if anyone would like to give me some advice
> about
> > getting started with a confocal training program.
> >
> > We do EM and LM already, thinking of adding LSCM. Any advice about must
> > includes,  good practice specimens , techniques etc?
> >
> > How about insights into instrurments, how basic can we go?, what cost
> range
> > should we be thinking.
> >
> > Thanks
> >
> > Jon
> >
>

[Sarah S. Subaran]

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

On the Zeiss LSM510 Meta, the stage crashes when you hold down the load
position for more than 2 seconds.


On Tue, Mar 19, 2013 at 4:56 PM, Arvydas Matiukas <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Philippe,
>
> thanks for the details how you train your users. My experience
> is very similar to yours  that a new user requires about 10hrs of
> training/work with confocal before he/she can start productively
> work on their imaging project independently. On the other
> hand about 3hrs of  practical training (i.e showing which buttons
> to push and which icons to click) is fairly enough to prevent
> users from breaking the equipment. Most of my users assume
> that this is enough of me wasting their time and typically
> prefer as a first choice to ask their previously trained coworkers which
> buttons
> to push. I kind of understand their quick pace which (as you wrote) often
> is imposed
> by a lab leader requesting quick results. Moreover, usually sample
> preparation
> takes much longertime than imaging and still does not guarantee good
> picture.
>
>
> Unfortunately, until now our campus did not have theoretical
> course on bioimaging/microscopy but I am working hard to change
> this. It that respect your experience is very useful.
>
> Finally, I wonder how it is possible to crash a stage (never saw or heard
> anything of such kind). Our inverted Axiovert 200M has a button to set the
> top
> limit of objective advancement which protects from crashing lens. However,
> the caveat is if somebody pushes that button at wrong z-position (while
> objective
> is outside working distance) then it is not possible to focus without
> restarting
> the microscope power.
>
> Best,
> Arvydas
>
>
> >>> phil laissue <[hidden email]> 3/19/2013 12:14 PM >>>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Arvydas,
>
> thanks for the feedback. I run four lectures (basics, dynamic range,
> colour, 3D & time-lapse) of 1.5 to 2 hours each (depending on questions) at
> the start of each term. I also have a three-day course each year with four
> 1.5h lectures followed by practicals. The third day is used entirely for
> samples the users have prepared. A strong point of the lectures is the
> practical aspect. I talk about the focal planes, lazy oil, and teach them a
> standard procedure to find their cells. This sounds very pedestrian, but
> I've seen often how PhDs spend half their time at the 'scope looking for
> their cells. We've also had two crashed stages (but luckily no objectives
> were hurt) in the first half year of having the new instruments, and the
> new way avoids that. It would be good to have parking sensors on stages. I
> still have to figure out if it can be done software-wise with NIS-Elements.
> I fully agree that many users could possibly care less, but not by a very
> large margin. I run the courses with interested students in mind, but also
> have the lectures on a training website, so they can be downloaded and
> studied in advance of a practical session. Printouts and crib sheets
> complement that. What does help with motivation is to make students
> understand that bioimaging is a hot area and increasingly desired on CVs.
> And so I end up with about one third interested, good, careful users, one
> third routine (but responsible) users, and one third of intermittent (and
> rather bored) users.
> David Piston, and John, are of course right. Rigorously speaking, you can
> probably only truly understand (and appreciate) a system if you've built
> one yourself. But multi-user facilities with all members having such
> insight and skills must be extremely rare. For many, it's just an
> instrument they want to use. So to some degree, I have the lectures for
> myself. Just showing the users which button to push is not fun. However, in
> defense of the students, it seems that the pressure and lack of
> understanding for the complexity of the matter (or the time it requires to
> do it properly) comes in some cases from the group leader. So another point
> I put forward is that it only makes sense to embark on a bioimaging project
> if they're willing, ready and able to put in the time; sample optimisation,
> proper image acquisition, and image analysis being aspects that can swallow
> a lot of time. But there's no 'quick & dirty' way to do it really, unless
> all they want is a pretty picture for a publication. So the minimum time
> charged for confocal training is 10h. Alison North's article (Seeing is
> believing? A beginners' guide to practical pitfalls in image acquisition)
> really helps to drive that point home as well. And when a user gets the
