Phototrophic autofluorescence detection

[Sam Albers]

Hello all,

I am about to begin a confocal analysis of biofilms in a salmon stream. I am interested in the composition of these biofilms. I am using are Syto9 to visualize bacteria, Triticum Vulgaris lectin conjugated with TRITC to visualize EPS and the autofluoroescence signal in the far red of chlorophyll a to visualize algae. My concern/ hope is that the biofilm will contain a significant cyanobacterial population. Cyanobacterial pigments (phycobilins) give a strong emission signal in the red part of the visible spectrum. My concern is that my TRITC lectin also gives an emission signal in the red part of the spectrum. According to Neu et al. (2004), "a TRITC labelled lectin may be employed and detected in the red channel together with the autofluorescence of cyanobacteria".

To do this would I use the same laser and record two different emission signals on different channels? This doesn't seem quite right as it seems like there might be a significant amount of bleed-through here. Is this where I would use colocalization techniques to resolve the difference? Alternatively, if I don't choose to measure cyanobacterial abundance, will this affect my conclusions from the TRITC labelled lectin?

So to restate, I would like to detect/ account for the presence of cyanobacteria in my samples while still being able to detect the TRITC labelled lectin. How do I separate those two signals?

My system is an Olympus Fluview 1000 with 458/488/515nm all from a multiline argon gas laser, and 543nm and 633nm from independent HeNe gas lasers.

Any thoughts on this would be much appreciated.

Thanks in advance.

Sam

--
*****************************************************
Sam Albers
Geography Program
University of Northern British Columbia
3333 University Way
Prince George, British Columbia
Canada, V2N 4Z9
phone: [phone]
*****************************************************

[Rietdorf, Jens]

Hello Sam,

 

If you have enough a priori knowledge about the spectra you might think about using spectral un-mixing to separate these. FluoView software offers algorithms for that. At the very least you should be able to separate the TRITC signal from the bulk of red signal.

 

Best, jens

 

---
dr. jens rietdorf[hidden email]
head microscopy
novartis research foundation
friedrich-miescher-institute, wro1066.2.16
maulbeerstr.66, 4058 basel, switzerland
txt: rietdorf at fmi dot ch

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Albers
Sent: Sunday, June 28, 2009 10:23 PM
To: [hidden email]
Subject: Phototrophic autofluorescence detection

 

Hello all,

I am about to begin a confocal analysis of biofilms in a salmon stream. I am interested in the composition of these biofilms. I am using are Syto9 to visualize bacteria, Triticum Vulgaris lectin conjugated with TRITC to visualize EPS and the autofluoroescence signal in the far red of chlorophyll a to visualize algae. My concern/ hope is that the biofilm will contain a significant cyanobacterial population. Cyanobacterial pigments (phycobilins) give a strong emission signal in the red part of the visible spectrum. My concern is that my TRITC lectin also gives an emission signal in the red part of the spectrum. According to Neu et al. (2004), "a TRITC labelled lectin may be employed and detected in the red channel together with the autofluorescence of cyanobacteria".

To do this would I use the same laser and record two different emission signals on different channels? This doesn't seem quite right as it seems like there might be a significant amount of bleed-through here. Is this where I would use colocalization techniques to resolve the difference? Alternatively, if I don't choose to measure cyanobacterial abundance, will this affect my conclusions from the TRITC labelled lectin?

So to restate, I would like to detect/ account for the presence of cyanobacteria in my samples while still being able to detect the TRITC labelled lectin. How do I separate those two signals?

My system is an Olympus Fluview 1000 with 458/488/515nm all from a multiline argon gas laser, and 543nm and 633nm from independent HeNe gas lasers.

Any thoughts on this would be much appreciated.

Thanks in advance.

Sam

--
*****************************************************
Sam Albers
Geography Program
University of Northern British Columbia
3333 University Way
Prince George, British Columbia
Canada, V2N 4Z9
phone: [phone]
*****************************************************

[Stanislav Vitha]

Hallo Sam,

Chlorophylls have an emission peak around 680nm and are easily excited by
the 488nm laser.
I normally use the 488 nm laser line for imaging GFP and simultaneously collect
the chlorophyll fluorescence signal in the second channel (Emission basndpass
set to ~660 to 720nm on our FV1000 confocal). The same should work for
your Syto9 + chlorophyll detection.

The TRITC has absorption peak close to the 543nm wavelength of your HeNe
laser, emisssion peak is around 580 nm.

So I would set up a sequential scan with Syto9 (detector 1) + chlorophyll
(detector 3) in one group, and TRITC (detector 2) in the second group.

