Re: Question about larger issue (was: Checking for flat field illumination)

[cromey]

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Robert, here is what I tell people. 

Doug

 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Robert Peterson
Sent: Friday, June 19, 2009 6:39 AM
To: [hidden email]
Subject: Question about larger issue (was: Checking for flat field illumination)

 

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.

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/