Fluorescence detection in living cells and ways to improve your image

Introduction

Fluorescence imaging of living cells can provide important data regarding the function and localization of proteins and other bio-molecules within a cell or tissue. These images give insight into fundamentally important biological processes and improve our knowledge about transient interactions we might not be able to detect otherwise. An added benefit is some simply remarkable pictures of colorized cells which are just fun to look at. Although in theory, fluorescence microscopy is simple, obtaining suitable images is difficult. Problems with cell health can occur due to long incubations in D-PBS in an attempt to reduce auto-fluorescence. Cell death due to light intensity or photo-bleaching can be problematic and need to be overcome. If you are having problems with signal:noise or cell health or obtaining the best image possible now is your chance to ask the expert.

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Question 1

What molecules in media present the biggest problems with fluorescence imaging?

Many of the chemicals in media fluoresce when the right wavelength light is present. Fluorescence microscopy is often difficult since getting the correct signal:noise is dependent on the components that make up the media. Often incubations in PBS are used to reduce auto-fluorescence but there are problems with that since the nutritional content of PBS … Continued

Question 2

What kind of system would you recommend for live-cell imaging? Would you use live-cell incubation chambers or something else. Also what are the advantages of live-cell imaging?

There are lots of good chamber systems for live imaging. If imaging is occurring for a few hours you might be able to get away without an incubation chamber but if your incubations are longer a more sophisticated system would be necessary. Here is a good website from Nikon that talks about them (http://www.microscopyu.com/articles/livecellimaging/culturechambers.html). Obviously … Continued

Question 3

How can we use cell imaging information to inform and improve our cell culture production?

Wow this a good question. With the latest technology there are many ways to measure cell health and other things that will ultimately make cell culture production better. For example Molecular Probes makes some great fluorescent dyes for just those purposes. pHrodo Indicators for example get onto cells and change colors depending on the cytosolic … Continued

Question 4

What would you recommend for label-free imaging and do you see genetic expression of fluorescent proteins being a challenge to imaging?

I am not sure that I understand the questions I am thinking that you are asking about something like GFP or one of the variants. To view GFP, most people use FITC filter sets and you will also need fluorescence capabilities. From your second question I don’t think it is too challenging to express GFP … Continued

Question 5

What do you recommend for evaluating cell viability with fluorescent imaging?

It depends on what you want to do. Newer fluorescent assays for cell viability work the same way as the older non-fluorescent counterparts. Viability assays work by taking advantage of membrane permeability loss in compromised cells. Therefore the dyes are influxed into the cell where they bind DNA. Unbound dyes do not fluoresce. An example … Continued

Question 6

What do you recommend for imaging of non-adherent cells?

You can do imaging on suspension cells. If you are using phase-contrast you won’t get much since the light reflects off of the cells in suspension, but you can get images using other types of microscopy. Confocal and fluorescent imaging is possible. Often fixations are necessary. The trick is using an ultra-thin layer of cells … Continued

Question 7

What methods are best for looking at intracellular events?

Hi I have to tell you there are many methods and probes for looking into intracellular events. I could not do the topic justice in a short answer. Here is a link to the Molecular Probes Handbook which will have all the answers to your questions. http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook.html Have a look. Total: 0 0 0 0