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Endocytosis of Quantum Dots

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Dries Vercauteren

Endocytosis of Quantum Dots

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

an easy question;

are free (unconjugated) Quantum Dots normally not taken up via endocytosis? Or is this dependent on charge, avidin coating, ...?

Thanks for the response in advance!

Dries

--
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone: +329/264 80 49
Mobile: +32485/30 69 80
E-mail: [hidden email]
[hidden email]

Rietdorf, Jens

Re: Endocytosis of Quantum Dots

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Dries,

indeed, my experience is such that uncoated QDs are not taken up, at least not into fibroblast or osteosarcoma cell lines. Lectin coated QDs are excellent markers for the plasma membrane, because they are not taken up either, unlike many other lectin-dye conjugates. These experiences date back some time (2 years?) and things may have changed.

Cheers, jens

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dries Vercauteren
Sent: Tuesday, June 17, 2008 4:08 PM
To: [hidden email]
Subject: Endocytosis of Quantum Dots

Ignatius, Mike

Re: Endocytosis of Quantum Dots

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Dries,

And from our studies as well, the uncoated Quantum Dot nanocrystals are not taken up by cells. Nor are our PEG coated dots designed for in vivo blood flow studies. Thus the QTracker line of reagents were designed to deliver material using poly-arg peptides on the outside.

There is one study I am aware showing smaller, unconjugated dots made of Cd/TE that aren't commercially available can be taken up by phagocytic cells.

<A href="javascript:AL_get(this, 'jour', 'Nano Lett.');">Nano Lett. 2007 Nov;7(11):3452-61. Epub 2007 Oct 19.Nonfunctionalized nanocrystals can exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Nabiev I, Mitchell S, Davies A, Williams Y, Kelleher D, Moore R, Gun'ko YK, Byrne S, Rakovich YP, Donegan JF, Sukhanova A, Conroy J, Cottell D, Gaponik N, Rogach A, Volkov Y.

Mike

Mike Ignatius, Ph.D. Molecular Probes labeling and detection technologies
INVITROGEN | 29851 Willow Creek Road, Eugene, OR 97402-9132
Tel: 541-335-0414 | Cell: 541-510-1736 [hidden email]

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rietdorf, Jens
Sent: Wednesday, June 18, 2008 1:09 AM
To: [hidden email]
Subject: Re: Endocytosis of Quantum Dots

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Dries,

Cheers, jens

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dries Vercauteren
Sent: Tuesday, June 17, 2008 4:08 PM
To: [hidden email]
Subject: Endocytosis of Quantum Dots

John Oreopoulos

Using an excitation filter as an emission filter

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Listserver,

One of my collaborators came to me the other day wanting to image a membrane protein tagged with YFP. Our lab did not have a YFP filter cube on hand for our Olympus epifluoresence microscope, but after rummaging around I realized that one of the excitation filters from another filter cube would make an excellent emission filter for YFP when exchanged for the emission filter on one of our green cubes for TRITC. As long as I orient the filter correctly with the arrow on the side pointing towards the dichroic mirror (chroma convention), this should work, correct? Is there any other reason why an excitation filter cannot be used as an emission filter and vice versa that I'm not aware of?

Thank you

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Eli Rothenberg

Re: Using an excitation filter as an emission filter

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi John,

Would probably work just fine, exciters and emitters
have virtually the same properties (at least when one takes
their spectrum).

Eli

---- Original message ----

>Date: Fri, 20 Jun 2008 18:59:43 -0400
>From: John Oreopoulos <[hidden email]>
>Subject: Using an excitation filter as an emission filter
>To: [hidden email]
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Dear Listserver,
> One of my collaborators came to me the other day
> wanting to image a membrane protein tagged with YFP.
> Our lab did not have a YFP filter cube on hand for
> our Olympus epifluoresence microscope, but
> after rummaging around I realized that one of the
> excitation filters from another filter cube would
> make an excellent emission filter for YFP when
> exchanged for the emission filter on one of our
> green cubes for TRITC. As long as I orient the
> filter correctly with the arrow on the side pointing
> towards the dichroic mirror (chroma convention),
> this should work, correct? Is there any other reason
> why an excitation filter cannot be used as an
> emission filter and vice versa that I'm not aware
> of?
> Thank you
>
> John Oreopoulos, BSc,
>
> PhD Candidate
>
> University of Toronto
>
> Institute For Biomaterials and Biomedical
> Engineering
>
> Centre For Studies in Molecular Imaging
>
> Tel: W:416-946-5022

