Just my two cents:
In most cases, the electronic document is now the publication of record, so
arguments about contrast of paper, etc. have been replaced about arguments
about compression, color mapping, etc. Same general problem, different
tech.
IMHO, Ken Yamada and Mike Rossner have done the best job of delineating what
is reasonable in image data presentation in a publication. See
http://jcb.rupress.org/cgi/content/full/166/1/11. Note most other journals
now have similar guidelines. See for example,
http://www.nature.com/authors/editorial_policies/image.html.
Ideally, data would be published alongisde a publication, and held in a
public repository. One step towards this is the JCB DataViewer:
http://jcb-dataviewer.rupress.org/. DECLARED CONFLICT:
2009/6/23 Guy Cox <[log in to unmask]>
> The same is true of digital enlargement and reduction. In fact there are
> considerable differences between different algorithms for enlarging and
> reducing. (See my chapter in Jim Pawley's Handbook). But the fact is no
> paper journal can reproduce your image pixel for pixel, and the printing
> process will have a very different gamma from your screen, so what's the
> issue? If you print on grade 3 paper instead of grade 1 you will have very
> different intensity values in your image. So should we insist on grade 2
> (so-called normal)? But the contrast difference between a cold-cathode
> enlarger (very low), a condenser enlarger (high) and a point-source enlarger
> (very high) are equally substantial.
>
> Numerical measurements should be made from unmodified images. But the
> printed image is by definition modified, so our aim should be to get it to
> look like what we see.
>
> Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Jeremy Adler
> Sent: Tuesday, 23 June 2009 6:22 PM
> To: [log in to unmask]
> Subject: Re: Question about larger issue
>
> Digital rotation, unless by an integer multiple of 90 degrees, does differ
> from rotating film. In a digital rotation there is not an exact mapping
> between the original and rotated pixels and 'new' intensities are created
> using the some combination of the original neighbouring pixels.
>
>
> Dr Jeremy Adler
>
> F451a
>
> Cell Biologi
>
> Wenner-Gren Inst.
>
> The Arhenius Lab
>
> Stockholm University
>
> S-106 91 Stockholm
>
> Sweden
>
> tel +46 (0)8 16 2759
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Guy Cox
> Sent: den 23 juni 2009 09:13
> To: [log in to unmask]
> Subject: Re: Question about larger issue
>
> What I nowadays put in the Materials and Methods section is something along
> the lines of "contrast adjustment and image scaling are not specifically
> noted but other manipulations are mentioned in the figure captions."
>
> The rationale as I see it is that in the days of photography one always
> adjusted contrast by grade of paper, and set the enlarger to give the size,
> and maybe rotation, you needed. So doing these things digitally is no
> different.
>
> A median filter, on the other hand (which I often use) is not part of
> standard expectations and so should be noted. (I did use to do unsharp
> masking photographically, but I would always mention that whether done
> digitally or photographically).
>
> One interesting point which isn't often noted is that if one scans a
> photographic image (which even now I quite often have to do with EM
> pictures) the scan software will by default use a sharpening filter without
> telling you. I turn it off (if I remember) but I wonder how many others
> even think of it?
>
> Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit,
> Madsen Building F09, University of Sydney, NSW 2006
> ______________________________________________
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Johan Henriksson
> Sent: Tuesday, 23 June 2009 4:49 PM
> To: [log in to unmask]
> Subject: Re: Question about larger issue
>
> Robert Peterson wrote:
> > Hi,
> >
> > First, I would like to thank Johan for the response. He was correct
> > that in cases where hardware is the issue, doing post-capture
> > processing
> would not help.
> > For example, when our stage and stage insert were not well aligned we
> > had different focal depths across the sample. We would see something
> > like an axon running from right to left across 4 or 5 tiles where in
> > the right hand side of each tile it was in focus, but then it would
> > dip out of focus by the left hand side of that tile. Then, it would
> > pop back into focus at the right-hand edge of the next tile.
> >
> > On the other hand, I like the idea of the range adjustment for a
> > shading problem I have been having on a different system.
> >
> > However, Johan's suggestion brought up something I had been wanting to
> > ask for quite some time. How much post-capture image manipulation do
> > you
> think is okay?
