Numericalapertureof microscope

Numericalapertureunit

I much prefer open source software, hardware and documentation for scientific transparency. The companies can still make money selling their expensive stuff - open source doesn’t mean they have have to give their kit (or software) away for free.

Hi, Going through a .czi metadata to extract some information I ran into both the NA 0.95 of the lens and a value set as the effective Numerical aperture a smaller value at 0.8. I wonder if anyone here can help me find this difference and from there to know which one theoretically is the one to be considered in the computatation of the point spread functio.

Hello and welcome to the Forum! I cannot give you a definitive answer but an opinion. This is because Zeiss ZEN is very much NOT open source software and you need to get a license just to get access to their file format details but maybe someone here already has this or knows the answer more definitively.

This shows five typical battery-powered lasers, along with their eye and skin hazard distances, and the distances at which they can cause visual interference to persons such as aircraft pilots. Click to enlarge.

Numericalapertureof lens

Numericalapertureformula

This diagram shows various ways to help reduce laser pointer incidents. These include: pilot training and glasses, arrests and prosecutions, laser labeling, user education, and new laws & restrictions. Click to enlarge.

“Dumb Ways to Blind” is a 2014 public service video that warns the Internet generation about the many ways lasers can be misused. As of February 2024, this has had almost 10 million views on YouTube.

Numericalapertureformula with refractive index

Numericalapertureof optical fiber

Public domain photo from the U.S. FAA, showing how a laser beam spreads over long distances and can fill the windscreen. The FAA’s highest-resolution version is here.

In April 2019 the U.S. Federal Aviation Administration posted this animated powerpoint. The narration says "…pointing a laser at an aircraft can distract or temporarily blind the pilot, potentially putting the lives of many innocent passengers at risk. That's why it's a federal crime with serious consequences like fines up to $250,000 or up to five years in jail…"

Numericalapertureof objective lens

The NA of the lens is the maximum NA rating it can practically handle using the immersion medium for which is was designed (air, water, glycerol, DIN oil, etc.). You can easily reduce this in practice for any experiment by (e.g.) stopping down the condenser iris and/or using a different mounting and immersion medium to the one for which the NA of the lens was measured at during the lens design and manufacture stage (e.g. using water or glycerol immersion instead of DIN oil with a lens designed for DIN oil immersion will reduce the effective NA of the experiment with that lens to below its rated standard).

The ED50 distance is about 1/3 of the NOHD. At the ED50 distance, there is a 50/50 chance of the laser causing the smallest medically noticeable retinal lesion under laboratory conditions where both the laser and the eye are stationary. Often such a lesion will heal, in the same way that a minor skin burn can heal. Beyond the ED50 distance, the chance of having a retinal lesion is further reduced.For a 5 mW 1 mrad laser, the ED50 distance is 16 feet. For a 499 mW 1 mrad laser, the ED50 distance is about 164 feet.This helps explain why a laser that is nominally hazardous at a certain distance (the NOHD) is extremely unlikely to cause even a small injury at that distance.There is more information about the NOHD and the ED50 distance on this page.

In most incidents, the beam only directly hits the aircraft windscreen a few times. That’s because it is almost impossible to hand-hold a laser onto a target hundreds or thousands of feet away. For example, these frames show 8 seconds of a laser illumination. Only in one of the frames — 1/2 second out of 8 — does a direct hit obscure the pilot’s vision. Of course, the waving beam is a distraction.

This diagram shows the hazard distances of a 5 mW green laser pointer. It is an eye hazard up to 52 feet from the laser, causes temporary flashblindness to 260 feet, causes glare and visual disruption to 1,200 feet, and is a distraction to 11,700 feet (2.2 miles). Click to enlarge.

That’s why a laser beam that can pop balloons or light cigarettes up close, could be eye-safe hundreds of feet away.Now, an eye-safe beam may still be very bright. It could cause temporary flashblindness, or vision-blocking glare, or be a distraction. This would be a hazard to persons such as pilots during critical phases of flight. The second part of the calculator above lists the distances for those visual interference hazards.

Having said that, from a purely scientific point of view (and it would be interesting to learn if the ‘official’ Zeiss .czi format definition differs from this), the ‘effective’ numerical aperture would be the angle of acceptance of illumination (refractive index of the medium taken into account as per the Abbe formula) that was permitted during the experiment regardless of the maximum NA specs of the lens.

A helicopter being deliberately targeted by a laser pointer. The light is a distraction and, if bright enough, can cause temporary flashblindness. It is NOT likely to cause any permanent injury. A video of this incident is here.

This movement may also be due to the illuminated person moving their head and eyes to avoid directly staring into the beam.In both cases, the laser light does not have as much time to stay on one spot on the retina. This reduces the chance of causing a lesion or burn.Of course, if a laser is powerful enough and a person is close enough — say, within the NOHD and certainly within the ED50 distance — a brief 1/4 second exposure can cause a retinal lesion.But for laser illuminations of pilots at aircraft distances of many hundreds or thousands of feet, even powerful lasers are considered by experts to be unlikely to cause serious or permanent harm.