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Water Refraction Experiment

Ever noticed how objects look different when you see them in water? It is all to do with how light travels from air into water, the light slows down and bends slightly. This bending is called refraction. Demonstrate how refraction of light occurs in water with this simple water refraction experiment. All you need are a few simple supplies, and our printable project!

Explore Water Refraction For Physics

Let’s keep it basic for our junior scientists. Physics is all about energy and matter and the relationship they share with one another. Like all sciences, physics is about solving problems and figuring out why things do what they do. Kids are great for questioning everything!

In our  physics activities , you will learn a little about static electricity, Newton’s 3 Laws of Motion, simple machines, buoyancy, density, and more! All with easy household supplies!

Encourage your kids to make predictions, discuss observations, and re-test their ideas if they don’t get the desired results the first time. Science always includes an element of mystery that kids naturally love to figure out!

Learn about the refraction of light in water with this hands-on physics experiment below.

Why not pair this simple refraction experiment with other fun water experiments !

• Printable templates (see below)

Instructions:

STEP 1: Print out the template designs and cut them out.

STEP 2: Fill a clear glass to the top with water.

STEP 3: Place each design behind the glass of water and stand back a bit to look at it through the glass. What can you see?

STEP 4: Try moving the designs closer to the glass and then further away. Record what you see.

Refraction Of Light In Water

Refraction of light in water is when light changes direction as it passes from air into water. When light enters water at an angle, it slows down and bends. This bending of light is called refraction .

This is why objects in water can appear distorted or appear to be in a different place than they really are.

The amount of refraction depends on the angle at which the light enters the water and the difference in the density of air and water. Learn more about density.

Refraction of light in water is important in many fields, including underwater photography and the design of eyeglasses and cameras.

This simple water refraction experiment below shows just how light refracts in water. You may not believe your eyes!

Get Your FREE Printable Water Refraction Project!

More Fun Light Activities

Make a color wheel spinner and demonstrate how you can make white light from different colors.

Explore refraction of light when you  make rainbows  using a variety of simple supplies.

Set up a simple  mirror activity  for preschool science.

Learn more about the color wheel with our  printable color wheel worksheets .

Explore the constellations in your own night sky with this fun constellation activity .

Make a DIY spectroscope and split visible light into the colors of the spectrum.

Helpful Science Resources

Here are a few resources to help you introduce science more effectively to your kiddos or students and feel confident when presenting materials. You’ll find helpful free printables throughout.

• Best Science Practices (as it relates to the scientific method)
• Science Vocabulary
• 8 Science Books for Kids
• What Is A Scientist
• Science Supplies List
• Science Tools for Kids
• Scientific Method for Kids
• Citizen Science Guide
• Join us in the Club

Printable Science Projects For Kids

If you’re looking to grab all of the printable science projects in one convenient place plus exclusive worksheets, our Science Project Pack is what you need!

Subscribe to receive a free 5-Day STEM Challenge Guide

~ projects to try now ~.

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Light Refraction Experiment

March 30, 2020 By Emma Vanstone Leave a Comment

This light refraction experiment might be one of the most simple to set up science experiments we’ve ever tried. It is a bit tricky to explain, but impressive even if you can’t quite get your head around it!

If you like this activity don’t forget to check out out our other easy science experiments for kids .

Materials for Light Refraction Experiment

Paper or card

Instructions

Fill the glass almost to the top.

Draw arrows on one piece of of card or paper. Place the paper behind the glass and watch as the arrow points the other way.

Now try to think of a word that still makes sense if you put it behind the glass.

We tried bud , the green ( badly drawn ) plant is on the opposite side when the paper is not behind the glass.

NOW works well too 🙂

How does this work?

Refraction ( bending of light ) happens when light travels between two mediums. In the refraction experiment above light travels from the arrow through the air, through the glass, the water, the glass again and air again before reaching your eyes.

The light reaching your eye (or in this case our camera) coming from the arrow is refracted through the glass of water. In fact the glass of water acts like a convex lens (like you might have in a magnifying glass). Convex lenses bend light to a focal point . This is the point at which the light from an object crosses.

