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Amscope...

Posted by Steve Sorrell  
avatar Re: Amscope...
March 27, 2012 11:06PM
    
Owen,

I forgot to mention that I have never known of anyone who thinks that they get better resolution from the camera than from the good old eyeball. To be fair, when comparing the view through the eyepiece with that of a captured image, it must be compared at the same scale. This is not easy to do, as the eyepiece view is measured in apparent angle of view, which means that the digital image must be compared at a distance where it has that same apparent angle of view.

Gene
avatar Re: Amscope...
March 28, 2012 03:23AM
Hi Gene,

Mineralogical Research Company Wrote:
-------------------------------------------------------
> Hi Owen,
>
> By calculation, at 62X, you should have a
> horizontal field of view of ~3.6mm on an APS-C
> sensor, if your relay lens is indeed 1X. If you
> measure 0.75mm, then something is definitely
> wrong.

Can we look at that for a moment? I'm thinking that the circular FOV is ~ 3.6mm and that's what my measurements seem to show, roughly supporting the manufacturer's spec. If that is so then only about one third of the FOV area (pi * (3.6^2)) is focussed onto the APS-C sensor? This is an important foundation to where I think I need to be going - so I had better have it right! smiling smiley

> I am assuming that there are no other
> optics between the relay lens and the sensor.

That's what I'm told (and think I can see). But the setup is delivering ~ x5 of 'empty' magnification. of which about sqrt 2 'mag' is accounted for by the cropping of the FOV falling on the sensor.It further seems that if I opt to utilise only the central square 3456x3456 pixels of the sensor, then a x0.5 relay lens should just about maximise the amount of the scope's FOV that can be captured on the 3456 pixel square. Make sense?

> Have you tired removing the relay lens and
> projecting directly onto the sensor? Depending
> upon the microscope optics, the relay lens may be
> necessary to correct field curvature, or even
> chromatic
> aberration. This is because, in some microscopes,
> the final correction is done in the eyepieces.

I'm not equipped to drop the lens out. I don't believe that with this pod (a MZS 1065T, which is closely similar in design and build to the more common MZS 1045 (T)) that correction for field curvature and chromatic aberration at the final stage (eye-piece/relay lens) is a requirement.

> I look at the aspect ratio issue from a different
> perspective and always run at sensor’s native
> aspect ratio. My reasoning is this. Many
> objectives will fully illuminate an APS-C sensor
> (see attachment). So, the notion of best
> matching to a circular field doesn’t enter into
> my thinking. It is the circular field that is
> actually cropped by the sensor. I want to capture
> all of the information that is possible and then
> crop out what I don’t want later.

Agreed. However, isnt photomigrography a special case, in that the MP count of modern cameras is way ahead of anything that the resolving power of an optical microscope can utilise? From memory, 5 MP is about the optimum effective pixel count for photomicrography, above which one simple gets over-sampling. Which is why USD **** digital cameras purpose-designed for optical photomicrography (which no DSLR is) so often have a surprisingly low pixel count and yet can produce marvelous photomicrographic images?

The 600D's sensor is 18 MP. If I choose to work with a square format that is reduces to ~12MP (useable), even then I have a margin in hand (and still some image over-sampling).

> especially important in stacking because of the
> spatial drift of each image in the stack with
> respect to the last. These artifacts have to be
> cropped out after processing, which further
> reduces effective sensor area.

One thing at a time Gene... one thing at a time. First I need to optimise the resolution of the recorded images. Then I'll turn to other issues smiling smiley For the while, I just experiment with some simple stacks (and early results are encouraging). Since my interest is in gem crystals, my stacks are mainly confined to just a few millimetres of Z axis. But one thing at a time. Until I've got the resolution is maximised, all else will be building on sand.

So, returning to microscope resolution (especially in Greenough stereo zoom designs) do you have any micrometer images that show the resolution capabilities of one or more other makes/models of microscope - or know of a web link where such can be found? I'd like to be as well armed as possible before beating up on the adapter manufacturer to rectify the unit sold to me.

Similarly, if anyone would like my complete set of micrometer images, to make comparisons of their own I'd be very happy to supply.

