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.

Which corresponds to barely more than a second of arc. This is an impressive feat! The pulse would be technically invisible by a human eye on the Moon, and its photons must still travel all the way back to Earth, undergoing further divergence. Catching a reflected photon is as hard as looking for a needle in a haystack. Luckily, scientists send a lot of photons at the same time, making detection possible.

Gaussian beamdivergencecalculator

Inside the chamber, you can see two mirrors on opposite and parallel sides. One of them (the one at the opening of the resonator) is partially transparent, while the other is fully reflective.

🙋 Lasers always have a divergence, even if we can tweak some parameters to make it as small as possible. A laser with extremely small divergence is called a collimated beam.

Gaussian beamdivergence

🙋 The diffraction limit is an important concept in optics (or whenever you can find an oscillatory phenomenon). It defines the maximum resolution obtainable, fixing a lower limit to the detection capabilities of sensors and such. In a laser, the diffraction limit defines the smallest possible spot of a beam: the wavelength of the light would make it impossible to go lower than that!

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…

“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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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.

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).

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.

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…

Laser beamdivergenceand spot size

You can reduce the divergence of a laser by acting on a single parameter, the initial diameter of the beam. Increasing it helps increase both the Rayleigh range (thus increasing the coherence length) and the divergence.

Lasers are devices that emit a highly coherent beam of monochromatic light thanks to a process of amplification of a single wavelength inside an energized medium.

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

Between the two mirrors, you can find a medium. This material constitutes the core of the device. If pumped with energy (either electricity or light), it can amplify the light of a specific wavelength. After bouncing back and forth many times, the photons escape the chamber, amplified and coherent.

The beam expands on its way to the Moon, reaching the surface of our satellite with a diameter of roughly 2 km2\ \text{km}2 km. The telescope sending the pulse has a diameter of 3.5 m3.5\ \text{m}3.5 m. Considering the mean value of the distance between Earth and Moon (384,400 km)(384,400\ \text{km})(384,400 km), we can calculate the divergence of the beam:

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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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?

Eventually, the problems were found, which allowed the lasers to find applications in many high-technology sectors. The devices are now irreplaceable in medicine, optics, astronomy, manufacturing... you name it!

Meet a laser. It looks like a uniform opaque cylinder from the outside, but you may notice a different material on one of the bases. That's where the radiation escapes the chamber: don't look at it when the device is on!

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

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

What is beamdivergence

Say this to an astronomer, and he will surely disagree. When the distances increase, lasers reach their limits while still maintaining an edge over traditional light sources.

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.

Divergence angleformula

In the further properties section, you can find the fields for the optional variables wavelength, waist diameter, and quality factor of the beam. If you insert them, we will fire a warning if your calculations will return a value below the minimal theoretical limit of divergence. If you leave them empty, the only alert that will fire is if the divergence calculation returns a negative value.

Divergence anglecalculator

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…

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…

The divergence of a laser beam is limited by the physical characteristic of the laser itself. For ideal beams (propagating in a Gaussian beam with quality factor M2=1M^2=1M2=1), the divergence can't be lower than twice the value given by the formula:

Physicist holding a 1st class degree and a member of the Institute of Physics. Creator of the UK vaccine queue calculator, and featured in many publications, including The Sun, Daily Mail, Express, and Independent. Tenacious in researching answers to questions and has an affection for coding. Hobbies include cycling, walking, and birdwatching. You can find him on Twitter @OmniSteve. See full profile

You can easily see this effect by observing the laser beam divergence formula and varying the value of the initial diameter Di:

The divergence of a laser beam is the measure of the increase in the beam diameter over distance. Even though lasers have a high directionality, the light propagates following a Gaussian beam, expanding over time.

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.

The previous formula for the divergence of a laser beam arises from geometrical considerations. As you can see in the diagram above, we ignore the structure of the Gaussian beam and only consider rays propagating in straight lines.

Physicist by education, scientist by vocation. He holds a master’s degree in complex systems physics with a specialization in quantum technologies. If he is not reading something, he is outdoors trying to enjoy every bit of nature around him. He uses his memory as an advantage, and everything you will read from him contains at least one “I read about this five years ago” moment! See full profile

Lowdivergencelaser

The beam reaches its smallest size at a particular point in the resonating chamber. We call that point the waist of the beam. Once the beam passes the waist, it starts expanding in a cone. The angle of expansion is the divergence of the beam. Let's analyze it in detail.

The diameter of the beam is measured at the 1/e21/e^21/e2 intensity point: the distance from the peak at which the intensity drops to 1/e21/e^21/e2 of the maximum value. The cone described by the divergence angle contains 86%86\%86% of the total power of the laser.

Lasers are good but not perfect: learn how — and why — a pointer can't work from the Earth to the Moon with our laser beam divergence calculator!

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

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.

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…

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…

The beam acquires its properties inside the optical resonator. We are talking about the three features listed above plus the way it propagates. Ideal lasers are Gaussian beams, which means that they move along a primary direction — or axis — with a cross-sectional intensity profile following a Gaussian curve.

The properties of laser radiation made the technology fundamental in the second half of the 20th century. However, when firstly devised, lasers were too "advanced": a scientist said of them that they were "a solution seeking a problem".

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.

Divergence angleof beam

Consider the process of lunar ranging, the measurement of the distance between Earth and Moon through the reflection of laser pulses on the reflectors left by space missions on the surface of our satellite.

This value is small but not that small for lasers. You can use our calculator in reverse too: insert the distance, the initial diameter, and the divergence, and find out the final diameter of the beam. In this case, at the distance of 1 km1\ \text{km}1 km the beam would be more than 35 cm35\ \text{cm}35 cm in diameter.

In a typical lunar ranging experiment, a telescope collimates a laser (thus achieving a small divergence) and shoots the beam in the direction of the Moon, trying to hit the area of the reflector. This task is not like shooting at the broad side of a barn: the reflectors are incredibly tiny, and even though their locations are known, the sheer distance between them and us makes every hit a success.

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).

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…

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.

Our laser beam divergence calculator calculates the divergence of the beam in the far-field limit. You only have to input the values of the diameters at the initial and final points and the distance between the two. We will calculate the rest.

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

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.”

Let's now calculate the divergence angle of a laser beam. Consider a beam with initial diameter Di=4 mmD_{\text{i}}= 4\ \text{mm}Di​=4 mm. Let's move away from the source by a distance l=10 ml=10\ \text{m}l=10 m, and measure the diameter again. We find Df=7.5 mmD_{\text{f}}=7.5\ \text{mm}Df​=7.5 mm. This information is all we need to calculate the divergence of that beam. Input the values in the LASER beam divergence calculator.

All of the calculations for the divergence of a laser beam rely on the far-field approximation. Take a look at the diagram below. Close to the waist, the beam expands following a smooth curve. If you measured the divergence around that area, you would get the wrong result, underestimating the actual value. If you move far from the waist, the beam diameter increases almost linearly with the propagation direction. This situation is what we call far-field approximation.

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,…

The lasers we see normally operate at relatively small distances, and we may be bound to think that they can propagate for an arbitrarily long distance without losing their characteristic "dot" shape.

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.

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…

The divergence of a laser beam measures how much the beam spreads with the distance, that is, the rate at which the laser diameter increases.