> imaging bug, all that effort seems worth while...
>
> Best regards
>
> Philippe
>
>
> _____________________________________
> Philippe Laissue, PhD, Bioimaging Manager
> School of Biological Sciences, Room 4.17
> University of Essex, Colchester CO4 3SQ, UK
> (0044) 01206 872246 / (0044) 07842 676 456
> [hidden email]
> privatewww.essex.ac.uk/~plaissue
>
>
> On Tue, Mar 19, 2013 at 1:23 AM, Arvydas Matiukas <[hidden email]
> >wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Philippe,
> >
> > I am impressed by the content and scope of your general lectures.
> > I wonder how many hours they take. I wish I  could implement
> > something similar, however, I am 99% positive that biomedical grad
> > students that are typical users will get bored to death. On the
> > other side, without a  proper prerequisite of math/physics/optics
> > users woudn't be able to in depth grasp the basic microscopy
> > elements.
> >
> > Regarding Jon's question I would like to suggest to use Zeiss or
> > Leica's confocal training presentations (just google them).
> > They contain a well balanced mixture of theory/practise/applications, and
> > take about 3-4  hrs to cover.
> > Regarding training cost, basic trainingin my Core  is below $100 ( the
> > policy is
> > to atract a new customer without scaring him/her with high price).
> >
> > Best regards,
> > Arvydas
> > ---------------------------------
> >
> >
> >
> > Arvydas Matiukas, Ph.D.
> > Director of Confocal&Two-Photon Imaging Core Facility
> > Department of Pharmacology
> > SUNY Upstate Medical University
> > 766 Irving Ave., WH 3159
> > Syracuse, NY 13210
> > tel.: [phone]
> > fax: [phone]
> > email: [hidden email]
> >
> > >>> phil laissue  03/18/13 8:40 PM >>>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi there,
> >
> > it really depends on the targeted users - needs differ much depending on
> > the place/lab. I tailor practical sessions usually to individual needs,
> but
> > have general lectures at the start. Basic lectures include: when to use a
> > confocal, dynamic range, histograms, saturation, pixels, LUTs + real
> > colour, basic filter settings, bleed-through; z-stack, zoom, Nyquist,
> > step/pinhole/image sizes, scan speed, laser power vs detector gain, SNR,
> > averaging/summing, considerations for experimental setup and live cell
> > imaging, sample quality control; PSF deconvolution, visualisation
> > (tiling/projections, scalebars, contrast) and preparing figures/movies
> for
> > presentation/publication. Basics in practicals include finding your cells
> > without crashing the lens (for automated XY stages), adjusting channels,
> > bleaching, taking a z-stack. We don't have too many users wanting
> advanced
> > techniques, so I keep information on TIRF/spectral/resonant/FRAP et
> > al./advanced image analysis options to a 'this is what it can do' level,
> > and get into details if they want to/need to use it.
> > For basic use, I hear that the LSM 700 does the job, but myself would
> > consider a Thorlab kit (although that again depends on the user basis) or
> > second-hand model (510, SP2, Radiance in good shape). I'm happy with our
> A1
> > workhorse.
> > Samples depend on the users in question, but transiently transfected
> cells
> > with varying intensities, strongly autofluorescent samples, diatoms,
> beads,
> > bulky whole mounts and poor slides (e.g. with air bubbles or poor signal)
> > all come in handy to make certain points...
> > I'm sure I forgot a few things, but hope this helps for starters.
> >
> > Kind regards
> >
> > Philippe
> >
> > _____________________________________
> > Philippe Laissue, PhD, Bioimaging Manager
> > School of Biological Sciences, Room 4.17
> > University of Essex, Colchester CO4 3SQ, UK
> > (0044) 01206 872246 / (0044) 07842 676 456
> > [hidden email]
> > privatewww.essex.ac.uk/~plaissue
> >
> >
> > On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp  wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Greetings
> > >
> > > Just a quick note to ask if anyone would like to give me some advice
> > about
> > > getting started with a confocal training program.
> > >
> > > We do EM and LM already, thinking of adding LSCM. Any advice about must
> > > includes,  good practice specimens , techniques etc?
> > >
> > > How about insights into instrurments, how basic can we go?, what cost
> > range
> > > should we be thinking.
> > >
> > > Thanks
> > >
> > > Jon
> > >
> >
>

*

*

[Christian-103]

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

In the next few weeks, I have a user who will need FRET analysis performed in Arabidopsis intact leaves.  He is using the new Clover/Ruby constructs which express very well in the plant material. 

The problem quite bluntly, is that I have not had any FRET training in 10 years except a brief webinar, which was advertised on this list-serv.  In any case, I work on a Nikon A1 with the FRET package in NIS-Elements, but also have a spectral detector.  If anyone would like to offer tips here or off list, I'd be very appreciative.  Any tutorials would be very beneficial as well. 

Thank you!

Christian