There are many options to do 5 color multiplexing and even more colors have been achieved. Do you work on fixed cells? If not, options may be a bit more limited, but also that depends on the speed of you confocal and the speed of the structures you try to record. Your first option to detect more dyes is, as Regina mentioned already, sequential scanning. Then, you may give linear unmixing a try, i.e. you record lambda scans for all dyes individually and a lambda scan of you multi stained probe. Linear unmixing calculations are then able to separate the dyes. The finer the lambda scans, the better the result. Finally, the easiest option is probably to give AF700 and AF750 a try. They both should work well with 633 excitation. Or look for other near infrared (NIR) dyes with large stokes shifts. Last but not least, DAPI may not be you best friend as it has a pretty broad emission spectrum. SYBR Green, DRAQ5, or the whole POPO, BOBO, YOYO, TOTO, JOJO, TO-PRO range of dyes may be better alternatives to stain DNA in your application.

I have done it

but it depends what kind of lasers does your confocal have

if your confocal has 561 yellow-green laser then you can do 594 too that makes it 5 colors. In addition you can do a lot

It is to important to note that some fluorophores have an overlapping emission spectra. So sequential acquisition is more indicated in this cases, mainly if you have colocalization experiment. You can acquire simultaneously the fluorophores with no emission overlapping and sequentially the others which have crosstalk between the channels

There is some large stokes-shift dyes (google this) on the market, like some DyLights (http://www.piercenet.com/product/dylight-long-stokes-shift-specialty-dyes). Moreover I could estimate to use quantum dots, where single excitation wavelength can be used to elicit emission of different quantum dot formulations. To separate the 5 dyes, filter based setups will not be successful - a spectroscopic approach, like with a Meta or Quasar detector (Zeiss) will become essential.

josef

There are many options to do 5 color multiplexing and even more colors have been achieved. Do you work on fixed cells? If not, options may be a bit more limited, but also that depends on the speed of you confocal and the speed of the structures you try to record. Your first option to detect more dyes is, as Regina mentioned already, sequential scanning. Then, you may give linear unmixing a try, i.e. you record lambda scans for all dyes individually and a lambda scan of you multi stained probe. Linear unmixing calculations are then able to separate the dyes. The finer the lambda scans, the better the result. Finally, the easiest option is probably to give AF700 and AF750 a try. They both should work well with 633 excitation. Or look for other near infrared (NIR) dyes with large stokes shifts. Last but not least, DAPI may not be you best friend as it has a pretty broad emission spectrum. SYBR Green, DRAQ5, or the whole POPO, BOBO, YOYO, TOTO, JOJO, TO-PRO range of dyes may be better alternatives to stain DNA in your application.

Thanks a lot for all your suggestions!

Im basically working with fixed blood vessels crossections and our leica microscope has the 405, 488, 561 and 633 lasers.

Im going to acquire the fluorophores sequentially and/or use the liner unmixing.

I have tried with AF594 in addition and next time I would try with AF405 (this last one I never used before)... Regarding DAPI, thanks! Im going to try with other dyes.

I've not worked with 5 fluophores in the same sample but I did with 2 fluophores and I had sometimes superposed emission because our confocal does not have option to separate the lambda of emission. Did you get it with sequential scanning? Otherwise, for DNA dye which excited in blue and emits in red, I suggest Acridine Orange. DAPI is always in the blue.

Ideally, a dye right between 555 and 647 (such as Alexa Fluor 594) would be the best option in your setup.

The 'limitation' in your case is the availability of 'only' four lasers. Unmixing could indeed be your solution.

I've three additional tips:

1. Try to select proteins that are expressed in different cellular locations or cell types, that helps in discriminating signals.

2. Do you know about the Fluorescence Spectraviewer? It's a nice informative tool, you can select multiple dyes and compare their excitation and emission spectra:

http://www.lifetechnologies.com/europe-other/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

3. As an alternative to DAPI, you could try to use TO-PRO-3 (as Tobial suggested) as a general nuclear marker, it's emission is a bit like AF 647.

good luck!