________________________________
Eli Rothenberg, Ph.D.
Post Doctoral Research Associate,
Howard Hughes Medical Institute,
Department of Physics,
University of Illinois,
Urbana-Champaign. 61801.
Illinois, USA
Tel: +217-333-3393;
Fax: +217-244-7187;
Email: [hidden email]

Frederick Madison

Re: Using an excitation filter as an emission filter

In reply to this post by John Oreopoulos

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Your fine

Omega Labs

Frederick Madison

Program Manager

Delta Campus, Omega Drive

Brattleboro, VT 05301

802.251.7345 Direct Line

866.488.1064 x345 Toll Free (US)

[hidden email]

www.omegafilters.com

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Friday, June 20, 2008 7:00 PM
To: [hidden email]
Subject: Using an excitation filter as an emission filter

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Listserver,

Thank you

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Morrison, Ian E

Re: Using an excitation filter as an emission filter

In reply to this post by John Oreopoulos

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I do this sometimes. The only problem I have noticed is that excitation filters can be physically thicker than an equivalent emission filter, and with an epifluorescence setup you may find a greater chromatic shift when merging images from different filter cubes.

Ian

-----------------------------------------Dr. I.E.G.Morrison [hidden email]}----------------------------------------

                                         Dept.Biological Sciences, University of Essex
                                         Wivenhoe Park, Colchester, Essex CO4 3SQ
-----------------------------------------Tel: 01206-872246           Fax: 01206-872592----------------------------------------

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]On Behalf Of John Oreopoulos
Sent: Saturday, June 21, 2008 12:00 AM
To: [hidden email]
Subject: Using an excitation filter as an emission filter

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Listserver,

One of my collaborators came to me the other day wanting to image a membrane protein tagged with YFP. Our lab did not have a YFP filter cube on hand for our Olympus epifluoresence microscope, but after rummaging around I realized that one of the excitation filters from another filter cube would make an excellent emission filter for YFP when exchanged for the emission filter on one of our green cubes for TRITC. As long as I orient the filter correctly with the arrow on the side pointing towards the dichroic mirror (chroma convention), this should work, correct? Is there any other reason why an excitation filter cannot be used as an emission filter and vice versa that I'm not aware of?

Thank you

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Rietdorf, Jens

Re: [SPAM] Using an excitation filter as an emission filter

In reply to this post by John Oreopoulos

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear John,

though the given spectra of ex and em filters look very similar in a linear transmission plot, the blocking property of the em filters is typically much better, which is necessary, because the ex light is typically about 6 orders of magnitude stronger than the fluorescence generated. In a log plot you would immediately see the differences.

I suspect that using an exciter filter at the emitter position will reduce contrast significantly. It can still be good enough for some applications.

Best regards, jens

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Saturday, June 21, 2008 1:00 AM
To: [hidden email]
Subject: [SPAM] Using an excitation filter as an emission filter

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Listserver,

Thank you

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Michael Cammer

Re: [SPAM] Using an excitation filter as an emission filter

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Semrock claims that some of their emission/excitation filters are
interchangeable.

