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Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.

It is important to examine a fluorophore in terms of its excitation and emission spectra, which essentially indicate the probability that a molecule will emit a photon of a certain wavelength of light given an excitation photon of a given wavelength. Figure 1 below illustrates the excitation and emission spectra of FITC under conditions of 488 nm excitation.

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A more correct statement is “fluorophores emit photons based upon their emission spectrum, whose maximum is shifted to a higher wavelength than the maximum of the excitation wavelength.” These kinds of phenomena remind us that fluorescence is a lot more complicated than we usually give it credit for (and a lot more interesting, for that matter).

The spectra of PE at two different excitation wavelengths, shown in Figure 2, illustrate this phenomenon even more compellingly.

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It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

There you have it – this odd and commonly misunderstood phenomenon is real and most importantly doesn’t defy the laws of physics.

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

FITC’s emission maximum is around 530 nm. This means that if we excite this molecule, there is a very high probability that it will emit green photons. Thus, we choose bandpass filters on our flow cytometers that are centered on this region of the spectrum (e.g. 525/30) in order to capture as many photons from the fluorescence process as possible.However, FITC’s emission is not restricted to green photons; it also emits yellow, orange, red, albeit at lower probabilities.

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Shapiro, H. M. Practical Flow Cytometry. Hoboken, New Jersey: John Wiley & Sons. 2003.If you’re serious about flow cytometry and want to be a part of Expert Cytometry, click here to learn more.

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You may notice something curious about the spectrum if you look closely at the emission curve immediately around the excitation line. It appears that there is finite (but very low) probability that photons will be emitted below the laser excitation wavelength. This would mean that the photons we harvest from the molecule are higher in energy than the photons that we introduce.

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

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You may notice this in your flow cytomtetry data when measuring FITC in the presence of PE, especially if using wide filters (e.g. 525/50 BP instead of 525/30 BP). Any PE signal you see in the FITC PMT is independent of the laser line. In other words, you would see this signal regardless of whether you excited PE with 488 laser light (the emission in this case would be higher in wavelength than the excitation line) or the 561 nm laser line (the emission would be lower in wavelength than the excitation line).

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Note: This would only be observable on a 488/561 colinear system, where the emission light from both lasers are collected on the same optical path. Multi-spot or multi-pinhole instruments would not have a detector with FITC collection filters on it.

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

When we learn about fluorescence, the first thing we are told is that fluorophores emit photons that are higher wavelength than the photons that they absorb.

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

“Okay, you might say, but the excitation spectrum overlaps the emission spectrum. If the shape of the emission spectrum remains the same, no matter what the excitation wavelength is, wouldn’t that mean that we could get 500 nm emission from 5 10 nm excitation, seemingly violating the Law of Conservation of Energy? Well, we could get 500 nm emission from 510 nm excitation, if the molecule in question was already in a vibrational excited state when the 510 nm excitation photon arrived. The cost of electronic excitation remains the same, but the molecule itself is coming up with some of the money.”

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But wouldn’t this mean that we are getting more energy out than we are putting in and defying the Law of Conservation of Energy?

PE has more complex excitation state than does FITC. Most strikingly, it has two excitation maxima: one at around 488 nm and one at around 561 nm. Interestingly, under conditions of 561 nm excitation, there is an appreciable probability that emitted photons will be higher in energy than absorbed photons.

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So, the statement “fluorophores emit photons that are higher in wavelength than the photons they absorb” is actually incorrect.

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What this specifically refers to is the stokes shift, which results from non-radiative energy transfer during the fluorescence process. When a photon is absorbed by a fluorophore molecule, some of the resultant energy is lost in molecular vibration and movement (among other things) so that the energy released after fluorescence is lower than the energy absorbed. Since wavelength is inversely proportional to energy, this lower output energy light is higher in wavelength than the input light.

In fact, you would probably see more signal in the FITC detector when exciting at 561 than 488, as the PE molecule is more efficiently excited at 561 than at 488. Most critically, the shape of an emission curve is independent of the excitation wavelength.”

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Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…