Hi,

I'm trying to find a good article that explains the basic reason for
using white light in a white light confocal microscope; I mean the real
advantage and the theory behind it. I really really appreciate if
anyone can give me some direction on this. I'm trying to understand
the kind of noise that would be introduced in a white light confocal
due to the use of white light, which would help me understand the right
choice for image restoration under white light confocal microscope.
I appreciate any inputs.

Madhu.

From:	Madhu
Subject:	Re: White light confocal microscope
Date:	13 Dec 2004 15:28:16 -0800

Hello Aaron,

Thanks for the link. Looks like it has almost everything in there.
I'm reading the article on non-coherent light sources.
Thanks very much for the link.

Madhu.

From:	Aaron
Subject:	Re: White light confocal microscope
Date:	14 Dec 2004 16:55:52 -0600

Essentially, lasers produce coherent light and the XBO HBO lamps
produce "white light" that is not coherent.

So on a casual level, consider "non-coherent" and "white light" to be
references to the same subject.. The are other issues. Laser light
tends to be narrow band and lasers do not exist for all the
wavelengths. The XBO/HBO white light sources have energy at some
wavelenghts that are not available with lasers. Also confocal
microscopes can be used to produce bright field images. They are not
just for fluorescence techniques. All of this leads to confusion
because the equipment and the techniques are not clearly separated and
labled.

Aaron

On 13 Dec 2004 15:28:16 -0800, "Madhu"
wrote:

>Hello Aaron,
>
>Thanks for the link. Looks like it has almost everything in there.
>I'm reading the article on non-coherent light sources.
>Thanks very much for the link.
>
>Madhu.

From:	Madhu
Subject:	Re: White light confocal microscope
Date:	26 Dec 2004 15:49:15 -0800

Hi Aaron, Chiristian,

Thanks for sharing your experience. I tried few many things and then
played with CCD a bit; finally by making CCD average a couple of
frames before being sent to the imaging board, I was able to reduce
almost 90% of the scan lines and the contrast improved drastically.
Thanks for your help.
Madhu.

From:	Madhu
Subject:	Re: White light confocal microscope
Date:	16 Dec 2004 10:17:06 -0800

Thanks for the insight Aaron. I read that article and is quite
elaborate and explains the choice of non-coherent light source for a
confocal. I am in a lab where they built their confocal using
white-light and nipkow spinning disk; I see the following problems in
the images acquired using the CCD coupled to the confocal:

1. I see scan lines that move across the image at a certain frequency
and 2. Speckle noise

I can remove the noise using some filter. But the scan lines are a big
problem; if you are familiar with the fourier techniques, I tried to
remove certain frequencies (since there is a band of frequencies that
represent the scan lines), sometimes I end up loosing some structure of
the specimen in the image. I think I can do something to the confocal
by either tweaking the nipkow disk speed, synchoronizing the disk with
the CCD capture rate or doing something with the white-light. I'm
trying to learn the theory behind all these, so that I can fix the
confocal.

If someone has any inputs on this, have experienced similar things, I
really really appreciate sharing your experience and any inputs.
Thanks very much,
Madhu.

From:	Christian Wilms
Subject:	Re: White light confocal microscope
Date:	Fri, 17 Dec 2004 13:29:46 +0100

Madhu wrote:

> 1. I see scan lines that move across the image at a certain frequency
> and

A simple cause for this might be 50 or 60 cycle noise (depending on
which country you're in). I have seen lines moving across the image at a
certain frequency caused by such noise in a glavo-driven confocal. I
would suggest checking the grounding and making sure all instruments
connected to the setup are attached to a _common_ ground.

Another issue - which Aaron already mentioned - might be mechanical
stability. Often insufficient vibration isolation might cause
irregularities in images. The spinning disc is a supreme source for
vibration, so that might really be an idea ...

cheers, Christian

From:	Aaron
Subject:	Re: White light confocal microscope
Date:	16 Dec 2004 19:25:07 -0600

Hi,

I do not have enough experience to comment on your specific problems.
Someone else will have to help.

If I were troubleshooting this instrument, I would start by consulting
the original builders.

Then, I would try to work with a test slide of known content and see
if I could get the expected results.

White light inconjunction with Nipkow disks seem like the accepted
way to go. That said every instrument maker does it slightly
differently.