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear John,
>
>
>
> though the given spectra of ex and em filters look very similar in a
> linear transmission plot, the blocking property of the em filters is
> typically much better, which is necessary, because the ex light is
> typically about 6 orders of magnitude stronger than the fluorescence
> generated. In a log plot you would immediately see the differences.
>
>
>
> I suspect that using an exciter filter at the emitter position will
> reduce contrast significantly. It can still be good enough for some
> applications.
>
>
>
> Best regards, jens
>
>
>
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of John Oreopoulos
> Sent: Saturday, June 21, 2008 1:00 AM
> To: [hidden email]
> Subject: [SPAM] Using an excitation filter as an emission filter
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Listserver,
>
>
>
> One of my collaborators came to me the other day wanting to image a
> membrane protein tagged with YFP. Our lab did not have a YFP filter cube
> on hand for our Olympus epifluoresence microscope, but after rummaging
> around I realized that one of the excitation filters from another filter
> cube would make an excellent emission filter for YFP when exchanged for
> the emission filter on one of our green cubes for TRITC. As long as I
> orient the filter correctly with the arrow on the side pointing towards
> the dichroic mirror (chroma convention), this should work, correct? Is
> there any other reason why an excitation filter cannot be used as an
> emission filter and vice versa that I'm not aware of?
>
>
>
> Thank you
>
>
>
> John Oreopoulos, BSc,
>
> PhD Candidate
>
> University of Toronto
>
> Institute For Biomaterials and Biomedical Engineering
>
> Centre For Studies in Molecular Imaging
>
>
>
> Tel: W:416-946-5022
>
>
>
>
>
>
>
>

_________________________________________
Michael Cammer http://www.aecom.yu.edu/aif/

Stephen Cody

Re: Using an excitation filter as an emission filter

In reply to this post by John Oreopoulos

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

G'day John,

Yes. You must be absolutely sure that the dichroic and band pass filters will be of the appropriate wavelength to stop any of the excitation source reaching your eye. You really don't want to risk your damaging your eyesight. Otherwise using an excitation filter as a emmission barrier filter is probably OK. It may not have the level of through put that you normally would get.

Visa versa: You shouldn't use an emmission barrier filter as an excitation filter as it may not be designed to cope with the intense light and heat from the source.

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria, 3050
Australia
Tel: 61 3 9341 3155 Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm Type your signature here

________________________________

From: Confocal Microscopy List on behalf of John Oreopoulos
Sent: Sat 21/06/2008 8:59 AM
To: [hidden email]
Subject: Using an excitation filter as an emission filter

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Listserver,

One of my collaborators came to me the other day wanting to image a membrane protein tagged with YFP. Our lab did not have a YFP filter cube on hand for our Olympus epifluoresence microscope, but after rummaging around I realized that one of the excitation filters from another filter cube would make an excellent emission filter for YFP when exchanged for the emission filter on one of our green cubes for TRITC. As long as I orient the filter correctly with the arrow on the side pointing towards the dichroic mirror (chroma convention), this should work, correct? Is there any other reason why an excitation filter cannot be used as an emission filter and vice versa that I'm not aware of?

Thank you

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Knecht, David

Re: Endocytosis of Quantum Dots

In reply to this post by Ignatius, Mike

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Are you saying that fibroblasts etc. do not do fluid phase endocytosis? If they are, how could they exclude a quantum dot that is in the fluid phase? Dave

On Jun 18, 2008, at 6:17 PM, Ignatius, Mike wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Dries,

And from our studies as well, the uncoated Quantum Dot nanocrystals are not taken up by cells. Nor are our PEG coated dots designed for in vivo blood flow studies. Thus the QTracker line of reagents were designed to deliver material using poly-arg peptides on the outside.

There is one study I am aware showing smaller, unconjugated dots made of Cd/TE that aren't commercially available can be taken up by phagocytic cells.

<a href="javascript:AL_get(this, 'jour', 'Nano Lett.');" style="color: blue; text-decoration: underline; ">Nano Lett. 2007 Nov;7(11):3452-61. Epub 2007 Oct 19.Nonfunctionalized nanocrystals can exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Nabiev I, Mitchell S, Davies A, Williams Y, Kelleher D, Moore R, Gun'ko YK, Byrne S, Rakovich YP, Donegan JF, Sukhanova A, Conroy J, Cottell D, Gaponik N, Rogach A, Volkov Y.