> > I know there have been threads about this before, but I was wondering
> > specifically what people do and do not suggest to their
> users/students/postdocs.
> > In particular, I'm wondering what kind of processing you find yourself
> > doing fairly often. I have a feeling I err on the conservative side
> > and I am definitely willing to broaden my range.
> >
> my opinion, being a phd student but specializing on image processing:
>
> always think of the purpose of the image.
> * is it to show the quality of a new type of optics? minimal corrections
>
> * is it to show some biological features? any manipulation is ok.
> but 1. always keep raw data 2. always state exactly what you did.
> if a paper only says "image processed with photoshop" then it should go
> straight back for revision.
> ultimately I think software should have the ability to store down how
> things are done so it easily can be repeated like others, like this:
> http://endrov.net/images/a/ac/Flowwindow.png
> there is no doubt what has been done or how to redo it. no one can accuse
> you of research falsification if you're open about what you are doing.
>
> manipulation is easy to miss. e.g. storing your data as TIFF will remove
> valuable information about the recording conditions. it also cannot be
> avoided, writing an image on paper will resample it and change the colors.
>
> I'm also doubting people's success with #1. I have seen people store images
> on USB sticks only, with almost random names, who then have had trouble
> finding the images when I've asked for them.
> there are image server solutions but only discipline helps in the end.
>
> /Johan
> > Thanks in advance!
> >
> > Robert
> >
> >
> > Johan Henriksson wrote:
> > > this problem can be trivially fixed by software, just apply a range
> > > adjustment bi-linearly interpolated over the image. you can find the
> > > optimal parameters using linear least squares or manually. but
> > > before that, make sure the hardware setup is optimal. had this
> > > problem on a simpler microscope, in that case due to a bad
> > > connection making the light come in at a slight angle.
> > >
> > > /Johan
> > >
> > >> Robert Peterson wrote:
> > >>
> > >>
> > >>> Hi,
> > >>>
> > >>> We do a lot of tiling on multiple systems. When there is not a
> > >>> flat field of illumination you get a checkerboard effect that
> > >>> ruins your images. So, I have spent a fair amount of time trying
> > >>> to achieve a flat field illumination. With the Zeiss there are a
> couple things I would suggest.
> > >>>
> > >>> 1. As was mentioned below there is always an edge effect and we
> have been
> > >>> told to zoom to at least 2 with our Zeiss objectives to
> "cut-off" this
> > >>> edge effect.
> > >>> 2. The stage alignment and stage insert alignment have to be
> perfect. The
> > >>> Zeiss service tech in our area uses a slide with a grid
> > >>> along
> the entire
> > >>> length to make sure that it is in the same focal plane at
> > >>> all
> points. Or,
> > >>> as near to it as possible.
> > >>>
> > >>> Don't know if this will be helpful or not, but wanted to chime in
> > >>> with some personal experiences.
> > >>>
> > >>> Thanks,
> > >>> Robert Peterson
> > >>>
> > >>>
> > >>> James Pawley wrote:
> > >>>
> > >>>
> > >>>>> I have recently been trying to determine how flat the
> > >>>>> illumination field is in our spinning disk system to make sure
> > >>>>> things are "fairly well" aligned, but have had some strange
> > >>>>> results that I do not understand. I would like some feedback on
> > >>>>> what is acceptable, as I am new to these type of measurements.
> > >>>>> I started by using a fluor-ref slide from microscopy education,
> which I assume is a good diagnostic slide for such an operation.
> > >>>>> I will also mention that I was using a Zeiss C-apochromat
> > >>>>> 40x/1.2 water immersion lens with an adjustable collar. With the
> > >>>>> fluor-ref slide image, the field looks very flat, with only
> > >>>>> about a 3% intensity drop on the edges (determined via a line
> > >>>>> profile across the image). However, if I take a molecular
> > >>>>> probes bead and move it to different areas of the field, I get a
> > >>>>> very different answer. I find that I have a relatively large
> > >>>>> area (~20% of the total field) on the bottom left corner that
> > >>>>> registers a 22% drop in intensity via measuring the bead. I
> > >>>>> focused up and down to make sure I was at the brightest point of
> > >>>>> the bead, in case the focus had changed in a different area of
> > >>>>> the field, but that did not seem to make a difference. I have
> > >>>>> two questions I would be interested in getting feedback on. 1)
> > >>>>> What is the reason for the difference between the flou-ref slide
> > >>>>> and the beads? and
> > >>>>> 2) What kinds of percent changes in intensity over the field are
> > >>>>> considered acceptable?