Owen
avatar Re: Amscope...
March 28, 2012 05:23AM
Gene,

Our exchange is out of synch - but we'll manage. Your after thought is a good one (but I certainly don't look for *better* resolution in camera over eye - just, near as dammit, as good.

To the rest, can we come at it from a different direction?

From your diagram, in a properly set up rig, the scope's diametric FOV is the same length as the as the distance between diametrically opposed corners of a rectangular full frame sensor and some mathematically-determinable greater length than the slant range across an APS-C sensor. It follows that if, at any handily chosen mag setting for the scope, the full 1.00 mm of the stage micrometer covers the same proportion of the slant range across a full frame sensor as it does of the scope's diametric FOV then the two observations, eyeball and camera are identically scaled. To do the same with an APS-C sensor one simply has to apply a fixed factor to compensate for the differently sized sensor area.

So what one wants from the image transfer from scope to camera is an arrangement that, for known dimensions of a sensor plate will, at any scope's magnification setting, cause a known length (say 1mm) to represent the same fraction of the slant range across the sensor as it does across the microscope's field of view.

So what's the compensation factor to be applied in reducing the microscope image to fit optimally to a APS-C sensor? If one opts to utilise only the centre square of the sensor plate, then the slant angle to which the micrometer scale must be aligned for testing is 45 degrees to the horizontal. And what one wants, it seems to me, from one's camera adapter (beyond a sturdy physical mounting for the camera) is an arrangement whereby the microscope's FOV is reduced according to the following formulae:

X = 2*L/3 where X is the side of the square on the sensor to be utilised and L is the length of the APS-C sensor

then:

R = X/cosine 45 where R is the slant length across the square.

So, if you can tell me the dimensions of an APS-C sensor in millimeters we'll have a working basis on which I can go away, make a fresh set of observations (at a 45 deg slant) and re-calculate the misalignment of my present set up from the ideal. It's been a long night here but not a wasted one for me I think. A clear step forward which would not have been taken (at least as quickly) had you not chipped in, Gene.

Thanks,

Owen (now edited to correct 'schoolboy' maths error smiling smiley



Edited 1 time(s). Last edit at 03/28/2012 12:45PM by Owen Lewis (2).
avatar Re: Amscope...
March 28, 2012 10:13PM
    
Hi Owen,

Strangely, various manufacturer's APS-C sensors are not all the same size. Canon's APS-C sensors are 22.2mm X 14.8mm.

The diagram that I included in my last post was just to illustrate the relative geometry of sensors superimposed on the Microscope's FOV on the image plane. The relative sizes are not necessarily correct for a given objective. Some objectives will more than cover a full frame sensor and some will not. Most will, fully illuminate an APS-C sensor.

My thinking is to forget what you see through the eyepiece. The eyepiece only covers a portion of the actual FOV at the image plane (or sensor). Further, we don't use the eyepiece for photography, so why consider it?

My goal is to get as much of the subject of interest onto the sensor. That optimizes the use of sensor area. So as not to confuse matters even more, I won't mention Nyquist sampling now. So, what is the scale factor for the situation above? Lets take a look at your microscope. I believe that it has a range of 10X to 62X when using 10X eyepieces. So, the zoom objective by itself has a range of 1X - 6.2X. For example, using the highest magnification of 6.2X, we divide the horizontal dimension of the sensor by 6.2. So, 22.2mm / 6.2 ~ 3.6mm is the field width. That is, the image plane (or sensor), will see 3.6mm of the object plane. Of course, if your objective lens cannot fully illuminate the sensor, then the above is not applicable.

I'll stop here to see if this makes sense, or if I should lay off of the Margaritias.

Just for fun, and to keep in touch with the spirit of these discussions, I have added a recent Apatite image from my new setup. The field of view is approximately 1 mm wide. There is still plenty of room for improvement, but for now this is my best effort at this image scale.

Gene
Attachments:
open | download - Apatite 02a_proc.jpg (396.2 KB)
avatar Re: Amscope...
March 29, 2012 08:54PM
    
Hi Owen,

There was so much in your previous post that I am addressing it point by point, as I have time.

Quote
Owen
Agreed. However, isnt photomigrography a special case, in that the MP count of modern cameras is way ahead of anything that the resolving power of an optical microscope can utilise? From memory, 5 MP is about the optimum effective pixel count for photomicrography, above which one simple gets over-sampling. Which is why USD **** digital cameras purpose-designed for optical photomicrography (which no DSLR is) so often have a surprisingly low pixel count and yet can produce marvelous photomicrographic images?