The light that was at the tip of the arrow is now on the right side and the light on the right side is now on the left as far as your eye is concerned (assuming you are further away from the glass than the focal point.

If you move the arrow image closer to the glass than the focal point it will be the way around you expect it to be!

More Refraction experiments

Create an Alice in Wonderland themed version of this too!

Find out how to make your own magnifying glass .

We’ve also got a fun disappearing coin trick .

Or try our light maze to learn about reflection .

Last Updated on February 22, 2021 by Emma Vanstone

Safety Notice

Science Sparks ( Wild Sparks Enterprises Ltd ) are not liable for the actions of activity of any person who uses the information in this resource or in any of the suggested further resources. Science Sparks assume no liability with regard to injuries or damage to property that may occur as a result of using the information and carrying out the practical activities contained in this resource or in any of the suggested further resources.

These activities are designed to be carried out by children working with a parent, guardian or other appropriate adult. The adult involved is fully responsible for ensuring that the activities are carried out safely.

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Water Refraction Science Experiment

This water refraction science experiment has such a "wow factor" and is so quick and easy I can't believe my kids and I haven't done it before. It would fit right in with our DIY science camp series , too. Best of all, there is almost no set up, but once I showed it to the kids, they experimented on their own and debated the reasons for the results. You have the option of setting up the science experiment as we did, or doing it right now with the glass of water sitting next to you!

What you need:

• Jar or glass
• A paper with a design on it. If you wish, you can download and print our printable . It is two pages and includes the colored bars and two arrows.

Instructions:

Place a jar or glass about 6 inches in front of the colored bars or arrows. Pour in the water. What happens?

Watch the video!

To make it more fun: 

Ask your kids to keep their eyes on the bars/arrows as you slowly pour the water into the jar.

I told my kids I had a magic liquid. I'm pretty sure my 6 year old believed me, but my 10 year old unconvincingly said, "It's water, mom." But then after I performed the experiment, he said, "Now try it with water." Ha! So I guess I did fool him.

Explorations:

• Does it make a difference how close the water is to the paper?
• Does it make a difference if the jar is square or round? What about the size of the jar or glass?
• Draw a diagram of what you think is happening to the light rays. (See explanation below)
• Super nerdy kids ( I say that with love ) can learn more about refraction of light here .

The science behind the water refraction experiment:

Refraction is the bending of light. In this case, light traveled from the air, through the front of the glass jar, through the water, through the back of the glass jar, and then back through the air, before hitting the picture. Whenever light passes from one medium into another, it refracts.

In addition, the water acts as a magnifying glass, which bends the light toward the center. The light comes together at the focal point and beyond the focal point the image looks reversed because the light that was on the right is now on the left, and vice versa. Clear as mud? ( Note: I am not a science teacher and if you would like to correct or add to my explanation in the comments, I welcome it! Update: someone did! Read the comments for more scientific explanation of the refraction phenomena. )

Want another cool and magical water experiment?

• Find out how to make a coin jump from a bottle .
• Or find more fun indoor water activities for kids .

Reader Interactions

Maria Dermitzaki says

February 12, 2017 at 3:25 pm

I would like to receive mails

February 13, 2017 at 9:44 am

You can sign up here: https://www.whatdowedoallday.com/newsletter-sign

Eliana Nevarez says

February 04, 2018 at 10:59 pm

we filled the glass half with water and put the arrow behind the glass and when we moved the arrow to a particular distance behind the glass it makes the arrow look like its going the other way. When light passes from one material to another, it can bend or refract. In the third experiment before hitting the arrow light traveled from the air, through the glass, through the water, through the back of the glass, and then back through the air. Anytime that light passes from one thing into another, it refracts. When light went through the glass the light bent toward the center. That’s where the light all came together this is called the focal point, but beyond the focal point the image reversed because the light rays that were bent pass each other and the light that was on the right side is now on the left and the left on the right and that is what makes the arrow looks reversed.

