Hi, I'm trying to image fluorescent labeled cells on a gel. How deep am I able to see with a confocal microscopy? What's the maximum depth of confocal microscope? I would love to hear some experience on imaging 3D cell cultures.
thanks
Hi,
The depth depends on the microscope used and the lense (magnification, NA). Normally it is about a couple of hundreds of microns and less. If you need to see deeper, you can use multi-photon microscope.
Cheers,
3D Confocal analysis on a gel matrix depends on the Z depth and multichannel imaging. In my opinion, 3D cell culture requires multi-dimensional imaging (time, multipoint, Z series, and wavelength). Generally, decreasing pinhole diameter under a higher NA objective will enhance the z‑resolution. Recently, I have used extended depth of focus (EDF). Here, images of different Z axis are merged together to generate a combined virtual 3D image.
2D and 3D deconvolution of image stacks are important in 3D imaging. Blind depth-variant deconvolution is the current standard. However, most instruments do not support the real time deconvolution process (although they claim it). The data of certain weak signals will be lost due to out of focus in real time.
I am not sure the type of application you are intended to do. For faster analysis, spectral slicing is preferred. However, the resolution will be compromised in spectral imaging. The cost paid for the speed :-)
Have fun with the nuance of imaging techniques!
dear Leon Hsieh,
if i have to give a direct answer, simply i will agree with @Alexandra (Sasha). however, in past 5 yrs people imaged tissue depths upto milli meters by CLSM. sample need special mounting preparations and you might even need specially designed objective lens. these methods were tested on brain, skin, heart and other tissues but not in 3D cell cultures and are for only fixed samples.
names of those techniques in case you wish to gothrough:
1. CLARITY TISSUE CLEARING METHOD.
2. ScaleA2
3. PACT
are major methods.
I am operating laser scanning confocal microscopy and so far i have imaged the 3d cell cultured sample of height 500 micron to 2 mm with multidyes in it. But it is not so easy and failed many time due to technical or experimental problem. Because maximum time the sample got, don't follow protocol that need for imaging. So to get perfect image in confocal you need following property.
1. the bottom of you plate should be coverslip of height 0.17mm (Because you can't put your sample on slide and invert it)
2. Your matrix in which you are doing 3d cell culture, that should be at least 80-85% transparent in nature. if it is 100% transparent that would be best to image.
3. Use the dye but be sure that don't use dye that bind matrix and cell both.
4. Check that what ever matrix you use it don't have auto fluorescence in nature.
5. Use 1-2 dye at a time (possibly 1 for nucleus and second for the plasma membrane). But be sure that both dye has different excitation and emission range and emission rang of one dye should not be the excitation of another dye.
once you have all and you go for imaging tell them to get the image in following way.
1. To use panorama of at least 5 X 5 area with a z stack. (where the distance between each stack should be 0.3 micron)
2. As this kind of panorama take lot of time, tell them to use fast scanning method, i.e. scanning speed will be 800-1000. It will reduce your quality of pic by 10% but you can get bigger area and from that you can chose desire area you want to see.
3. It is important that it should use less leaser power. Because this kind of imaging lead to photo bleaching.
By this way you can get much better picture that can be publishable
I hope this answer will help you.
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