Actually, for a given objective, it is the size of the pixels that determines Nyquist sampling rate, and not the total number of pixels in the sensor. Think of it this way. If your smallest resolvable feature, on the image plane, is X um in extent, then you would need at least 3 pixels to cover the feature for critical sampling, so each pixel would need to be X um / 3 in size . Because of the Bayer filter, it takes ~3 pixels, rather than 2, to satisfy the Nyquist sampling theorem.

Example: You have an objective that is 5X and has a resolution of 3 um. On the image plane, the smallest feature of 3 um would be 3 um X 5 = 15 um in extent. Therefore, a pixel size of 15 um / 3 = 5 um would give the lowest sampling rate for maximum resolution on the image. The Canon 18MP APS-C sensor has 4.3 um X 4.3 um pixels, a sampling rate of ~3.5, so is just about ideal. An eyepiece camera with 14MP has 1.2 um pixels and for this example would have a sampling rate of ~12, which is far from ideal. On the other hand, a 1.3MP eyepiece camera has 3.6 um pixels and has a closer to ideal sampling rate. With the assumed objective, this would do very well, but at a reduced field of view due to the small number of pixels.

So, if the pixel size is set for best sampling, then the number of pixels just determines the amount of FOV captured. A small sensor and a large sensor, at the same sampling rate will have the same resolution, but the fields covered will be different. The limit on the maximum number of useful pixels then is a function of your objectives usable coverage at the image plane.

One last comment. If you consider one of the high quality objectives like a 4X Plan Apo with NA=0.2 and resolution of 1.6 um, the sampling rate using the Canon APS-C is 1.56, clearly under sampled. The camera is limited in this case. To get the highest resolution images, the objective must be properly matched to the objective lens.

I'll need another Margarita before I think about the rest of your post. :)

Gene
avatar Re: Amscope...
March 29, 2012 09:53PM
Hi Gene,

I spent some time yesterday reading up on sensors. Cutting a long story short, my experimentation of last weekend may well have been 'moon-beam chasing' and needs to be done again from the beginning. The bull points seem to be these:

- The crispness obtained in a captured image depends on only on the resolution obtained by the microscope and delivered (as undiminished as possible) to the object plane but also on the algorithmic processing of the small electrical signals created in the sensor's pixels. I may well be inadvertently degrading the resolution obtainable by over-processing of the sensor data through selecting to employ routines such as noise reduction.

- I need to get some definitive answers re. the match/mismatch of my camera adapter's lens view to the Canon APS-C sensor plate dimensions. This with a view to optimising the match if necessary and possible. The images I made on Sunday actually don't help with this, though it was a useful little self-training exercise and providing a reference set, against which similar images can be assessed for any improvement in resolution.

So, before spending more camera-time in creating new images concerned with obtaining the best resolution, I think I need first to correct or confirm my present understanding of what is the optimum match between the 'lens view' and image captured on the Canon APS-C (non-standard size) sensor plate. Which brings me to responding to your thought provoking last post.

Mineralogical Research Company Wrote:
-------------------------------------------------------
> Hi Owen,
>
> Strangely, various manufacturer's APS-C sensors
> are not all the same size. Canon's APS-C sensors
> are 22.2mm X 14.8mm.

That's what I read too. Whether those are the physical dimensions of the plate or the dimensions of the actual pixel area I can't find. Anyway, apart from noting that this may account for some minor level of discrepancy between theoretical calculations and practical observations, one can let it go for now.

> The diagram that I included in my last post was
> just to illustrate the relative geometry of
> sensors superimposed on the Microscope's FOV on
> the image plane. The relative sizes are not
> necessarily correct for a given objective. Some
> objectives will more than cover a full frame
> sensor and some will not. Most will, fully
> illuminate an APS-C sensor.

I understand. But the camera manufacturer designs his lenses and bodies to get consistently close to a best matching of lens view to a full frame sensor's dimensions. In the case of some 'Brand X' microscope optics being used instead of one of his own nice lens units, then, as you say, what the optical train in the 'scope presents to the camera's sensor may or may not be well fitted to the camera manufacturer's 'lens view''. If there is a gross mismatch in coverage, the selection and use of an appropriate relay lens in the camea adapter might well improve the match. Does that seem fair and correct - thus far?