Sauli Jämsä says

March 30, 2020 at 7:48 am

You should be talking about light traveling FROM the picture TO your eyes.

June 03, 2020 at 9:41 am

Light doesn't travel, it's just waves.

September 19, 2020 at 5:54 am

What is the prinsipal for this experiment

ayushi says

May 12, 2021 at 3:16 am

prinicipal - refraction of light.

person says

February 10, 2022 at 2:15 pm

you spelt principal wrong

February 10, 2022 at 2:16 pm

ranji spelt principal wrong*

February 22, 2021 at 3:58 am

It will just be same if the glass is not round?

February 22, 2021 at 3:15 pm

Might be a fun experiment to try it and find out. 😉

Ms. Right says

January 14, 2023 at 12:04 pm

Everyone spelled it wrong. Principal = the leader of a school. Principle = theory of reasoning

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Refraction of Light Experiment

Ever looked at a straw in a cup of water and wonder why it looks bigger and appears to be bent? Or look at something underwater and wondered why it looks so big? These are all a result of the bending of light or refraction. In today’s science experiment on the refraction of light, we’re going to take a closer look at how light bends.

What is refraction?

Refraction is the bending of light. This occurs when light travels through one material to another (ex: air, water, etc…) Unlike objects that reflect, objects that refract light look different. For example, when looking at a glass of water with a straw in it, the straw may appear to be broken. This happens because light moves more slowly in water than in air. As a result, the light bends as it passes from air to water, making the straw appear to be bent.

Materials for light refraction experiment:

• glass jar, container, or vase
• paper (or use the FREE printable Valentine message from down below)
• marker (to write your message if you’re using paper)

Experiment Instructions:

STEP 1: Fill the glass jar almost to the top with water.

STEP 2: If creating your own “secret message” fold the paper in half. Then write your message on one side of the paper. Make sure to write the message backward from right to left. You can create a second message on the back of the folded paper. To make things even easier, you can always download the Free Valentine printable from down below. It’s already done for you!

STEP 3: Next, place the glass jar on a flat surface. Place the folded paper about 3-4 inches behind the jar filled with water.

STEP 4: Looking through the front side of the glass of water, look at your secret message. What do you see?… Your message is not much of a secret anymore!

How does this refraction experiment work?

During the experiment, light travels from the secret message, through the air, through the glass, through the water, then through the glass again, and through the air one more time before finally reaching your eyes.

When light travels through different materials such as the glass jar, air, or water, it travels at different speeds. This causes the light to refract.

Light waves travel faster through the air than they do through water or glass because the air is less dense. It then slows down a little when traveling through the water and is at its slowest, when passing through the glass jar. This is what causes the light to refract or bend and make the secret message change direction. As a result, the message is no longer a “secret” and can be read.

The light that is refracted through the glass of water also acts as a magnifying glass. It makes the image appear larger than it really is. Try moving the image closer to the glass jar and see what happens.

Helpful Resources

If you like kid-friendly science resources and want to learn more about light energy & the other forms of energy, check out my complete energy unit perfect created with kids in mind.

… and if you’d like to use the “secret” messages I used above for the refraction lab, you can download it for free here.

(If you liked these tips, feel free to use this image to save this post to your Pinterest board. )

A third-grade teacher with a passion for creating time-saving classroom resources. She enjoys sharing her attempt to juggle it all... grading papers, lesson planning, student referrals, parent communication, test prep, and so much more all while managing a busy home life with two active teens.

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Refraction of light.

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Refraction is the bending of light (it also happens with sound, water and other waves) as it passes from one transparent substance into another.

This bending by refraction makes it possible for us to have lenses, magnifying glasses, prisms and rainbows. Even our eyes depend upon this bending of light. Without refraction, we wouldn’t be able to focus light onto our retina.

Change of speed causes change of direction

Light refracts whenever it travels at an angle into a substance with a different refractive index (optical density).

This change of direction is caused by a change in speed. For example, when light travels from air into water, it slows down, causing it to continue to travel at a different angle or direction.

