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Help for a beginner

Posted: 28.11.2007 20:54
by jonhudson
Hello can anyone help with what I am sure is a beginners lack of understanding.

I am using the trial version of Focus for series of images of spirogyra taken at 100X magnification with bright field illumination.

I am using a Canon G2 camera connected to a Wild M20 and can achieve good focused single images captured as RAW files onto the PC using Breeze systems remote capture software.

When I try a series of 10 20 30 40 or 50 images each taken with very small variations of the fine focus on the wild (which is reliable) and then process them the result is poor and looks like 10 etc images stitched side to side i.e multiple images overlapping not one image with added depth.

I am confident because of the set up that I am not moving the specimen relative to the camera between exposures.

I have tried lots of permutations of radius and smoothing and also tried processing the images in ascending and descending order.

I know from the inspirational work of Mr Krebs that Focus is capable of doing exactly what I want it to do so what am I doing wrong?

Any suggestions will be gratefully received.



Posted: 29.11.2007 07:46
by Ariel
Have you just tried stacking a few jpegs and see what the results were? Just to see...

Posted: 29.11.2007 18:52
by Dan Kozub
Seeing resulting image would help...

Posted: 29.11.2007 21:17
by jonhudson

This is the image

Posted: 30.11.2007 18:24
by Dan Kozub
No attachment present...


Posted: 30.11.2007 20:20
by jonhudson
I tried to post an image without success.

When I read the FAQ this seems impossible unless you have your own website which I haven't.



Posted: 08.03.2009 03:56
by alanj49
Hi... am am fairly new to this subject and am having some of the same problems. I am using a GX Optics LH1500BHTG Microscope with a Trinocular head, the camera is a Canon EOS 450D attached via a C Mount adaptor and T Ring. The few subjects that I have at the moment are all bottom lit with a variable halogen light source via a condenser with iris control. I have taken sequences of images at 40 & 100x magnification and have successfully combined them in Helicon Focus. I tried the Canon software, and that is a big pile of poo, so I am now trialing the Breeze Software which is markedly better and does not freeze or lose connection with the camera. All of the images were shot as RAW at maximum resolution, my system is a MAC Pro with Dual 3.2Ghz processors and 10Gb of RAM and I have not had any out of memory errors yet.

My problems are:

1. Getting even lighting across the image
2. I am having a lot of difficulty with focus/sharpness, initially on my monitor, which is connected as a HD monitor. It is almost there but I cannot get it pin sharp.

I have been at this for a few days now before I came to the forum, and I would be very appreciative of any help/suggestions regarding software and procedure before I pull out the remainder of what hair I have left :cry:

Posted: 08.03.2009 10:10
by jonhudson

In the end I found it was much better to use jpeg rather than raw. Try a stack of about 5 images to see the difference. I have been only really successfull using low powered i.e 3X microscope objective but I know others have success at higher powers.

The lighting and focus issues are common to all photomicroscopy. Except for the newest live view SLR's like the 40D few people manage to be able to focus via the monitor. What most people do is strive to get the ocular image nearly parfocal with the camera image and then make trial and error fine focus adjustments to get a sharp camera image.

Many people know a lot more than me about this. As a start look at the Charles Krebs site . He is the acknowledged master and his images are amazing and award winning. The site has much really good and hepfull advice.

The yahoo microscope group has a lot of expertise too and this subject receives a lot of attention.


Posted: 09.03.2009 01:49
by alanj49
Hi Jon, many thanks for the reply..I have spent most of today scrutinising Charles Krebs site, and like most people am in awe at his results. After a lot more trial and even more errors I think I have narrowed some of my problems to not being able to get the camera parfocal with the microscope, with the adaptors/spacers that I have I can get it just in or out of focus but not dead on, plus the relay eyepiece that I have is 5x which is probably a bit too strong. The 450D has live view with mirror lockup so I can view the image live on the screen, but it never seems to be pin sharp

I will definitely try your suggestion re using Jpeg as opposed to RAW, I used RAW in order not to lose the detail that is lost in Jpeg's.

I will let you know the results.... plus I will have to rig something up to sort out the parfocal issue.

Posted: 09.03.2009 10:38
by jonhudson

You could also take a look at this ... /micro.htm

I know it uses a 40D and some seriously expensive microscope objectives but the results seem to be what you are aiming for. I can only dream of such a set up. I use a Canon G2 which I bought off E bay for 50 euros.


Posted: 09.03.2009 19:28
by alanj49
Hi Jon,
I have had a bit of success since yesterday, I found out that there is a local firm that manufacture spacers, mounts, tubes etc especially for optical systems. I got a set of spacers which has got me nearer to my goal, but I think I will go back to him and get them to machine out one of the tubes on a lathe so that I have more flexibility as to where the relay objective is.

The website you gave me has much the same setup as I have now, except for the Microscope and the model of camera, my only gripe is that the Canon software is terrible, so I use the Breeze Systems software which is much more reliable.

I am gradually getting there. I just got things set up to run off a set of images and the camera battery went flat, that is my state of luck at the moment

Posted: 09.03.2009 23:22
by alanj49
I have had a bit of success, I have just taken a sequence of images to produce the image of the leg and hooks of a Housefly. The marks on the hooks are under the cover slide and were there when I was supplied with the slide, so not guilty on that one

This is my first posted image so I would be grateful for any constructive criticism.... Many thanks