Mike

Mike Ignatius, Ph.D. Molecular Probes labeling and detection technologies
INVITROGEN | 29851 Willow Creek Road, Eugene, OR 97402-9132
Tel: 541-335-0414 | Cell: 541-510-1736 [hidden email]

From: Confocal Microscopy List [[hidden email]] On Behalf Of Rietdorf, Jens
Sent: Wednesday, June 18, 2008 1:09 AM
To: [hidden email]
Subject: Re: Endocytosis of Quantum Dots

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Dries,

indeed, my experience is such that uncoated QDs are not taken up, at least not into fibroblast or osteosarcoma cell lines. Lectin coated QDs are excellent markers for the plasma membrane, because they are not taken up either, unlike many other lectin-dye conjugates. These experiences date back some time (2 years?) and things may have changed.

Cheers, jens

From: Confocal Microscopy List [[hidden email]] On Behalf Of Dries Vercauteren
Sent: Tuesday, June 17, 2008 4:08 PM
To: [hidden email]
Subject: Endocytosis of Quantum Dots

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

an easy question;

are free (unconjugated) Quantum Dots normally not taken up via endocytosis? Or is this dependent on charge, avidin coating, ...?

Thanks for the response in advance!

Dries

--
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone: +329/264 80 49
Mobile: +32485/30 69 80
E-mail: [hidden email]
[hidden email]

Dr. David Knecht

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)

John Oreopoulos

Playback of large movies with high frame rates

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear confocal listserver,

I just posted the following email to the ImageJ listserver, but I thought perhaps people in this community might be able to help too:

I have a small problem I was hoping the community might be able to advise me on. I have several long time-lapse live-cell image sequences captured using TIRF microscopy. 1000 images were captured with a 200 ms exposure time and a 500 ms delay in between exposures. A typical image sequence file size is about 500 MB. It is necessary to capture many images and with this rate of repetition since the the cells I am imaging express a fluorescent membrane protein that exhibits very interesting dynamics (both fast and slow motions) and over a long time period. I do not have the option of capturing fewer images over the same time interval since doing so will cause the software to miss recording very transient and fast vesicle fusion events with the membrane. We use ImageJ for analysis and we run the program on a powerful Linux computer with 4GB of RAM. To see the protein dynamics, it is necessary to play back the image sequence with a 300 ms frame rate on the computer screen in ImageJ, which is possible with the latest version of ImageJ (max frame rate 1000 fps or frame rate allowed by memory and processor speed). If played slower than this, then it's difficult for the human eye to discern a fusion event and notice any protein motion.

My problem is that I would like to make these movies portable for presentations (in Powerpoint or Quicktime for example) without too much compression that masks the features I'm trying to show and without slowing them down to a lower frame rate.

I usually save image sequences as .avi files, but I've noticed that if I do this when the display frame rate in ImageJ is greater than 100 ms, the avi is saved with a frame rate of 100 ms. Is this a bug or is it simply a limitation of the .avi format, or is it a limitation of the player I use to playback the .avi file (Quicktime)? I can find no mention of a maximum frame rate in the .avi writer plugin on the ImageJ website.

Even if I can save files properly with these high frame rates, I likely will not be able to embed them in Powerpoint and play them at that speed since the demands on my laptop for this file size and display rate are too great. Does anyone know of an alternative way for displaying large movies with high frame rates?

Thank you for your time.

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Julio Vazquez

Re: Playback of large movies with high frame rates

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,

I'm a bit confused... if you need a frame rate of 300 ms (~ 3 frames per second), then a avi's frame rate of 100 ms (10 frames per second) should be sufficient, no? As for imagej's 1000 frames per second (1 ms per frame), that would be about 10 times faster than most monitor's refresh rate (?)...

In any event, with QuickTime Pro, you can reformat your movies at speeds up to 60 frames per second (16 milliseconds per frame).