How much does light bend?

The amount of bending depends on two things:

• Change in speed – if a substance causes the light to speed up or slow down more, it will refract (bend) more.
• Angle of the incident ray – if the light is entering the substance at a greater angle, the amount of refraction will also be more noticeable. On the other hand, if the light is entering the new substance from straight on (at 90° to the surface), the light will still slow down, but it won’t change direction at all.

Refractive index of some transparent substances

All angles are measured from an imaginary line drawn at 90° to the surface of the two substances This line is drawn as a dotted line and is called the normal.

If light enters any substance with a higher refractive index (such as from air into glass) it slows down. The light bends towards the normal line.

If light travels enters into a substance with a lower refractive index (such as from water into air) it speeds up. The light bends away from the normal line.

A higher refractive index shows that light will slow down and change direction more as it enters the substance.

A lens is simply a curved block of glass or plastic. There are two kinds of lens.

A biconvex lens is thicker at the middle than it is at the edges. This is the kind of lens used for a magnifying glass. Parallel rays of light can be focused in to a focal point. A biconvex lens is called a converging lens.

A biconcave lens curves is thinner at the middle than it is at the edges. Light rays refract outwards (spread apart) as they enter the lens and again as they leave.

Refraction can create a spectrum

Isaac Newton performed a famous experiment using a triangular block of glass called a prism. He used sunlight shining in through his window to create a spectrum of colours on the opposite side of his room.

This experiment showed that white light is actually made of all the colours of the rainbow. These seven colours are remembered by the acronym ROY G BIV – red, orange, yellow, green, blue, indigo and violet.

Newton showed that each of these colours cannot be turned into other colours. He also showed that they can be recombined to make white light again.

The explanation for the colours separating out is that the light is made of waves. Red light has a longer wavelength than violet light. The refractive index for red light in glass is slightly different than for violet light. Violet light slows down even more than red light, so it is refracted at a slightly greater angle.

The refractive index of red light in glass is 1.513. The refractive index of violet light is 1.532. This slight difference is enough for the shorter wavelengths of light to be refracted more.

A rainbow is caused because each colour refracts at slightly different angles as it enters, reflects off the inside and then leaves each tiny drop of rain.

A rainbow is easy to create using a spray bottle and the sunshine. The centre of the circle of the rainbow will always be the shadow of your head on the ground.

The secondary rainbow that can sometimes be seen is caused by each ray of light reflecting twice on the inside of each droplet before it leaves. This second reflection causes the colours on the secondary rainbow to be reversed. Red is at the top for the primary rainbow, but in the secondary rainbow, red is at the bottom.

Activity ideas

Use these activities with your students to explore refration further:

• Investigating refraction and spearfishing – students aim spears at a model of a fish in a container of water. When they move their spears towards the fish, they miss!
• Angle of refraction calculator challenge – students choose two types of transparent substance. They then enter the angle of the incident ray in the spreadsheet calculator, and the angle of the refracted ray is calculated for them.
• Light and sight: true or false? – students participate in an interactive ‘true or false’ activity that highlights common alternative conceptions about light and sight. This activity can be done individually, in pairs or as a whole class .

Useful links

Learn more about different types of rainbows, how they are made and other atmospheric optical phenomena with this MetService blog and Science Kids post .

Learn more about human lenses, optics, photoreceptors and neural pathways that enable vision through this tutorial from Biology Online .

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Simple Science Experiments: Simple Light Refraction Experiment

December 28, 2017 by Selena Robinson 9 Comments

Sharing is caring!

We’re continuing with our simple science experiments this week by taking a look at light refraction. I found a great light refraction experiment video on YouTube and decided to try it with Tigger.

Full disclosure: I didn’t know that refraction was what this experiment demonstrated. I actually had to look it up first. But the experiment is super easy and quick, so that’s a big plus!

Check out this easy way to teach kids about light with this simple light refraction experiment !

And, if you like this one, try some of our other science activities, including how to make an egg float and our easy heat conduction experiment !

