Fluorescent microscopy uses the emissions of fluorescently labelled probes to generate an image.

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30 views

Hela live cell confocal laser scanning - reccommendations for good fluorophore that will show good movement

I've been doing a lot of live cell imaging lately mostly using hela cells expressing some EYFP based chimeric proteins. I'm building a video library for an art student here at the university who is ...
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1answer
27 views

What's the best way to measure/calculate the size of a light beam at the sample of a microscope?

I am using a microscope with an LED derived light through the epi-fluorescent port of a microscope. I know that the "field of view" for a given objective is equal to the field number/magnification ...
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6k views

Hoechst or DAPI for nuclear staining?

When is it best to use Hoechst vs. DAPI for nuclear staining? They seem to be very similar on paper. Are there situations where one is clearly preferable?
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26 views

ligand binding fluorescent protein

does anyone know of a fluorescent protein that upon binding of a substrate its fluorescence is activated? i've been looking on this and perhaps my keywords are not the right ones. Thanks in advance
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Assay for Beta-galactosidase activity in single cell microscopy

I'd like to be able to measure the activity of $\beta$-galactosidase in living cells with simple optical (maybe fluorescence) microscopy. Ideally I'd like to do a minimum of genetic engineering, and ...
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Removal of the Initial Methionine in Venus for FRET

I'm working on building some FRET reporters. In addition to a cleavage site (of varying composition from 15-18AA), a 1 AA linker, I'm using Venus and Cerulean. Initially I was worried that 18AA ...
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2answers
475 views

How does formaldehyde/PBS or methanol fixation of cells affect lysosomal pH?

The question is fairly simple - does formaldehyde or methanol fixation in preparation for immunocytochemistry/immunofluorescent staining affect the pH of the lysosomes? Some background: I'm trying to ...
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1answer
83 views

How do different tissue culture matrices affect background in fluorescent microscopy?

In response to my previous question, I've been reading up a little bit on poly-D-lysine, Collagen I, Collagen IV, laminin, and other tissue culture coatings that promote cell adhesion. I've always ...
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1answer
391 views

How to convince suspension cells to adhere more tightly?

I'm developing a cell-based assay in 96-well plates that requires adherent cells, as they need to be washed at least twice during the protocol. I'm using in-house strains of HT1080 cells (some ...
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1answer
223 views

How long does it take to stain cells?

I'm never going to run these type of experiments but I do need to have a good idea of their timescale. After I fix my cells, if I stained my cells with histology stains like DAPI, Fluorescein, and ...
8
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178 views

Spatial resolutions in optical microscopy

I have read that different optical imaging techniques such as such as wide-field microscopy, confocal microscopy or STED microscopy can theoretically achieve a different spatial resolution. ...
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67 views

Is there a photobleaching-resistant, cell-permeant viability stain in the far red part of the spectrum?

I am looking for a live-cell–impermeable dye for viability. (The cells cannot be permeabilized and fixed in this experiment.) I would prefer with excitation and emission spectra similar to Cy5, but I ...