Qdots and FISH

[Boswell, Carl A - (cboswell)]

Hi all,
A recent query came up here for which I had no answer.  Someone wants to do
multichannel FISH (up to 7 labels) and I immediately thought of quantum
dots.  However I don't know how easy it is to get Qdots into a fixed
bacterium.  The specimen will be paraffin embedded and sectioned gut of an
insect.  Any thoughts?

Thanks,
c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Joshua Larkin]

Carl,

 

I have recently entertained this idea but, seeing as there is limited report of success using Q-dots for FISH, I have not been adventurous enough to try it. I came across a paper (Ioannou et al., "Quantum dots as a new-generation fluorochromes for FISH: an appraisal", Chromosome Research, 17, 2009) that, in summary, reports successful indirect immuno-labeled FISH using Q-dots; however, direct-labeled FISH probes, using commercially available Q-dots, doesn't seem to produce favorable results. Now, the work in this report was on human cells, so maybe you would have better luck in bacteria.

 

Hope this helps,

Josh

 

--
Josh Larkin, Ph.D.
Post-doctoral Fellow
Sir William Dunn School of Pathology
University of Oxford
Oxford, UK, OX1 3RE
[hidden email]

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Carl Boswell [[hidden email]]
Sent: 08 June 2010 20:01
To: [hidden email]
Subject: Qdots and FISH

Hi all,
A recent query came up here for which I had no answer.  Someone wants to do
multichannel FISH (up to 7 labels) and I immediately thought of quantum
dots.  However I don't know how easy it is to get Qdots into a fixed
bacterium.  The specimen will be paraffin embedded and sectioned gut of an
insect.  Any thoughts?

Thanks,
c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[M. van de corput]

I have tried twice to get it to work. Both to get it to my DNA (which was a big mess in
the end) and using indirect with anti-bodies (sometimes ok but most of the time huge
background and a lot of aggregates). My experience is that the q-dots tend to aggregate
and stick to the membrane of the nucleus. With bacteria I would think accessibility might
be a problem. The paraffin does not help either but with thin enough sections I guess it
would be doable. I would try first one q-dot staining before doing multi-colour FISH. The
few times I was successful the signals were absolutely great, that was indirect using a
digoxigenin labeled probe. You can also try a combination of both q-dots (when you get one
or two to work in you FISH) and conventional dyes.

Good luck,
Mariette

on 08-06-2010 21:01 Carl Boswell said the following:

> Hi all,
> A recent query came up here for which I had no answer.  Someone wants to
> do multichannel FISH (up to 7 labels) and I immediately thought of
> quantum dots.  However I don't know how easy it is to get Qdots into a
> fixed bacterium.  The specimen will be paraffin embedded and sectioned
> gut of an insect.  Any thoughts?
>
> Thanks,
> c
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]

[George McNamara]

Hi Carl,

7 targets with small dyes by spectral immunofluorescence (and optionally DAPI - so could have 8 colors) has been done:

J Histochem Cytochem. 2000 May;48(5):653-62.

Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition.

Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T.

Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan. [hidden email]

Abstract

Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 and Cy5.5, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images.

PMID: 10769049

If the FISH probes are small and dim, I recommend multiple rounds of fluorescent tyramide signal amplification. See Van Tine 2004 for procedure (they used up to three rounds). For oligo FISH, this implies 7 rounds of HRP-oligo's (or HRP-peptide nucleic acids? - could be pricey) followed by HRP + fluorescent-TSA. If using haptens, with the right combination of species or isotypes, could get done in fewer rounds (e.g. first two oligo-hapten probes, HRP-anti-mouse, detect, kill HRP, HRP-anti-rabbit, detect, kill HRP, apply next round of probes).


Methods Mol Biol. 2005;292:215-30.

Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence.

Van Tine BA, Broker TR, Chow LT.

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Abstract

The use of tyramide-coupled immunofluorescence at the single cell level provides expedient, clean, and sensitive signals for detection of DNA, RNA, or proteins. The principle is based on the ability of horseradish peroxidase (HRP) to cleave tyramides into a free radical species with a very short diffusion radius. The free radicals are then covalently bound to electron-rich moieties such as tyrosine in proteins proximal to the targets. Here we present protocols for tyramide fluorescent in situ hybridization (T-FISH), which detects unique DNA species using DNA probes as short as approx 300-500 bp, or unique RNA species with probes as small as an oligonucleotide. We also present a protocol for tyramide immunofluorescence (T-IF) to detect protein antigens. By combining these protocols with several tyramide-coupled fluorophores, multiple targets can be detected simultaneously in situ, which is ideal for in-depth analyses at the molecular and cellular levels. Finally, we describe the detection of nascent viral RNA transcripts simultaneously with integrated viral genomes or chromosomal domains in single cells or tissue sections.

PMID: 15507711

At 03:01 PM 6/8/2010, you wrote:

Hi all,
A recent query came up here for which I had no answer.  Someone wants to do multichannel FISH (up to 7 labels) and I immediately thought of quantum dots.  However I don't know how easy it is to get Qdots into a fixed bacterium.  The specimen will be paraffin embedded and sectioned gut of an insect.  Any thoughts?

Thanks,
c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33136
[hidden email]
[hidden email]
305-443-8436 office
323-251-8878 cell