Simple Light Refraction Experiment

Watching the original light refraction experiment on YouTube will give you a great look at what’s involved in this activity. But you really only need four things:

• A sticky note (I used a Post-It)
• An empty transparent bottle

Draw two arrows on a sticky note. Make sure that each arrow points in a different direction. Stick the note to a blank wall.

Next, fill up the water bottle. Oh – put the lid on before you do this too! You don’t want water spilling out when you move the bottle around…lol.

The alternating arrows on the note point to the left and the right. Let the kids gradually move the water-filled bottle in front of the sticky note. As the bottle moves in front of the sticky note, something amazing happens.

The arrows appear to change direction! The top arrow, which points to the left, appears to point to the right. And the bottom arrow, which points to the right, appears to point to the left!

Move the bottle back to see the arrows return to their original directions.

So what exactly is going on? We learned that refraction occurs because light bends when it passes through substances, such as water and plastic.

As the light travels through a substance, it becomes concentrated into a focal point, usually near the center. After light passes through the focal point, the rays cross over each other and cause images to appear reversed.

Turns out you can’t believe your eyes after all! 🙂

Books with Simple Science Experiments:

If you liked this simple science experiment, take a look at these books with even more easy activities! (Affiliate links provided here for convenience. For details, see our Disclosure Policy .)

• Science is Simple: Over 250 Activities for Preschoolers
• 365 Simple Science Experiments with Everyday Materials
• The Everything Kids’ Science Experiments Book
• Safe and Simple Electrical Experiments

Don’t miss the rest of our Simple Science Experiments!

For more science homeschooling ideas, follow my It’s Science board on Pinterest!

P.S. Get more fun learning ideas in our email newsletter!

July 13, 2014 at 5:11 pm

I love this demonstration. Must do it again with my kids! Amazing how much you can learn and do with simple household objects!

August 3, 2014 at 1:43 am

Light refraction and how it moves is really so cool. Thanks for linking up to Science Sunday (even when I’m behind on commenting).

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[…] Simple Light Refraction Experiment from Look, We’re Learning!: This experiment is quick and easy to set up, and it’s a simple way to teach kids about light refraction in water using a water bottle, a post-it note, and a marker. […]

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Water Refraction Experiment (Video)

Have you ever noticed that things look strange when you view them through a glass of water?

It’s not just because of imperfections in the glass; there’s a fascinating scientific phenomenon at work.

When light waves pass from one transparent substance to another, such as from air to water or air to glass.

The speed of light changes as it enters a different medium. When light travels through denser materials like water or glass, it slows down.

This change in speed affects the direction of the light, causing it to bend. This bending of light is what we call refraction.

Refraction is the bending of light as it passes from one transparent medium into another ​1​ . 

Let’s explore a simple water refraction experiment using everyday materials like a glass of water placed in front of a sheet of paper.

In this experiment, we observe the phenomenon of refraction in action.

This fun and educational activity will help kids and adults understand how light bends through different mediums, such as air, water, and glass.

With just a glass of water, a few graphics, and some creativity, you can observe the bending of light and explore the scientific principles behind this phenomenon.

Here are two simple and fun water refraction experiments .

Refraction of Light in Water

Here is an easy science experiment you can do to "wow" your kids or friends. It's super simple and you can set it up within minutes.

• graphics you want to see the changes
• a clear glass or jar
• adult supervision

Instructions

• Place the glass in front of the graphics.
• Adjust the distance between the glass and the graphics until the image can be seen clearly through the empty glass.

Did you try this project?

Follow us on Pinterest and share a photo!

• Do the experiment again using different distances between the glass and the graphics
• Try glasses and jars of different shapes .
• Try using different liquids such as oil, juice, etc.

The Science of Light Refraction

Light refraction is a science key concept wherein light changes direction as it moves from one substance to another due to a difference in densities.

For instance, when light travels from air to water or glass, it slows down and bends. This bending of light causes objects viewed through these substances to appear distorted or displaced.

The angles at which the light enters and exits the substance play an important role in determining the level of distortion.

