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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
> [log in to unmask]
> privatewww.essex.ac.uk/~plaissue <http://privatewww.essex.ac.uk/%7Eplaissue>
> 
> 
> On Mon, Mar 18, 2013 at 9:56 PM, jmkrupp jmkrupp <[log in to unmask]> 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
>>