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Questions tagged [fluorescent-microscopy]

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

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Why does these confocal images show concentric circles?

I recently got this confocal microscopic image from my microscopy facility. I am unable to understand why these concentric circles are there. They look like out of focal fringes in conventional ...
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Live cell imaging using confocal microscope - Question about the microscope settings and laser wavelength

I am doing some calcium imaging to measure calcium levels in HeLa cells. To do this I have expressed cameleon calcium indicator (YC3.60) in the cells and I am using confocal microscope Fluoview for ...
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How to estimate calcium concentration from fluorescence microscopy?

Is there a way to get a rough estimate of a [Ca2+] based on fluorescence microscopy images (obtained using GCaMP for example)? Usually what we get is the ratio f/f0, but is there a way to estimate the ...
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Is colocalisation of a protein with a presynaptic marker sufficient evidence to say that the protein is a component of axon terminals?

I am reading journal papers about the subcellular localisation of the insulin receptor (IR) in neurons. I have read a paper stating that IR is highly enriched at synapses, localising to both the ...
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Which programming language is beneficial for a cell biologist who does extensive confocal microscopy and image analysis? Python or MATLAB?

I'm currently learning to acquire images using confocal microscopy and subsequently analyze the images using the Fiji software. I want to know how and where exactly Python or MATLAB is used by ...
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How to correct the different (background) fluorescence for different cell types?

I am differentiating mouse bone marrow macrophages in vitro. However, I found that the differentiated macrophages have higher background fluorescence (the unstained sample) in all channels, which ...
William Wong's user avatar
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Can you use calcein to stain milk somatic cells and observe them through a fluorescent microscope?

I am currently working on an image cytometer that could automatically count somatic cells in milk samples. Can I use Calcein (i.e., C30H26N2O13) to stain the somatic cells? If not, what dye can I use ...
Gaell Gelacio's user avatar
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Suggestion on program/algorithm to segment nuclei

I've got some images such as the one in this post, obtained from the neuroepithelium of a chick embryo in a confocal microscope, where nuclei have been fluorescently labeled. I'd like to be able to ...
Santiago Bosch's user avatar
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Why is there a λ⁴ in the spectral overlap integral in FRET calculations

The Förster resonance energy transfer (FRET) spectral overlap integral looks like: $$ J=\int_{0}^{\infty}\bar{F}_{D}(λ)ε_{A}(λ)λ^4 dλ $$ Where $λ$ is the wavelength, $\bar{F}_{D}(λ)$ is the normalized ...
Weiwei's user avatar
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Are antibodies labelled with fluorescence that have not attached to an antigen visible under light microsocopes?

I came across this thought while studying histology, what happens to fluorescence labelled antibodies that do not bind with an antigen, can we see them? or are antibodies activated upon antigen ...
Doe Pual's user avatar
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How to measure the pH of a bacterial species?

I would like to calculate the pH of a certain bacteria species before after an experiment. I was reading about the pH cell of bacteria and I found out about Bacterial Intracellular pH which I ...
Anwar Elhadad's user avatar
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What is the process by which fluorescent proteins in two photon microscopy are optically stimulated by membrane depolarization?

How does the flow of calcium ions through neurons cause the dyes to activate? The voltage is extremely small, so the dyes have to be extremely close to the neurons, which would disrupt the cells, so ...
C-Consciousness's user avatar
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In fluorescence microscopy images what is meant by the term "puncta"?

I am reading papers where confocal fluorescence microscopy images were analysed. In many of the papers I see the term "puncta" being used when researchers analyse the colocalisation between ...
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Does it make sense to say that microtubules are arranged in a ring around the cell periphery?

I am reading a journal paper and it is about how the organisation of microtubules are altered in CHO cells that overexpress microtubule-associated protein Tau. In the paper, the authors found that ...
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Can Ni-NTA-Atto Conjugates bind to single His-tag

Can I label a protein with a single His-tag with Ni-NTA-Atto conjugates? Papers generally use this technique to label 6His-tag.
Antoine Roland's user avatar
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Why are microtubules absent in and around the nucleus?

I was going through fluorescence microscopy and found these four images. The top left corner shows the actin cytoskeleton (rhodamin phalloidin) and the bottom left image shows the microtubule ...
The Limit Does Not Exist's user avatar
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colocalization by IFA?

I have done IFA (immunofluorescence assay) previously but i targeted only one protein or i tagged my endogenous protein with GFP/RFP then i performed IFA. I want to do the IFA in parental cell line (...
Rengaraj's user avatar
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What does it mean to "cure" microscope slides?

I am reading a journal paper and I have come across the following statement in the materials and methods section regarding the preparation of microscope slides: Coverslips were washed once more with ...
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When should I use cryofixation and chemical fixation?

We know that the technique used in TEM sample preparation involves multiple steps, one of the most important of them is fixation. Fixation can be of two types: Cryofixation, that suggests that the ...
Ahmed's user avatar
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is it possible to visualize coronavirus infection and what type of assays are routinely used?

There has been some electron micrograph of SARS-COV-2 published; but are there any fluorescent/ confocal-fluorescent image of them? or is it possible to do them? I know that viruses are usually much ...
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Nuclear vs Cytoplasmic Fluorescence [closed]

When staining transciption factor proteins with fluorescent antibodies (Alexa, etc.), why are the fluorescence signals stronger in the nucleus as compared to fluorescence signals in the cytoplasm (in ...
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Need help with ImageJ thresholding

I'm trying to measure the colocalization of two proteins in a .tiff image taken with a wide-field fluorescence microscope. In ImageJ I have split the image into the two channels (red and green), and ...
betelgeuse's user avatar
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How does Line Integrate work on a laser scanning confocal microscope?

Most microscope software provides options for line average or line integrate as a function that helps the signal to noise ratio when taking an image. The math behind line average seems straight ...
Eric McGhee's user avatar
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1 answer
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Quick overview of how to capture microscope images of histology plate or fluorescent plate

I am looking at these two: I would like to DIY take photographs of plant and animal cells using a cheap microscope such as this: I don't know too much about microscopes or the techniques to do the ...
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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. ...
Carmen Sandoval's user avatar
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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.
Bio314's user avatar
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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-...
uhoh's user avatar
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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 ...
Jyoti Sharma's user avatar
3 votes
1 answer
91 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 ...
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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: ...
Kapil Singh Rawat's user avatar
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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 ...
Dziban N's user avatar
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1 answer
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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 ...
Koreanraichu's user avatar
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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.
Bio's user avatar
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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 ...
Bio's user avatar
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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 ...
J. Doe's user avatar
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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 ...
amd1972's user avatar
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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 ...
naandep's user avatar
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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.
aksagar's user avatar
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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?
The Nightman's user avatar
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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 ...
rhill45's user avatar
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2 votes
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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 ...
Macond's user avatar
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2 answers
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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 ...
rhill45's user avatar
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2 votes
0 answers
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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 ...
Katz's user avatar
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3 votes
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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 ...
Parham's user avatar
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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-...
Cymatical's user avatar
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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 ...
Amanda's user avatar
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1 answer
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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?
user2002858's user avatar
2 votes
1 answer
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 ...
Chastain's user avatar
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1 answer
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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?
MaxJ's user avatar
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6 votes
4 answers
699 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 ...
bio's user avatar
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