Example of the spectraviewer (attachment):

The Five-Color-Imaging problem, albeit for the case of using an arc lamp rather than lasers, has recently also been addressed in Kurt's Microscopy Blog (http://nic.ucsf.edu/blog/?p=813). I can more than recommend Kurt's Blog.

I guess four would be the best. I haven't used 5 colors. It depends on the filters what you have. Please check your Microcope filters.

Already >15 years ago, Multi-color-FISH approaches used 7 colors in addition to DAPI. (not confocal, there it was restricted to 5-6). Combinatorial labeling then allowed to label all 24 different chromosomes in a specific color combination. Here are some of those papers:

Nat Genet. 1996 Apr;12(4):368-75. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Speicher MR, Gwyn Ballard S, Ward DC.

PLoS Biol. 2005 May;3(5):e157. Epub 2005 Apr 26. Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. Bolzer A, Kreth G, Solovei I, Koehler D, Saracoglu K, Fauth C, Müller S, Eils R, Cremer C, Speicher MR, Cremer T.

Cytogenet Genome Res. 2006;114(3-4):367-78. Towards many colors in FISH on 3D-preserved interphase nuclei. Walter J, Joffe B, Bolzer A, Albiez H, Benedetti PA, Müller S, Speicher MR, Cremer T, Cremer M, Solovei I.

Fluors that have been used at the time: DEAC, FITC, Cy3, Cy3.5, Cy5, Cy5.5., Cy7.

Now, some of those are not available any more (Cy5.5., Cy7) or became obsolete due to better ones (FITC-> Alexa488). In your case, Cy3.5 or the spectrally similar TexasRed should do. I am not sure if AF555 is well separated from those, you may have to replace it with Cy3, but have a look at the spectra. I have successfully used FITC, Cy3, TexasRed and Cy5 on the confocal in addition to Dapi. Dapi always was my friend, since no other useful blue label was available anyway nor did we have have problems with bleed-through to other channels.

Alternatives (or in addition).

+Alexa 633 can be separated from Cy5 by spectral unmixing.

+The already mentioned Quantum dots can be excited with blue light and may emit >600 nm due to the large Stokes Shift.

In any case, before you do the multi-color labeling I'd suggest trying separated fluorochromes to get well defined color channels with the spectral detector, to avoid bleed-through.

Good luck!

If you are interested in co-localization, you could consider a FRET approach. Eseentially you use two fluos with overlapping emission excitation wavelengths, excite with the lower wavelength and look for the longest wavelength. No co-localization, no fluorescence, very close (!) co-localization, fluorescence.

I've used 4 and the best way I find is to image with each laser in turn starting with the longest wavelength and working backwards i.e. Far Red-Red-Green-Blue, this minimized bleed through between fluorophores. As said earlier, the Molecular Probes Spectra Viewer is a microscopists best friend for selecting fluorophores.

Hi Julian,

Sorry to be late to the conversation, but I just came across this thread...

BD Bioscience has a license for the Brilliant Violet polymer fluorophores originally developed by Sirigen.  They are also developing their own versions of these fluors in-house (after purchasing Sirigen).  See link below.

These fluors are incredibly bright and the first two - Bv421 and BV480 allow for 5-color imaging with bright, photostable fluors.  BV480 is probably more than 20-fold brighter than DEAC, but spectrally similar, and photostable.

The imaging scheme we've worked out here at Chroma, together with BD Biosciences is to use the blue "gap" in the spectrum, starting with BV421, then BV480, then AlexaFluor 488 (or equivalent), AlexaFluor 568 (or equivalent) and AlexaFluor 647, 660, or 680 (or equivalent).

That's 5 colors attainable with a very basic light source, basic camera and no specialized equipment or heroic efforts - apart from the labeling process itself!

Here's an additional link to a slide deck I put together for this purpose.

Good luck!

http://www.bdbiosciences.com/us/applications/research/cellular-imaging/m/745135/sampledata#search=bv480

https://www.chroma.com/sites/default/files/CHROMA%20%26%20BD%205-COLOR%20IMAGING.pdf

What's the best 5-color schemes to show the images? (Red/Blue/Grey/Green..?)