Re: Poor man's confocal - HiLo method

[Daniel James White]

Hi Christophe,

Begin forwarded message:

>
> Date:    Thu, 27 May 2010 11:19:45 +0200
> From:    Christophe Leterrier <[hidden email]>
> Subject: Re: Poor man's confocal
>
> There is at least another cheap and smart method to get optical
> sectioning from a widefield microscope, called "HiLo" microscopy:
>
> Wide-field fluorescence sectioning with hybrid speckle and
> uniform-illumination microscopy.
> Lim D, Chu KK, Mertz J.
> Opt Lett. 2008 Aug 15;33(16):1819-21.
>
> The idea is to take a speckled image and a non speckled one, from
> which high and low frequencies of the images are computed and merged
> to from the final image.

looks very simple.... i wonder if you really need a laser illumination ? Would a metal halide lamp also work?
I dont see why not? Ot does it need to be a coherent beam?

> I don't think there is a commercial version
> of it, but I've seen two other labs using it and it seems quite simple
> to implement.

do you know how what kind of diffuser they use and where to get one?

how did they move the diffuser fast without vibrations?

cheers

Dan


Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[lechristophe]

On Fri, May 28, 2010 at 10:31, Daniel James White <[hidden email]> wrote:

Hi Christophe,

Begin forwarded message:

>
> Date:    Thu, 27 May 2010 11:19:45 +0200
> From:    Christophe Leterrier <[hidden email]>
> Subject: Re: Poor man's confocal
>
> There is at least another cheap and smart method to get optical
> sectioning from a widefield microscope, called "HiLo" microscopy:
>
> Wide-field fluorescence sectioning with hybrid speckle and
> uniform-illumination microscopy.
> Lim D, Chu KK, Mertz J.
> Opt Lett. 2008 Aug 15;33(16):1819-21.
>
> The idea is to take a speckled image and a non speckled one, from
> which high and low frequencies of the images are computed and merged
> to from the final image.

looks very simple.... i wonder if you really need a laser illumination ? Would a metal halide lamp also work?
I dont see why not? Ot does it need to be a coherent beam?

I'm not sure but I think you need to form a speckled illumination pattern. Does this requires a coherent beam ?

> I don't think there is a commercial version
> of it, but I've seen two other labs using it and it seems quite simple
> to implement.

do you know how what kind of diffuser they use and where to get one?

 

Don't laugh, but the setup I've seen working used a bit of transparent (invisible "crystal") adhesive tape as the diffuser...
 

how did they move the diffuser fast without vibrations?

I think the adhesive tape bit was just put in a motorized filter wheel or something like that.

 

cheers

Dan


Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net       BioImageXD
http://pacific.mpi-cbg.de               Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk               Dan's Homepages
https://ifn.mpi-cbg.de                  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

Cheers,

Christophe

[David Coder-2]

Potentially another option for a relatively inexpensive structured
illumination add-on is one of the first systems developed by Pio Benedetti
at the CNR in Pisa.   The system is described by a collaborator, Rainer
Heintzmann, in Chap. 13 of  Jim Pawley,  Handbook of Biological Confocal
Microscopy, 3rd Ed. The system is distributed by a small company (Biomedica
Mangoni in Pisa) described for Nikon microscopes, but probably adaptable to
any.  No pricing for optics and software alone (only a system to which you
add a microscope; 36K Euros) Here’s a link to the English description:
http://www.biomedicamangoni.it/Inglese/Products/Depliant/BMViCo_e.htm 

Since the company is in Pisa, it may more convenient for European  users;
there appears to be no US distributor.
================
David M. Coder, PhD
Irvine, CA
Cell phone: [phone] begin_of_the_skype_highlighting              949
233 2641      end_of_the_skype_highlighting
Skype: dave.coder
Email: [hidden email]

[Shalin Mehta]

Hi Christophe and Daniel,

For HiLo microscopy, potentially any method that can impose high
frequency variations on the specimen fluorescence will work. It seems
there are two 'ingredients' to the sectioning produced by the HiLo
method:

- High spatial-frequencies in the specimen are sectioned even in usual
wide-field illumination, i.e., fast spatial variations of fluorescence
get blurred rapidly as we defocus.
- Low spatial-frequencies, however, are not sectioned that strongly.
But, if we artificially turn them into high-variation structure by
illuminating with high-frequency illumination, we gain the sectioning
much like the above case.