Turning then to a Canon camera using the APS-C sensor. This is substantially smaller than a full frame sensor plate. This in turn has the effect of cropping the 'lens view'. Canon publishes this 'crop factor'/focal length multiplier/empty magnification (all three terms referring to the one same effect) as 1.6. I take this to mean that, taking the corner to corner diametric distance of an APS-C sensor as being 1, on that same scale, the diameter of the lens view will be 1.6. In other words the lens view captured on the sensor plate is cropped linearly by about 1/3rd as a result of the physical dimensions of the sensor plate. That seem correct to you?

> My thinking is to forget what you see through the
> eyepiece. The eyepiece only covers a portion of
> the actual FOV at the image plane (or sensor).
> Further, we don't use the eyepiece for
> photography, so why consider it?

It's important (to me) only that, in a perfect world, I would want my camera to record as closely as possible the image I see through an eyepiece. This is a wish only. My intent is to find out how close to that I can get with the instruments I have.


> My goal is to get as much of the subject of
> interest onto the sensor. That optimizes the use
> of sensor area.

Ditto to the first part.

To my thinking, the second introduces a conflict of interest. Let's agree for a moment that potential image quality is maximised by utilising the entire sensor plate (rectangular area). But more of a circular field of view can be captured on a square snug-fitted to the circle circumference than can be captured in a rectangle of any other dimensions. So why don't camera manufacturers use square sensor plates? Two reasons I'd suggest:

- The 'letter box' has a smaller area, therefore fewer pixels and therefore saves them money.
- A large majority of the public prefer to frame their pictures as rectangles (there are good aesthetic reasons for this). Accordingly, providing them with a square (or even circular) sensor area that they would only choose to crop away anyway could not make economic sense.

However *if* one allows that modern pixel counts are actually not useful for photomicrography (pace 'stacking' concerns) because of the resolution limitations on objective lenses, one comes to my line of thought which is to:

- Utilise only the central square part of the APS-C sensor (the design of the camera firmware permits this). This effectively reduces the useful pixel count from 18 MP to 12 MP (still more than is necessary for photomicrography) but puts in place the basis for a maximum capture of the 'lens view'.

- Employ a reducing lens as the relay lens in the camera adapter to match as closely as possible the lens view focussed on the now square format-cropped objective plane and thus capture as much as possible of what the microscope objective gathered in the first place. Look Mum, no more enforced crop factor smiling smiley

> So as not to confuse matters even
> more, I won't mention Nyquist sampling now.

Yes, lets leave Messrs Nyquist and Shannon sleeping between the covers for now.

> Lets take a look at your microscope. I believe
> that it has a range of 10X to 62X when using 10X
> eyepieces. So, the zoom objective by itself has a
> range of 1X - 6.2X. For example, using the
> highest magnification of 6.2X, we divide the
> horizontal dimension of the sensor by 6.2. So,
> 22.2mm / 6.2 ~ 3.6mm is the field width. That is,
> the image plane (or sensor), will see 3.6mm of
> the object plane.

That is true for my eyepiece view (checked by means of a stage micrometer slide and an eyepiece graticule with a prepared graticule calibration table for x0.5 steps in zoom setting).

My understanding is that it *should* also be approximately true for the image plane on the sensor - except for the manufacturer's designed in 1.6 crop factor where the sensor is an APS-C. Are we together on this - or am I still missing a trick?

> Of course, if your objective
> lens cannot fully illuminate the sensor, then the
> above is not applicable.
>
> I'll stop here to see if this makes sense, or if I
> should lay off of the Margaritias.

In theory at least, my camera seems to provide the inverse situation. in which the object plane image over spills the sensor area in the diameter ratio of 1.6:1. What it does in practice (my tests of last Sunday now being in the trash can) remains to be verified, once I've finished getting the fundamentals fully sorted out with your good self. My thanks again for bearing patiently with this old man

> Just for fun, and to keep in touch with the spirit
> of these discussions, I have added a recent
> Apatite image from my new setup. The field of
> view is approximately 1 mm wide. There is still
> plenty of room for improvement, but for now this
> is my best effort at this image scale.