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N., mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org/

On Jun 23, 2008, at 11:29 AM, John Oreopoulos wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear confocal listserver,

I just posted the following email to the ImageJ listserver, but I thought perhaps people in this community might be able to help too:

I have a small problem I was hoping the community might be able to advise me on. I have several long time-lapse live-cell image sequences captured using TIRF microscopy. 1000 images were captured with a 200 ms exposure time and a 500 ms delay in between exposures. A typical image sequence file size is about 500 MB. It is necessary to capture many images and with this rate of repetition since the the cells I am imaging express a fluorescent membrane protein that exhibits very interesting dynamics (both fast and slow motions) and over a long time period. I do not have the option of capturing fewer images over the same time interval since doing so will cause the software to miss recording very transient and fast vesicle fusion events with the membrane. We use ImageJ for analysis and we run the program on a powerful Linux computer with 4GB of RAM. To see the protein dynamics, it is necessary to play back the image sequence with a 300 ms frame rate on the computer screen in ImageJ, which is possible with the latest version of ImageJ (max frame rate 1000 fps or frame rate allowed by memory and processor speed). If played slower than this, then it's difficult for the human eye to discern a fusion event and notice any protein motion.
My problem is that I would like to make these movies portable for presentations (in Powerpoint or Quicktime for example) without too much compression that masks the features I'm trying to show and without slowing them down to a lower frame rate.
I usually save image sequences as .avi files, but I've noticed that if I do this when the display frame rate in ImageJ is greater than 100 ms, the avi is saved with a frame rate of 100 ms. Is this a bug or is it simply a limitation of the .avi format, or is it a limitation of the player I use to playback the .avi file (Quicktime)? I can find no mention of a maximum frame rate in the .avi writer plugin on the ImageJ website.
Even if I can save files properly with these high frame rates, I likely will not be able to embed them in Powerpoint and play them at that speed since the demands on my laptop for this file size and display rate are too great. Does anyone know of an alternative way for displaying large movies with high frame rates?

Thank you for your time.

John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

John Oreopoulos

Re: Playback of large movies with high frame rates

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal My mistake, I should have written "fps" for the playback frame rate instead of "ms". I got my units mixed up in the second paragraph of my email.

John

On 23-Jun-08, at 2:48 PM, Julio Vazquez wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,

I'm a bit confused... if you need a frame rate of 300 ms (~ 3 frames per second), then a avi's frame rate of 100 ms (10 frames per second) should be sufficient, no? As for imagej's 1000 frames per second (1 ms per frame), that would be about 10 times faster than most monitor's refresh rate (?)...

In any event, with QuickTime Pro, you can reformat your movies at speeds up to 60 frames per second (16 milliseconds per frame).

--
Julio Vazquez, PhD
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N., mailstop DE-512
Seattle, WA 98109-1024

http://www.fhcrc.org/

==

On Jun 23, 2008, at 11:29 AM, John Oreopoulos wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear confocal listserver,

I just posted the following email to the ImageJ listserver, but I thought perhaps people in this community might be able to help too:

I have a small problem I was hoping the community might be able to advise me on. I have several long time-lapse live-cell image sequences captured using TIRF microscopy. 1000 images were captured with a 200 ms exposure time and a 500 ms delay in between exposures. A typical image sequence file size is about 500 MB. It is necessary to capture many images and with this rate of repetition since the the cells I am imaging express a fluorescent membrane protein that exhibits very interesting dynamics (both fast and slow motions) and over a long time period. I do not have the option of capturing fewer images over the same time interval since doing so will cause the software to miss recording very transient and fast vesicle fusion events with the membrane. We use ImageJ for analysis and we run the program on a powerful Linux computer with 4GB of RAM. To see the protein dynamics, it is necessary to play back the image sequence with a 300 ms frame rate on the computer screen in ImageJ, which is possible with the latest version of ImageJ (max frame rate 1000 fps or frame rate allowed by memory and processor speed). If played slower than this, then it's difficult for the human eye to discern a fusion event and notice any protein motion.
My problem is that I would like to make these movies portable for presentations (in Powerpoint or Quicktime for example) without too much compression that masks the features I'm trying to show and without slowing them down to a lower frame rate.
I usually save image sequences as .avi files, but I've noticed that if I do this when the display frame rate in ImageJ is greater than 100 ms, the avi is saved with a frame rate of 100 ms. Is this a bug or is it simply a limitation of the .avi format, or is it a limitation of the player I use to playback the .avi file (Quicktime)? I can find no mention of a maximum frame rate in the .avi writer plugin on the ImageJ website.
Even if I can save files properly with these high frame rates, I likely will not be able to embed them in Powerpoint and play them at that speed since the demands on my laptop for this file size and display rate are too great. Does anyone know of an alternative way for displaying large movies with high frame rates?