Our FV1000 has two spectral detectors, so if your is the same configuration,
the third detector is filter based. Make sure you use a suitable long-pass filter
(e.g., 650nm) in front of the third detector to block the TRITC signal that may
be excited by the 488 nm.
Also , for the 2nd detector (TRITC signal), set the detection with enough
separation from the chlorophyll peak (something like 560 - 600nm) so that the
chlorophyll signal (680nm peak) is not detected in this channel.  
   
Your controls ("no-TRITC", "no SYTO"), should tell you if your detection
scheme is specific enough.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

 
When working with plants, I
On Sun, 28 Jun 2009 13:23:11 -0700, Sam Albers
<[hidden email]> wrote:

>Hello all,
>
>I am about to begin a confocal analysis of biofilms in a salmon stream. I am
>interested in the composition of these biofilms. I am using are Syto9 to
>visualize bacteria, *Triticum Vulgaris* lectin conjugated with TRITC to
>visualize EPS and the autofluoroescence signal in the far red of chlorophyll
>a to visualize algae. My concern/ hope is that the biofilm will contain a
>significant cyanobacterial population. Cyanobacterial pigments (phycobilins)
>give a strong emission signal in the red part of the visible spectrum. My
>concern is that my TRITC lectin also gives an emission signal in the red
>part of the spectrum. According to Neu et al.
>(2004)<http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-

4B5435C-
3&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_se
archStrId=942123313&_rerunOrigin=scholar.google&_acct=C000050221&_versio
n=1&_urlVersion=0&_userid=10&md5=28fd37bbe4ea8d0364d9f7dcd9624379>,

>"a TRITC labelled lectin may be employed and detected in the red channel
>together with the autofluorescence of cyanobacteria".
>
>To do this would I use the same laser and record two different emission
>signals on different channels? This doesn't seem quite right as it seems
>like there might be a significant amount of bleed-through here. Is this
>where I would use colocalization techniques to resolve the difference?
>Alternatively, if I don't choose to measure cyanobacterial abundance, will
>this affect my conclusions from the TRITC labelled lectin?
>
>So to restate, I would like to detect/ account for the presence of
>cyanobacteria in my samples while still being able to detect the TRITC
>labelled lectin. How do I separate those two signals?
>
>My system is an Olympus Fluview 1000 with 458/488/515nm all from a

multiline

>argon gas laser, and 543nm and 633nm from independent HeNe gas lasers.
>
>Any thoughts on this would be much appreciated.
>
>Thanks in advance.
>
>Sam
>
>--
>*****************************************************
>Sam Albers
>Geography Program
>University of Northern British Columbia
>3333 University Way
>Prince George, British Columbia
>Canada, V2N 4Z9
>phone: [phone]
>*****************************************************
>

[Ignatius, Mike-2]

In collaboration with Betsey Pitts at the Center for Biofilm Engineering, Montana State University, Bozeman
we have just launched a series of dyes for biofilm based detection.  Dyes for the microbes and the deposited biofilm itself.

 

For all the details see:  http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-and-Tissue-Analysis/Microbiological-Analysis/FilmTracer-Biofilm-Stains.html

 

For example as you are concerned about red emission overlap, there are green and even violet alternatives for staining cells. 

 

Mike Ignatius

 

Molecular Probes/Invitrogen

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Albers
Sent: Sunday, June 28, 2009 1:23 PM
To: [hidden email]
Subject: Phototrophic autofluorescence detection

Hello all,

I am about to begin a confocal analysis of biofilms in a salmon stream. I am interested in the composition of these biofilms. I am using are Syto9 to visualize bacteria, Triticum Vulgaris lectin conjugated with TRITC to visualize EPS and the autofluoroescence signal in the far red of chlorophyll a to visualize algae. My concern/ hope is that the biofilm will contain a significant cyanobacterial population. Cyanobacterial pigments (phycobilins) give a strong emission signal in the red part of the visible spectrum. My concern is that my TRITC lectin also gives an emission signal in the red part of the spectrum. According to Neu et al. (2004), "a TRITC labelled lectin may be employed and detected in the red channel together with the autofluorescence of cyanobacteria".

To do this would I use the same laser and record two different emission signals on different channels? This doesn't seem quite right as it seems like there might be a significant amount of bleed-through here. Is this where I would use colocalization techniques to resolve the difference? Alternatively, if I don't choose to measure cyanobacterial abundance, will this affect my conclusions from the TRITC labelled lectin?

So to restate, I would like to detect/ account for the presence of cyanobacteria in my samples while still being able to detect the TRITC labelled lectin. How do I separate those two signals?

My system is an Olympus Fluview 1000 with 458/488/515nm all from a multiline argon gas laser, and 543nm and 633nm from independent HeNe gas lasers.

Any thoughts on this would be much appreciated.

Thanks in advance.

Sam

--
*****************************************************
Sam Albers
Geography Program
University of Northern British Columbia
3333 University Way
Prince George, British Columbia
Canada, V2N 4Z9
phone: [phone]
*****************************************************