Aaron

On 16 Dec 2004 10:17:06 -0800, "Madhu"
wrote:

>Thanks for the insight Aaron. I read that article and is quite
>elaborate and explains the choice of non-coherent light source for a
>confocal. I am in a lab where they built their confocal using
>white-light and nipkow spinning disk; I see the following problems in
>the images acquired using the CCD coupled to the confocal:
>
>1. I see scan lines that move across the image at a certain frequency
>and 2. Speckle noise
>
>I can remove the noise using some filter. But the scan lines are a big
>problem; if you are familiar with the fourier techniques, I tried to
>remove certain frequencies (since there is a band of frequencies that
>represent the scan lines), sometimes I end up loosing some structure of
>the specimen in the image. I think I can do something to the confocal
>by either tweaking the nipkow disk speed, synchoronizing the disk with
>the CCD capture rate or doing something with the white-light. I'm
>trying to learn the theory behind all these, so that I can fix the
>confocal.
>
>If someone has any inputs on this, have experienced similar things, I
>really really appreciate sharing your experience and any inputs.
>Thanks very much,
>Madhu.

From:	Aaron
Subject:	Re: White light confocal microscope
Date:	16 Dec 2004 20:35:11 -0600

Hi,

I had one last thought. It probably is not worth much, but if the
dichroic mirror that directs the output to the CCD had some small
amount of play, that might cause the speckle and scan lines.
Otherwise I can't see anyother way for that type of signal to reach
the CCD.

Let me know if that is the issue.

Aaron

On 16 Dec 2004 19:25:07 -0600, Aaron wrote:

>Hi,
>
>I do not have enough experience to comment on your specific problems.
>Someone else will have to help.
>
>If I were troubleshooting this instrument, I would start by consulting
>the original builders.
>
>Then, I would try to work with a test slide of known content and see
>if I could get the expected results.
>
> White light inconjunction with Nipkow disks seem like the accepted
>way to go. That said every instrument maker does it slightly
>differently.
>
>Aaron
>
>
>
>On 16 Dec 2004 10:17:06 -0800, "Madhu"
> wrote:
>
>>Thanks for the insight Aaron. I read that article and is quite
>>elaborate and explains the choice of non-coherent light source for a
>>confocal. I am in a lab where they built their confocal using
>>white-light and nipkow spinning disk; I see the following problems in
>>the images acquired using the CCD coupled to the confocal:
>>
>>1. I see scan lines that move across the image at a certain frequency
>>and 2. Speckle noise
>>
>>I can remove the noise using some filter. But the scan lines are a big
>>problem; if you are familiar with the fourier techniques, I tried to
>>remove certain frequencies (since there is a band of frequencies that
>>represent the scan lines), sometimes I end up loosing some structure of
>>the specimen in the image. I think I can do something to the confocal
>>by either tweaking the nipkow disk speed, synchoronizing the disk with
>>the CCD capture rate or doing something with the white-light. I'm
>>trying to learn the theory behind all these, so that I can fix the
>>confocal.
>>
>>If someone has any inputs on this, have experienced similar things, I
>>really really appreciate sharing your experience and any inputs.
>>Thanks very much,
>>Madhu.

From:	Madhu
Subject:	Re: White light confocal microscope
Date:	17 Dec 2004 09:23:28 -0800

I'll check both the dichroic mirror and as well as the grounding.
Also, I noticed that the article on non-coherent light sources
(http://www.olympusconfocal.com/theory/noncoherentsources.html
) has a section on "Coherence"; in the third paragraph, they mentioned
that "If the coherence of the illuminating light is too high, images
develop fringes caused by interference of the coherent wavefronts
reflected from any of the optical surfaces, including the lenses,
mirrors, dust windows and in particular, the cover pages. This complex
interference pattern can appear as defined rings, but more commonly, it
appears as a high-contrast granular speckle".

I checked the white light source being used: its a 300 w xenon lamp.
How do I know the coherence is high / normal?

Madhu.

From:	Aaron
Subject:	Re: White light confocal microscope
Date:	12 Dec 2004 14:44:12 -0600

Hello Madhu,

Two wonderful and extensive sources of informamtion on all aspects of
microscopy are available on the web at the "Molecular Expressions" and
the "Nikon MicroscopyU" websites. This is the first place I go for
background on issues with which I am not familiar..

Here is a discussion of exactly the topic you requested

http://www.olympusconfocal.com/theory/noncoherentsources.html

For other general information on confocal theory, see

http://www.olympusconfocal.com/theory/index.html

and

http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html

Aaron

On 11 Dec 2004 09:08:46 -0800, madhu.balasubramanian@gmail.com wrote:

>Hi,
>
>I'm trying to find a good article that explains the basic reason for
>using white light in a white light confocal microscope; I mean the real
>advantage and the theory behind it. I really really appreciate if
>anyone can give me some direction on this. I'm trying to understand
>the kind of noise that would be introduced in a white light confocal
>due to the use of white light, which would help me understand the right
>choice for image restoration under white light confocal microscope.
>I appreciate any inputs.
>
>Madhu.