Hi,

as you are citing my GIT-article I may give some additional comments: The advantages in bioluminescence microscopy concerning autofluorescence etc. are real. But on the other hand and I also mention this in the article the signals are MUCH dimmer than fluorescence and this is a big challenge. We have started doing this by setting up a microscope with an EMCCD camera in a totally black room. Later we invested in the LV200 microscope from Olympus. To my knowledge still the only instrument really dedicated to bioluminescence microscopy. I am happy to answer additional questions.

Yes, I suppose several groups are working on this topic: see e.g. http://www.nature.com/nmeth/journal/v5/n11/full/nmeth.f.226.html

When we are working with live cells autofluorescence is always present. We can avoid its detection by using fluorofores with emission that is easily separated from the autofluorescence signal. Most of the autofluorescnce is "green" because of nad(p)h and flavoproteins. Lipid oxdation products are also a cause of autofluorescence. Light is always going to cause some damage to the cells observed. Certainly, if UV (short wavelength) light is used for excitation, more damage is caused. So, choosing fluorofores that are excited in the red and emit in far red may help. Bioluminescence of luciferin has its disadvantages too. For example, how do you put luciferin and luciferase inside your live cells? There is a lot of work on preparing a system that has all the elements working in live cells. The methods complement each other, it is not a competition.

According to a question of W. Muss, I answer my own question!!

As retired microscopist but reader of old and new publications, I read sometimes that researchers have some problems with photobleaching and autofluorescence (papers and on Research Gate!)

It is a PERVERSION for a microscopist when he still works with old PROBLEMS and he doesn´t read the new publications !

BE AWAKE and SEND COMMENTS. Thank you in advance!

JRG

For example:

http://www.news-medical.net/news/20130205/Bioluminescence-microscopy-an-interview-with-Robert-Schmalzigaug-Olympus-America.aspx,

http://www.omicsonline.org/fractionation-of-biologically-active-compounds-extracted-from-propolis-by-nanofiltration-2155-9589.1000109.pdf,

best wishes and regrads,

WM

Dear Michal, Dear Andre, Dear Wolfgang,

thank you very much for the posted answers. I know the papers cited in your answers and I hope to see and read more papers about bioluminescence in the future .

Successful research for Michal, Andre and Wolfgang.

JRG

I came across this post while researching how to capture bioluminescence from luciferase in live, cultured mammalian cells. I am having difficulty configuring my microscope to image light from the luciferase/D-luciferin reaction.

On a related note, I'd like to mention that there is one significant disadvantage for luciferase/ bioluminescence. As far as I know, it is impossible to detect bioluminescence via flow cytometry, which is much more powerful than microscopy in terms of throughput. In order to image luciferase via flow cytomerty, the cells must be fixed, premeabilized, and stained with a dye-conjugated antibody (the intracellular protein cytometry, ICC, method). This is a very laborious process that is prone to artifacts. It would be absolutely wonderful if my assertion about flow cytometry and luciferase were incorrect and there were a protocol or machine that I'm just unaware of...

Hi,

as you are citing my GIT-article I may give some additional comments: The advantages in bioluminescence microscopy concerning autofluorescence etc. are real. But on the other hand and I also mention this in the article the signals are MUCH dimmer than fluorescence and this is a big challenge. We have started doing this by setting up a microscope with an EMCCD camera in a totally black room. Later we invested in the LV200 microscope from Olympus. To my knowledge still the only instrument really dedicated to bioluminescence microscopy. I am happy to answer additional questions.

Dear Christoph,

thank you for your answer and hints (LV200 Microscop Olympus). I hope that young researchers will use this new technique. Congratulations to your GIT article. It was for me very interesting.

I wish you a successful research and a nice week.

JRG

Dear all:

just to point to some more references:

http://cshprotocols.cshlp.org/content/2012/8/pdb.top070607.abstract

(only Abstract, Article only PPV=pay per view),

http://www.imaging-git.com/science/light-microscopy/bioluminescence-microscopy

(in G.I.T. Imaging&Microscopy: Bioluminescence Microscopy / New Avenues in Live Cell Imaging, Nov. 12, 2013, Charna Dibner Christoph Bauer Jerome Bosset),

http://onlinelibrary.wiley.com/doi/10.1111/jmi.12109/abstract :

Bioluminescence microscopy using a short focal-length imaging lens

K. OGOH, R. AKIYOSHI, MAY-MAW-THET, T. SUGIYAMA, S. DOSAKA, Y. HATTA-OHASHI, H. SUZUKI;

JOURNAL OF MICROSCOPY (Early View (Online Version of Record published before inclusion in an issue) © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society),

http://www.focusonmicroscopy.org/2012/PDF/182_Bauer.pdf (only Abstract.

Best regards, Wolfgang

Thank you Wolfgang!

Another reference is:

Bioluminescence Microscopy /New avenues in Live Cell Imaging

in GIT Laboratory Journal EUROPE 3-4/ 2014 and refs (as complete list). in www.imaging-git.com.

Authors C. R. Bauer et al

JRG

Christoph Bauer, I am wondering how much your imaging improved when you started using the LV200 compared to the the EMCCD in a black room .  We want  to start with this but like you will make our first attempt with a EMCCD.  How dark was was it?  Do you have any advice for us?  Many thanks in advance.

We have been using the LV200 to detect weak luminescent signals induced by radioactive decay (so called radioluminescence). The LV200 is highly sensitive, and it would not be possible to get the same level of sensitivity just by swapping in an EMCCD. This is because the LV200 employs a custom-made 36 mm tube lens, whereas a standard Olympus microscope uses a 180 mm tube lens. There are some drawbacks to the short focal length, such as distortions around the edges, but it provides the highest sensitivity for a given magnification. Now if you don't mind long exposures, you could probably image bioluminescence with a standard microscope, but keeping room lights to the absolute minimum will be key (due to the long required exposure time).

You can see more on our work here (OA):

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046285

I totally agree with Guillems comments and remarks. The LV200 is still the best instrument for low light applications if you need microscopic resolution (single cells or similar).