A nice explanation/illustration is on Jerome Mertz's website:
http://biomicroscopy.bu.edu/r_hilo.htm

The sectioned low spatial-frequencies can be extracted by low-pass
filtering of the image taken with high-frequency illumination (e.g.
speckle) imposed. I think speckle is a nice high-frequency
illumination for this purpose, because it is nearly random (low but
finite chances of artifacts) and because one can apply a fast variance
filter to extract the in-focus low-pass regions.

I have seen speckles during transmission imaging when using narrowband
filter after halogen lamp (20 nm width around 546nm) and using the
illumination aperture of around half that of the imaging aperture. One
can possibly do the same with the mercury arc-lamp at the cost of
brightness.

As a side-note, I just found that under right conditions the sun-light
reflected from fingernail  produces speckle:
http://www.sciencenewsforkids.org/pages/puzzlezone/muse/muse0705.asp/

best
Shalin

blog: shalin.wordpress.com

Bioimaging Lab, Block-E3A, #7-10
Div of Bioengineering, NUS Singapore 117574
website: http://www.bioeng.nus.edu.sg/optbioimaging/colin/people.asp#shalinm



On Fri, May 28, 2010 at 5:42 PM, Christophe Leterrier
<[hidden email]> wrote:

> On Fri, May 28, 2010 at 10:31, Daniel James White <[hidden email]> wrote:
>>
>> Hi Christophe,
>>
>> Begin forwarded message:
>>
>> >
>> > Date:    Thu, 27 May 2010 11:19:45 +0200
>> > From:    Christophe Leterrier <[hidden email]>
>> > Subject: Re: Poor man's confocal
>> >
>> > There is at least another cheap and smart method to get optical
>> > sectioning from a widefield microscope, called "HiLo" microscopy:
>> >
>> > Wide-field fluorescence sectioning with hybrid speckle and
>> > uniform-illumination microscopy.
>> > Lim D, Chu KK, Mertz J.
>> > Opt Lett. 2008 Aug 15;33(16):1819-21.
>> >
>> > The idea is to take a speckled image and a non speckled one, from
>> > which high and low frequencies of the images are computed and merged
>> > to from the final image.
>>
>> looks very simple.... i wonder if you really need a laser illumination ?
>> Would a metal halide lamp also work?
>> I dont see why not? Ot does it need to be a coherent beam?
>>
> I'm not sure but I think you need to form a speckled illumination pattern.
> Does this requires a coherent beam ?
>
>
>> > I don't think there is a commercial version
>> > of it, but I've seen two other labs using it and it seems quite simple
>> > to implement.
>>
>> do you know how what kind of diffuser they use and where to get one?
>>
>
> Don't laugh, but the setup I've seen working used a bit of transparent
> (invisible "crystal") adhesive tape as the diffuser...
>
>
>> how did they move the diffuser fast without vibrations?
>>
>
> I think the adhesive tape bit was just put in a motorized filter wheel or
> something like that.
>
>
>>
>> cheers
>>
>> Dan
>>
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>> Senior Microscopist / Image Visualisation, Processing and Analysis
>> Light Microscopy and Image Processing Facilities
>> Max Planck Institute of Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> [phone] (German Mobile)
>> [phone] (Work phone at MPI-CBG)
>> [phone] (Fax MPI-CBG LMF)
>>
>> http://www.bioimagexd.net       BioImageXD
>> http://pacific.mpi-cbg.de               Fiji -  is just ImageJ (Batteries
>> Included)
>> http://www.chalkie.org.uk               Dan's Homepages
>> https://ifn.mpi-cbg.de                  Dresden Imaging Facility Network
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>
> Cheers,
>
> Christophe
>