Thank you for your time.

John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Mario-2

Re: Endocytosis of Quantum Dots

In reply to this post by Dries Vercauteren

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: Endocytosis of Quantum Dots

Dries,

When we first published successful results using nanocrystal phosphors (CdSe/ZnS) for biological labeling (Marcel Bruchez Jr., et al (1998). Semiconductor nanocrystals as fluorescent biological labels. Science 281: 2013-2016), silanization was used to stabilize the surface, rendering the QDs inert yet dispersible in aqueous media. Resulting surface groups were either siloxanes with polar -OH groups, or other versions that displayed thiols or amino groups used for conjugation of IgG, avidin, other targeting molecules.

Followup experiments testing for endocytosis were done by adding polar siloxane coated QDs to cell growth medium and treating NIH-3T3 cells overnight or longer. The QDs did not appear toxic to cell growth. Further, the treated cells when imaged using confocal showed that QDs were clearly visible inside the cells with a tendency to cluster near the nucleus where one usually fines endoplasmic reticulum and lysosomes. This work never made it past the Biophysics 1999 meeting at least while I was involved. I am sure much has been done since then, but in any event, I do think it likely that QDs depending on the time of exposure, their specific surface groups, and the cell type you are using will have a significant effect as to whether you get endocytosis or not. If you are going to buy some you might want to make sure that they do or they don't for the particular lots that are available before investing a lot of money.

My two cents,

Mario M.

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

an easy question;

are free (unconjugated) Quantum Dots normally not taken up via endocytosis? Or is this dependent on charge, avidin coating, ...?

Thanks for the response in advance!

Dries

--
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone: +329/264 80 49
Mobile: +32485/30 69 80
E-mail: [hidden email]
[hidden email]

--

________________________________________________________________________________

Mario M. Moronne, Ph.D.

[hidden email]

[hidden email]
[hidden email]

Kevin Hodgson

Re: Endocytosis of Quantum Dots

In reply to this post by Dries Vercauteren

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Dries;

I'm not sure of the current state of the art of QD coatings, but we
found that phagocytosis of QD's, even when coated with PEG's, occurred
to some degree or another. We were able to take advantage of this
property, as the rates of systemic clearance of Q-Tracker dots, used
as an agioscopic dye, differed and depended upon the health of the
animal subject. Our realization was that "benign" QD's will be
endocytozed (a good thing for our analysis)... it's just a matter of
time.

There are two articles that may interest you in the Nov-Dec 2007 issue
of Biomedical Optics:

Endotoxemia increases the clearance of mPEGylated 5000-MW quantum dots
as revealed by multiphoton microvascular imaging
Bateman, et al
J. Biomed. Opt. Vol. 12, 064005 (Dec. 18, 2007)

Core-shell silica nanoparticles as fluorescent labels for nanomedicine
Jinhyang Choi, et al
J. Biomed. Opt. Vol. 12, 064007 (Dec. 28, 2007)

Hope this helps,

Kevin

On 17-Jun-08, at 7:07 AM, Dries Vercauteren wrote:

> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> an easy question;
>
> are free (unconjugated) Quantum Dots normally not taken up via
> endocytosis? Or is this dependent on charge, avidin coating, ...?
>
> Thanks for the response in advance!
>
> Dries
>
> --
> Dries Vercauteren, PhD student
> Master of Bioscience Engineering: Cell and Gene Biotechnology
>
> Ghent Research Group on Nanomedicines
> www.ugent.be/fw/en/research/biofys
> Faculty of pharmaceutical sciences, Ghent University
> Harelbekestraat 72, 9000 Ghent
> Belgium
>
> Phone: +329/264 80 49
> Mobile: +32485/30 69 80
> E-mail: [hidden email]
> [hidden email]