> > >>>>> Thanks,
> > >>>>>
> > >>>>>
> > >>>>> Sean Speese, Ph.D.
> > >>>>> UMASS Medical School
> > >>>>> Department of Neurobiology
> > >>>>>
> > >>>>>
> > >>>> Dear Sean,
> > >>>>
> > >>>> The fact that the intensity loss seems much worse in one corner
> > >>>> than in the other three does suggest an alignment issue. I am
> > >>>> only guessing but it might be that, in that corner, the laser
> > >>>> light is not being as accurately focused into the pinholes by the
> > >>>> microlenses. (But I have no idea how to adjust this.)
> > >>>>
> > >>>> However, one should always expect some drop off in the corners
> > >>>> for a variety of reasons.
> > >>>>
> > >>>> Aberrations and field curvature, that are corrected almost
> > >>>> perfectly on axis, are usually not corrected so well off the
> > >>>> axis. The result is a larger spot, and therefor a lower intensity
> > >>>> of excitation illumination. The higher aberrations also reduce
> > >>>> optical performance on the fluorescence side, making the larger
> > >>>> spot larger again, and causing more of it to be intercepted by
> > >>>> the pinhole. This will be more obvious with the beads than with a
> > >>>> bulk specimen where, to some extent, light originating nearby
> > >>>> will
> still make it to the image plane. With the beads, there is no fluorescent
> material to make "nearby"
> > >>>> light.
> > >>>>
> > >>>> Finally, large low-mag, high-NA lenses often suffer from
> > >>>> vignetting. This can be seen clearly in Fig 11.9 on page 246 of
> > >>>> the Handbook. The lower row of images represent performance in
> > >>>> the back focal plane of 4 objectives, with a point light source
> > >>>> that images 10mm off axis at the primary image plane. You can see
> > >>>> that the BFP in not fully filled (i.e., not circular, as the top
> > >>>> row is). The black area represents light that was not collected,
> > >>>> in
> this case probably 25% of the total for the NA 1.2 water lens.
> > >>>>
> > >>>> This last point fits in with the recent discussion on the "Re:
> > >>>> Recommendations for commercial multi-photon system purchase"
> > >>>> thread about an NA 1.2 20x objective. If there really isn't room
> > >>>> in the old RMS objective mount for all the light paths from the
> > >>>> edges of the field of view of an NA 1.2 40x, then the matter will
> > >>>> be 2x worse with a 20x. Therefore, one will need a much larger
> diameter tube and tube lens to capture all the data from such an objective.
> > >>>>
> > >>>> Originally such high-NA, low-mag objectives were not contemplated
> > >>>> because they were harder to make and besides one could not see
> > >>>> this much information with the unaided eye. Now that we decode
> > >>>> position information from either CCDs or mirror position, this is
> > >>>> no longer a limitation and, partially for this reason, we have
> recently seen the introduction a variety of "non-standard"
> > >>>> microscope configurations such as the the Agilent/TILL and the
> > >>>> new Olympus box scopes.
> > >>>>
> > >>>> The brave new world awaits.
> > >>>>
> > >>>> Cheers,
> > >>>>
> > >>>> Jim P.
> > >>>>
> > >>>> PS: We still have 3 open places at the UBC 3D Live-cells Course,
> > >>>> http://www.3dcourse.ubc.ca/
> > >>>>
> > >>>>
> > >>>
> > >>>
> > >>>
> > >>
> > >>
> > >
> > >
> > >
> >
>
>
> --
> --
> ------------------------------------------------
> Johan Henriksson
> MSc Engineering
> PhD student, Karolinska Institutet
> http://mahogny.areta.org http://www.endrov.net
>
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