Stack Exchange Network

Stack Exchange network consists of 175 Q&A communities including Stack Overflow, the largest, most trusted online community for developers to learn, share their knowledge, and build their careers.

Visit Stack Exchange

Questions tagged [fluorescent-microscopy]

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

0
votes
0answers
9 views

Re-stain slice using a different secondary antibody?

I stained a free-floating ~300uM brian slice using 4 different 1º antibodies (rabbit, chick, mouse, rat). For the mouse primary, I mistakenly used an Alexa-350 secondary, when I should have used 594. ...
0
votes
1answer
22 views

Can flow-FISH be used on metaphase cells?

I know flow-FISH can be used on interphase cells but was wondering if it only works on interphase cells or can work on cells regardless of stage in the cell cycle.
0
votes
1answer
36 views

How are light sheet microscopy 3D images and their computed sections built up from stacks of 2D images?

The iBiology Techniques video Microscopy: Dual-View Inverted Selective Plane Illumination (diSPIM) (Hari Shroff) describes light sheet microscopy and the improvement in resolution by introducing dual-...
1
vote
1answer
70 views

How to measure the length of mitochondria from z stack fluorescent microscopy image?

I have been working on yeast cells to analyse the effect of DNA damaging agents on mitochondrial structure. I have imaged my culture treated with MMS for a period of 6 hours and while observing the ...
3
votes
1answer
32 views

Does the use of UV light beam lead to poorer contrast in optical microscopy of cells than the higher wavelengths visible light?

I am reading about "diffraction-limited system" on Wikipedia, which mentioned "To increase the resolution, shorter wavelengths can be used, such as UV and X-ray microscopes. These techniques offer ...
0
votes
1answer
56 views

How to choose right volume of DNA and SYBR Green

I'm testing fluorescence level of a sample having dsDNA, SYBR Green and milli q water but I'm facing difficulty in choosing right volume of dsDNA and cons level of SYBR Green. Details of sample: ...
1
vote
0answers
116 views

Why there is no fluorescent light from my DAPI-stained Hibiscus Rosa Sinensis Pollen?

I currently work with fluorescence microscope to observe generative and vegetative cells within hibiscus rosa sinensis pollen. I used DAPI to stain pollens but there is no light that indicate the ...
-1
votes
1answer
153 views

Can streptavidin be conjugated with EDC-activated carboxylates?

Recently I conducted an experiment to conjugate amine-modified oligonucleotides with hydrogel particles having carboxyl groups. However, the fluorescence (from hybridized FAM-DNA) only appears on a ...
3
votes
0answers
53 views

Why do I see so many kinetochores?

I am analysing RPE-1 cells from humans and I do not understand why I see so many kinetochores by immunofluorescence (more than 100 in many cells). They are in prometaphase.
1
vote
1answer
37 views

Combination of different antibodies

How can I know if I can combine anti Cenp-C and anti Rod1 antibodies? I want to use them to label those protein and analysing cells by fluorescence microscopy. *Cenp-C is kinetochore protein and Rod1 ...
1
vote
0answers
35 views

Link between cell area and efflux pump?

I conducted an experiment involving a fluorescent F-actin stain called sir-actin which is removed by an efflux pump. After increased time and removal of the medium containing sir-actin, the cell ...
1
vote
0answers
37 views

Forgot to cool slides before washing

I just finished an immunofluorescence experiment and I'm wondering what went wrong. The tissues seem dimmer than they should be. One mistake I made was: I completed the antigen retrieval step, in a ...
1
vote
0answers
19 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 ...
3
votes
1answer
47 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.
0
votes
0answers
418 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?
1
vote
1answer
2k 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 ...
2
votes
1answer
249 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
votes
2answers
80 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
0answers
38 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 ...
3
votes
2answers
1k 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 ...
6
votes
1answer
118 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 in-...
3
votes
0answers
510 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 ...
1
vote
1answer
33 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?
2
votes
1answer
2k 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 ...
1
vote
1answer
46 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?
6
votes
4answers
596 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 ...
3
votes
1answer
301 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 ...
0
votes
1answer
124 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 ...
3
votes
1answer
119 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 ...
2
votes
1answer
64 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
votes
1answer
87 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
1
vote
3answers
557 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 ...
2
votes
0answers
68 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 ...
4
votes
2answers
2k 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
votes
1answer
286 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 ...
5
votes
2answers
3k 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 ...
13
votes
3answers
47k 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?
6
votes
1answer
873 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
votes
3answers
1k 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. However,...
3
votes
1answer
118 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 ...