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

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12
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3answers
20k 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?
8
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3answers
533 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. ...
6
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4answers
333 views

What kind of microscope for ML/biological research?

I am a computer science student, focusing on machine learning applications. I have been always interested in biology but I lack any training in it. Now, I had an idea that I could introduce myself to ...
6
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1answer
413 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 ...
5
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1answer
62 views

What Websites Have Image Libraries for Bacteria and other Microorganisms

What Websites Have Image Libraries for Bacteria and other Microorganisms? With age of cell phone microscopes and hand held spectrometers it would be interesting and valuable to be able to compare ...
4
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1answer
24 views

Is NADH found in all type of cell in human body?

Is NADH found in all kinds of cell in the human body? I am curious about microglia specifically.
4
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2answers
2k 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 ...
3
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2answers
350 views

How to set threshold in ImageJ for quantifying relative fluorescence?

I have taken images from two different samples that have uptaken EdU during DNA synthesis (S-phase). The experiment setup is to find which condition had uptaken more EdU. Therefore I am detecting EdU ...
3
votes
1answer
95 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 ...
3
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1answer
193 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 ...
3
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1answer
92 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 ...
3
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2answers
1k 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 ...
3
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0answers
227 views

How to prevent e coli from clumping (for FACS)?

I'm performing FACS on e coli, but the cells are clumping together so each event is multiple cells. I ran a control where I had one flask of e coli expressing GFP, and one flask expressing RFP. Run ...
2
votes
1answer
90 views

Monochromator in fluorescence microscopy [closed]

I'd like to ask why are there no fluorescence microscopes that use a monochromator instead of filters? If there are any, then I should change my question as, which ones use it and why are they so ...
2
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2answers
42 views

Possible to remove accumulated haze on fluorescence interference filters?

I have a few different fluorescent microscopes, the two that use Mercury lamps are older than 7 years. I notice on those that the thin film filters in the filter cubes (green, red, blue) have ...
2
votes
1answer
472 views

Does methanol fixation deform cultured cells?

I use methanol fixation (@ -20⁰C for 10 minutes) when performing immunofluorescence assays on cultured cells. Generally speaking, this results in very good antibody staining. However, the cells tend ...
2
votes
1answer
147 views

Why are some scorpion species fluorescent under UV light?

It's known for some scorpion species such as Pandinus imperator, Heterometrus Petersii etc. to be shining under UV light. That makes them easier to capture and collect by humans. Is there any ...
2
votes
1answer
46 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 ...
2
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1answer
52 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
2
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0answers
27 views

background extraction in microscope analysis

I have taken microscope pictures for proteins in HeLa cells, in order to quantify the intensity (immunofluorescence). The proteins are both at the nuclear envelope, and in order to quantify the ...
2
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0answers
57 views

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 ...
1
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1answer
197 views

Calculation of final magnification when using a CCD camera

I'm trying to empirically calculate the approx final magnification (mag) of digital images rendered through our confocal microscope. Using a micrometer and the microscopy equipment I have, the ...
1
vote
1answer
27 views

Could the proteome of E. coli be fluorescently labelled?

What proportion of the total number of proteins in E. coli could be fluorescently labelled for PALM/STORM imaging?
1
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1answer
34 views

Exclusive microtubule minus-end labeling

Like the title explains I am looking for a way to exclusively label microtubule minus end in vivo. Looking through the literature I could not find any techniques yet. Do you have an idea?
1
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3answers
317 views

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 ...
1
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0answers
14 views

Red transcriptional reporter in Psudomonas putida

I need to build a transcriptional reporter in P. putida with high excitation wavelength,in order to decrease phototoxicity (I have been using mNeonGreen until now, and I would like to try something ...
0
votes
1answer
80 views

Enzyme kinetics [closed]

I can't understand how to study enzyme kinetics. Say I have a lipase and want to study the kinetics of this lipase using a fluorogenic substrate, how would I do this? From what I understand I would ...
0
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0answers
10 views

What does bone look like while fluorescently staining nerves

I am working on a project involving changes in innervation in the subchondrial bone. Nerves were stained fluorescently for PGP9.5 and I have to analyze the samples. However, I don't have much of a ...
0
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0answers
39 views

Effects of Fixation on Cell Membrane

Why does fixing of cells with something like formaldehyde disrupt the membrane enough for probes, antibodies, and dyes to get into the cell?