Kevin Hodgson
BioImaging Facility, Botany
UBC Vancouver Canada
www.emlab.ubc.ca
604-822-6996

Bill Oliver-3

Re: Playback of large movies with high frame rates

In reply to this post by John Oreopoulos

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

An excellent freeware program for such manipulations is mplayer (and in its format manipulation mode, aka mencoder). This is the most prominent open source easy video format encoding app. It is native to Linux, but has ports to Windoze and MacOSX. See:

http://www.mplayerhq.hu/design7/news.html

for the general page, and

http://www.mplayerhq.hu/design7/projects.html#windows

for Windoze ports.

Because of the huge number of options, you need to read the documentation if you want to play with all the paramaters. It is written for command-line control, but there are a number GUIs to act as more friendly front ends. You also want to download the complete codec sets.

billo

On Mon, 23 Jun 2008, John Oreopoulos wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> My mistake, I should have written "fps" for the playback frame rate instead
> of "ms". I got my units mixed up in the second paragraph of my email.
>
> John
>
> On 23-Jun-08, at 2:48 PM, Julio Vazquez wrote:
>
>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-
>> bin/wa?S1=confocal Hi John,
>>
>> I'm a bit confused... if you need a frame rate of 300 ms (~ 3 frames per
>> second), then a avi's frame rate of 100 ms (10 frames per second) should be
>> sufficient, no? As for imagej's 1000 frames per second (1 ms per frame),
>> that would be about 10 times faster than most monitor's refresh rate (?)...
>>
>> In any event, with QuickTime Pro, you can reformat your movies at speeds up
>> to 60 frames per second (16 milliseconds per frame).
>>
>> --
>> Julio Vazquez, PhD
>> Fred Hutchinson Cancer Research Center
>> 1100 Fairview Ave. N., mailstop DE-512
>> Seattle, WA 98109-1024
>>
>>
>> http://www.fhcrc.org/
>>
>> ==
>>
>>
>> On Jun 23, 2008, at 11:29 AM, John Oreopoulos wrote:
>>
>>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/
>>> cgi-bin/wa?S1=confocal Dear confocal listserver,
>>>
>>> I just posted the following email to the ImageJ listserver, but I thought
>>> perhaps people in this community might be able to help too:
>>>
>>>
>>> I have a small problem I was hoping the community might be able to advise
>>> me on. I have several long time-lapse live-cell image sequences captured
>>> using TIRF microscopy. 1000 images were captured with a 200 ms exposure
>>> time and a 500 ms delay in between exposures. A typical image sequence
>>> file size is about 500 MB. It is necessary to capture many images and with
>>> this rate of repetition since the the cells I am imaging express a
>>> fluorescent membrane protein that exhibits very interesting dynamics (both
>>> fast and slow motions) and over a long time period. I do not have the
>>> option of capturing fewer images over the same time interval since doing
>>> so will cause the software to miss recording very transient and fast
>>> vesicle fusion events with the membrane. We use ImageJ for analysis and we
>>> run the program on a powerful Linux computer with 4GB of RAM. To see the
>>> protein dynamics, it is necessary to play back the image sequence with a
>>> 300 ms frame rate on the computer screen in ImageJ, which is possible with
>>> the latest version of ImageJ (max frame rate 1000 fps or frame rate
>>> allowed by memory and processor speed). If played slower than this, then
>>> it's difficult for the human eye to discern a fusion event and notice any
>>> protein motion.
>>> My problem is that I would like to make these movies portable for
>>> presentations (in Powerpoint or Quicktime for example) without too much
>>> compression that masks the features I'm trying to show and without slowing
>>> them down to a lower frame rate.
>>> I usually save image sequences as .avi files, but I've noticed that if I
>>> do this when the display frame rate in ImageJ is greater than 100 ms, the
>>> avi is saved with a frame rate of 100 ms. Is this a bug or is it simply a
>>> limitation of the .avi format, or is it a limitation of the player I use
>>> to playback the .avi file (Quicktime)? I can find no mention of a maximum
>>> frame rate in the .avi writer plugin on the ImageJ website.
>>> Even if I can save files properly with these high frame rates, I likely
>>> will not be able to embed them in Powerpoint and play them at that speed
>>> since the demands on my laptop for this file size and display rate are too
>>> great. Does anyone know of an alternative way for displaying large movies
>>> with high frame rates?
>>>
>>> Thank you for your time.
>>>
>>>
>>> John Oreopoulos, BSc,
>>> PhD Candidate
>>> University of Toronto
>>> Institute For Biomaterials and Biomedical Engineering
>>> Centre For Studies in Molecular Imaging
>>>
>>> Tel: W:416-946-5022
>>>
>>
>
>