That's a lovely picture, Gene, with some interesting stacking effects. You are demonstrably getting better resolution that I can obtain for the moment. More experimentation from me (real soon now) once the basics are sorted. I'll first switch off all the optional processing of pixel information and see what improvement (if any) that alone can deliver. Once that question mark is disposed of, a return to chasing after the resolving capabilities of my scope can be resumed. And along the way I'll be establishing in *practice* what the true crop factor for my current setup is.

Best,
Owen



Edited 1 time(s). Last edit at 03/30/2012 12:41PM by Owen Lewis (2).
avatar Re: Amscope...
March 29, 2012 10:57PM
    
Owen,

I think you missed the second of my two consecutive posts that suggests that most modern DSLRs do not have too many pixels for photomicroscopy, and in fact can produce under sampled images.

Cheers,
Gene
avatar Re: Amscope...
March 30, 2012 03:53PM
Gene,

My last mail had just been dispatched before I saw yours, to which I now respond. Sorry. No discourtesy by seemingly ignoring it was intended. It was simply unfortunate timing.

Anyway, I respond to it now (by ducking winking smiley ) See below.

Mineralogical Research Company Wrote:
-------------------------------------------------------
> Hi Owen,
>
> There was so much in your previous post that I am
> addressing it point by point, as I have time.
>
>
> Agreed. However, isnt photomigrography a special
> case, in that the MP count of modern cameras is
> way ahead of anything that the resolving power of
> an optical microscope can utilise? From memory, 5
> MP is about the optimum effective pixel count for
> photomicrography, above which one simple gets
> over-sampling. Which is why USD **** digital
> cameras purpose-designed for optical
> photomicrography (which no DSLR is) so often have
> a surprisingly low pixel count and yet can produce
> marvelous photomicrographic images?
>
>
> Actually, for a given objective, it is the size of
> the pixels that determines Nyquist sampling rate,
> and not the total number of pixels in the sensor.
> Think of it this way. If your smallest resolvable
> feature, on the image plane, is X um in extent,
> then you would need at least 3 pixels to cover
> the feature for critical sampling, so each pixel
> would need to be X um / 3 in size . Because of
> the Bayer filter, it takes ~3 pixels, rather than
> 2, to satisfy the Nyquist sampling theorem.

I have managed to start so many hares running that if I chase after them all at once I'm likely to bag none of them.

The position you set out cogentlyis not fully agreed by all in every situation. There is at least one senior academic (Prof, working in optical physics) who has net-published an explanation as to why high megapixel cameras must over-sample in (some) photomicrographic applications.

This would be an interesting matter to pursue to a conclusion at some future point - and perhaps we shall - but it is not necessary for me to progress with my immediate concerns.

So, for the purposes of our present discussion, that of my testing soon to follow and of reporting thereon, let me drop entirely my 'square is better than rectangle' proposition so we can proceed on a mutually agreed playing field, that of the best fit of lens view to a full Canon APS-C pixel count. For the purposes of this thread, all my further testing and reporting with be on that basis.

> Example: You have an objective that is 5X and has
> a resolution of 3 um. On the image plane, the
> smallest feature of 3 um would be 3 um X 5 = 15 um
> in extent. Therefore, a pixel size of 15 um / 3 =
> 5 um would give the lowest sampling rate for
> maximum resolution on the image. The Canon 18MP
> APS-C sensor has 4.3 um X 4.3 um pixels, a
> sampling rate of ~3.5, so is just about ideal. An
> eyepiece camera with 14MP has 1.2 um pixels and
> for this example would have a sampling rate of
> ~12, which is far from ideal. On the other hand,
> a 1.3MP eyepiece camera has 3.6 um pixels and has
> a closer to ideal sampling rate. With the assumed
> objective, this would do very well, but at a
> reduced field of view due to the small number of
> pixels.
>
> So, if the pixel size is set for best sampling,
> then the number of pixels just determines the
> amount of FOV captured. A small sensor and a
> large sensor, at the same sampling rate will have
> the same resolution, but the fields covered will
> be different. The limit on the maximum number of
> useful pixels then is a function of your
> objectives usable coverage at the image plane.
>
> One last comment. If you consider one of the high
> quality objectives like a 4X Plan Apo with NA=0.2
> and resolution of 1.6 um, the sampling rate using
> the Canon APS-C is 1.56, clearly under sampled.
> The camera is limited in this case. To get the
> highest resolution images, the objective must be
> properly matched to the objective lens.

Gene, that's very good stuff which I file carefully and thank you for. As above - and sorely tempted as I am to do otherwise - I plead 'noli contendere' for the time being.

I look forward to knowing your thoughts on the rest.

As a preliminary to running a first set of tests to determine effect on resolution by the algorithmic processing of pixel signals, I've just combed out my Canon 600D settings to those I *think* will require the least signal processing. Your view (if any) of these new base-line settings would be welcome. The settings now are:

- Manual control of shutter speed and focus. Selected
- ISO 100. - Selected *
- Flash - Disabled.
- No exposure compensation.
- Picture style - Faithful.*
- White balance - Incandescent light.
- Auto lighting optimiser - Off. *
- Single shot shooting - Selected
- Spot metering selected (I'd switch the metering off as it is irrelevant for these tests but that does not seem possible).
- White balance Shift/bracket - None (auto correction disabled). *
- Aspect ratio 3:2 - Selected.
- Mirror lock-up - Enabled.
- Long exposure noise reduction - Off *
- High ISO speed noise reduction - Off *
- Highlight tone priority - Off. *
- Image recording quality. - Large/Fine. JPEG only. 18MP. 6.4 Mb file size.

The plot is to get the best image resolution I can, using the above settings, at both 1.0 and 6.5 (nominal) 'scope zoom settings as a baseline reference to image resolution. Then for the settings above that are marked with an asterisk, to return them, one as a time, to my usual preferred setting and record for each change any discernible degradation in image resolution. If there is no discernible difference (or even an improvement) then I'll leave the new setting selected, else I'll retain the basic setting and pass on to testing with the next parameter change.

Hopefully, this procedure will eliminate any cause for degradation of image resolution that may occur in the camera's signal processing and is unrelated to my microscope's performance.

That precursor being done and dusted, I should be ready to return to improving (if I can) and recording the best resolution that can be achieved by my microscope at each 0.5 step of microscope zoom. In this second series of tests I will also be collecting the information required to calculate the observed crop factor that exists between my relay lens view and the Canon APS-C sensor whilst utilising the maximum (3:2 aspect ratio) pixel count.

> I'll need another Margarita before I think about
> the rest of your post. :)


So you're Margarita man, huh? With a salt rather than a sugar rim, I'll be bound! I was introduced to that seductive drink some 30 years ago during a series of stays I made in Texas at that time. I became very partial to Margaritas and every summer of mine since has been marked by the consumption of quantities of the same. But I think one needs those sundowns after blazing hot days though and, in my damp little island home, we don't see enough of those. So my first Margarita of 2012 is yet to come.

Best,
Owen
avatar Re: Amscope...
March 30, 2012 07:35PM
    
Do not use the JPEG setting for your files! If the camera has a higher bit density than 8 bits per sample, use it. On my Nikon D300, I use 14 bits/ sample and the RAW output format. JPEG is restricted to 8 bits/sample in the achromatic channel and 4 bits/sample in the yellow-blue and red-green channels. Any type of post processing on JPEG images can have VERY delterious effects on your images. JPEG is good for saving file size after all image processing has been accomplished.
avatar Re: Amscope...
March 30, 2012 09:09PM
    
Hi Owen,

I agree that some applications, such as confocal microscopy and very low contrast subjects can benefit from oversampling. However, we are mainly concerned with large high contrast (relatively speaking) subjects that are illuminated by reflected light. In this case, critical or slightly oversampled is all that is required. Here is a quote from

Quote
University of Florida in collaboration with Optical Microscopy, National Magnetic Field Lab.
To ensure adequate sampling for high-resolution imaging, an interval of 2.5 to 3 samples for the smallest resolvable feature is suggested.

A majority of digital cameras coupled to modern microscopes have a fixed minimum sampling interval, which cannot be adjusted to match the specimen's spatial frequency. It is important to choose a camera and digitizer combination that can meet the minimum spatial resolution requirements of the microscope magnification and specimen features. If the sampling interval exceeds that necessary for a particular specimen, the resulting digital image will contain more data than is needed, but no spatial information will be lost.