billo
http://www.billoblog.com/billoblog

Ricardo Figueroa

Re: Playback of large movies with high frame rates

In reply to this post by John Oreopoulos

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Since the actual frame rate that is displayed on your average monitor can be no more than ~80 fps how about merging every 5 slides ie. a 1000 frame stack --> 200 and runing the movie at a frame rate of 60 fps (actually this is probably what is happening in imagej in real time the images are simply written on top of each other creating a blur until the buffer is dumped to the screen).

This macro (ImageJ) should do the trick just modify stuff to suit to your needs

title = getTitle();
slices = nSlices();
n = 4;
selectImage(title);
run("Z Project...", "start=1 stop="+n+" projection=[Average Intensity]");
rename("AVG Projection of "+title+" every " + n + " slices");
setBatchMode(true);
for(i = 2*n; i <= slices; i = i+n){
    selectImage(title);
    run("Z Project...", "start="+ i-n +" stop=" + i +" projection=[Average Intensity]");
    run("Select All");
    run("Copy");
    close();
    selectImage("AVG Projection of "+title+" every " + n + " slices");
    setSlice(nSlices());
    run("Add Slice");
    run("Paste");
}
setBatchMode(false);

//Ricardo Figueroa

John Oreopoulos wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear confocal listserver,

I just posted the following email to the ImageJ listserver, but I thought perhaps people in this community might be able to help too:

I have a small problem I was hoping the community might be able to advise me on. I have several long time-lapse live-cell image sequences captured using TIRF microscopy. 1000 images were captured with a 200 ms exposure time and a 500 ms delay in between exposures. A typical image sequence file size is about 500 MB. It is necessary to capture many images and with this rate of repetition since the the cells I am imaging express a fluorescent membrane protein that exhibits very interesting dynamics (both fast and slow motions) and over a long time period. I do not have the option of capturing fewer images over the same time interval since doing so will cause the software to miss recording very transient and fast vesicle fusion events with the membrane. We use ImageJ for analysis and we run the program on a powerful Linux computer with 4GB of RAM. To see the protein dynamics, it is necessary to play back the image sequence with a 300 ms frame rate on the computer screen in ImageJ, which is possible with the latest version of ImageJ (max frame rate 1000 fps or frame rate allowed by memory and processor speed). If played slower than this, then it's difficult for the human eye to discern a fusion event and notice any protein motion.

My problem is that I would like to make these movies portable for presentations (in Powerpoint or Quicktime for example) without too much compression that masks the features I'm trying to show and without slowing them down to a lower frame rate.

I usually save image sequences as .avi files, but I've noticed that if I do this when the display frame rate in ImageJ is greater than 100 ms, the avi is saved with a frame rate of 100 ms. Is this a bug or is it simply a limitation of the .avi format, or is it a limitation of the player I use to playback the .avi file (Quicktime)? I can find no mention of a maximum frame rate in the .avi writer plugin on the ImageJ website.

Even if I can save files properly with these high frame rates, I likely will not be able to embed them in Powerpoint and play them at that speed since the demands on my laptop for this file size and display rate are too great. Does anyone know of an alternative way for displaying large movies with high frame rates?

Thank you for your time.

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022