But, this is theory and in practice we can only hope to get results that approach the theoretical limits of resolution. As an example, I am not coming close to these limits for other more problematic and less understandable reasons. Vibration, is the limiting factor in my work and has been mentioned by better micro photographers than I on this forum in the past. At my higher magnifications, I can see constant jitter in the order of a few pixels with the system sitting on a desk. The next step will be to move the setup to the basement concrete floor. Even on the concrete floor there will still be resonant modes, caused by the stepper motor and shutter, in the mechanical setup itself that will have to be examined and mitigated.

For now, I rest my case and retire to make more modifications and adjustments, in hopes of asymptotically reaching those elusive theoretical limits.

Gene
avatar Re: Amscope...
March 30, 2012 10:01PM
Hi Ronald,

Thanks for that. As you may surmise, I'm on a steep learning curve with a new and decent DSLR and ots application to photomicrography. I have a 'to do' list longer than my arm and with items getting added faster that they are crossed off. Mastering to ins and outs of RAW file processing is somewhere on the list.

So for my upcoming series of tests, I shall (following from your post) capture all images simultaneously in JPEG and RAW formats (the camera supporting that option). In this way and without having to repeat all the tests the images in RAW format will be available for for direct comparison with the JPEGs as and when there's time. So thanks for the thought.
avatar Re: Amscope...
March 31, 2012 10:23PM
Gene,

Later, my dear sir; later, I promise smiling smiley I have to prioritise. Priority one is to determine - in the environment I have - what deleterious effect, if any, my preferred camera settings may be having on my test shots. That done the second equal priorities are:

- To determine the crop factor of the Canon APS-C sensor in its basic 3:2 configuration to the lens view of my camera adapter. If it is ~ 1.6, well, that is what the great god Canon seems to have ordained. If it is > 2, then I'm gunning for an improvement through tweaking the lens view as projected onto the object plane.

- The best resolution I can obtain of simple straight edge targets at a known separation and magnification (resolution varying with magnification).

I have the setup I have. My aim is simply to determine the best resolution I can obtain from it. In turn, that knowledge will (I hope) usefully inform my photomicrographic attempts and improve them.

I intend to tackle both of these tomorrow.

Thank you for adding to my misery by starting yet another hare for me to chase after, that of vibrational effects. That's another discussion we might have down the road. But, 'cos I'm a sport, let me send a hip-shot after it right now.

Diminution of vibrational effects is at the root, not only of my microscopic endeavours but also the regular use of a Mettler 0.1mg analytical balance. I'm fortunate enough (very) to live in a rural situation where only about three vehicles an hour pass withing 300 metres (all light vehicles). My work is done on the ground floor of my house, the floor being a concrete slab laid straight on the dirt with only the cushion of a waterproof membrane. I live alone, so if I don't move and hold my breath, nothing moves. My microscope base and supports are both massive and rigid (think Gemolite Mk10/Kruess KSW 7000). With these factors in my favour I have thus far not detected a positive need for me to invest in a good anti-vibration table - but, should occasion arise, one will surely be put in.

This leaves the vibrational effects created within the DSLR itself. Mirror inertia and bounce is looked after by mirror lock-up, leaving some small and irreducible vibrational effect from the shutter operation. With the excellent rigidity of my setup and, typical exposure times in the range 1 - 20 secs, prima facie, chasing after shutter vibration does not seem the top priority, sitting stock-still being rather more important - but we shall see in due course, after the more crass and likely causes of less-than-optimal resolution have been chased out.

Not that the current results are all that poor. It's simply that I know that they might be better and wish to see what improvement in my system performance I can obtain through careful testing and adjustment.

Owen



Edited 1 time(s). Last edit at 04/01/2012 12:00AM by Owen Lewis (2).
avatar Re: Amscope...
April 02, 2012 05:20PM
    
Hi Owen,

My compulsive notion that good enough is never good enough often overshadows my sense of civility. If I have added misery and/or frustration to your priorities, then I apologize profusely. My intent was only to alert you to possible system problems that you may eventually have to deal with, as I am currently having to do. Vibration is a real system problem, albeit one that is easily overlooked. In fact, a great deal of research and effort has gone into to quantizing its effects. Accelerometers have been mounted on various parts of the photomicroscopy system in an effort to study the impulses created by mirror and shutter action that then give rise to resonant modes in the system, which turn out to be of significant magnitude. To make this story short, the results of the tests reveal several methods to mediate this problem.

1. Lengthen exposure time so that the resonant modes are short in comparison to the total exposure. That's what you are doing now and you may have already solved the problem. Exposures longer than one second, and up to several seconds, work well.

2. Use of flash illumination effectively stops the movements.

3. Decouple the camera from the microscope. This reduces apparent movement at the image plane by a factor of the objective magnification.

Apparently, there are a multitude of physical effects that conspire to prevent us from approaching diffraction limited images. However, I believe that every discussion, such as this one, may lead us to a better understanding and direction towards that end. And finally, at least for me, collaboration keeps the interest high.

Kind regards,
Gene
avatar Re: Amscope...
April 03, 2012 01:45AM
Gene,

You know the old prayer, 'Lord, give me the strength to change those things I can, the patience to endure those things I cannot change and the wisdom to know the difference'? I find I need to remind myself of it regularly.

Sunday's session with the microscope did produce some concrete advances in knowledge, certainty (and a little in image quality) It also raised clear reasons for more learning and research. On the plus side, I think finally to have quantified the mismatch I have between microscope view and image capture on the camers's APS-C sensor. Here's my take:



Here is an image of a 1mm graticule that is sub-divided into divisions of 0.01mm (10 micron). The image was made with the scope set to max (6.2) zoom, giving an FOV of ~3.6mm. With the crop factor of 1.6 designed into the camera by Canon, I think the FOV captured across diametrically opposed corners of the complete APS-C pixel field should be 3.6/1.6 ~ 2.2mm? The shot shows that the maximum image capture along the maximum dimension of the APS-C sensor is only ~1.3mm, or only just over 1/3 of the ideal match to the FOV. So.... if we put an x0.5 relay lens in the adapter, replacing the x1 presently fitted, that should give an approx. a 1.8mm lens view, reducing the crop factor to 1.8/1.3 (about 1.3). This is better than the 1.6 crop factor which Canon inflict on those who use their APS-C fitted cameras as a microscope imager. Does that sound right to you? If so, and if one could obtain 0.3 relay lens, than the match of FOV to image capture dimensions would be close to the best possible.

Other main points of interest are that for this very limited type of imaging, neither ISO setting (at least up to 1600) nor long exposure noise reduction nor high ISO noise reduction has a significant impact on resolution. Similarly, there is no apparent effect on resolution by capturing the image as RAW or by using the camera's JPEG algorithm - there is, however, marked benefit in processing RAW files to improve other image qualities before saving as a JPEG.

I now understand 4-5 micron to be about the maximum resolution that a microscope of this general specification is capable of and this my setup is delivering from about x6 zoom upwards. Interestingly, Airy ring artefacts of approx 5 micron diameter are appearing in the image field at a zoom factor of 5.0 and above but these can only be clearly identified as such (concentric rings of light and dark) at the maximum zoom and resolution.

Just one more session at this I think to maximise the image quality and then into discussions with the camera adapter manufacturer to discuss the way ahead.

Many thanks for your further thoughts on vibration. As well as this, there must also be a point at which even small thermal change results in pixel switchings? Perhaps one's level of concern is determined by the level of accuracy at which one needs to work. To continue with my previous comparison with the use of an analytical grade of scale. With a gm 4DP level of accuracy both vibration and temperature change require control and sometimes compensation. With care, I can manage well enough in *my* home environment, thanking my lucky star that I do not have a gm 6DP scale, for I know that to use such to its full capability would require me to install expensive levels of environmental monitoring and control. As regards photomicroscopy, I am still discovering the environmental controls I require. To date - and as you commented - long exposures seem to cope satisfactorily with shutter vibration. It's also free and easy to manage smiling smiley Thermal disturbance of the uniformity of RI in hydrocarbon immersion fluids I have yet to crack. Commonsense tells me that the answer must good quality IR filtering- but it remains another thing on the long 'to do' list.

Best,
Owen
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