Transparent materials like lenses and prisms can also influence light refraction, and a simple glass of water can be a great example to explore these principles.

Our eyes and corrective lenses utilize the same refraction principles to help us focus and perceive the world more clearly.

Exploring Refraction and the Bending of Light with Water and Glass

The Water Refraction Experiment used a clear glass or jar filled with water and placed in front of the graphics you want to see the changes.

Adjusting the distance between the glass and the graphics allows us to observe how the image changes as the light passes through the water and the glass.

Trying glasses and jars of different shapes or using various liquids, such as oil or juice, can further enhance this exploration of light refraction.

When we look at objects through a glass cup of water, the light coming from those objects goes through multiple refractions.

As the light enters the cylindrical glass from the surrounding air, it bends or refracts due to the change in density between the air and the glass.

This bending of light continues as it passes from the glass into the water, encountering another change in density.

When the light exits the water and enters the air or a shallower layer and then again passes from the glass with water to the air, it undergoes additional refractions.

These repeated light refractions create interesting effects we can observe during the experiment.

The Role of Density and Temperature

The density and temperature of the medium through which light travels impact how light bends or refracts.

When light passes through a denser material, such as water, it slows down and changes direction more significantly.

This phenomenon is influenced by the density of the water and the transition from air into water.

Likewise, changes in air temperature or the temperature of liquids can affect the refraction angle. As temperature alters the density of the medium, it can lead to different degrees of light bending.

Exploring the Water Refraction Experiment with cold water or liquids at different temperatures can provide fascinating insights into how temperature, density, and light refraction are interconnected.

Additional Observations and Experiments

The Magical Water Experiment presents exciting opportunities for further investigation.

By adjusting the angle at which light enters the glass or jar, we can observe different refraction angles and more pronounced changes in the images.

Exploring transparent mediums, such as prisms or substances with different densities, allows us to understand how light bends and how density influences refraction.

The fun water refraction experiments provide valuable hands-on learning experiences that allow us to explore the intricate relationship between light and matter.

The best part is that these experiments can be conducted using simple, affordable, and easily accessible materials.

The experiment offers a fantastic way to delve into the captivating world of light refraction.

A fun, educational, and engaging activity, the Water Refraction Experiment brings the magic of science to life for kids and adults alike.

Science Kits And Books On Light

• 1. Jiang W, Chen RT, Lu X. Theory of light refraction at the surface of a photonic crystal. Phys Rev B . June 2005. doi: 10.1103/physrevb.71.245115

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Refraction of Light : Play & Learn Activity for Kids

• July 15, 2019
• 10 Minute Science , 5-6 Year Olds , Household Items , Physics , Popular , Rainy Day Science , STEAM

Today we are going to learn about some simple physics by doing simple and easy science activity i.e. Refraction of light.

This activity is so quick and I really wondering how I missed this experiment this long to explain or show my kids and make them Wowww!! This is one among the 10 min STEAM activity . This activity is perfect for 5-7 years old kids (since they are in budding stage to understand physics), however all the kids can understand the science behind light refraction. Children under 5 years may not understand the science but they will enjoy the experiment results. The best part is, it is an experiment which is listed under experiments done with items available at home . 

As mentioned it is suitable for all kids. However, 5-7 year-old kids can understand the science behind this. These children will know light and can relate to refraction. Smaller ones will enjoy but may not understand the details and the concept.

In the beginning, my daughters were like scratching their heads and saying “what Mamma!! What are you going to do only with a glass of water”? “We don’t find much ingredients except water which is boring!!” There is a reason why kids were disappointed seeing the ingredients because this experiment or activity is so quick and best part is that it does not need much ingredients. In addition, this activity doesn’t need much set up as well since we can set it up within minutes.

But after seeing the magic that light does in water, my kids were amazed. Let us see what made them surprised in this activity.

What is refraction of light?

When light travels from one transparent medium (air) to another transparent medium (water), the speed of the light slows down and when it hits water it changes its direction slightly. This change in the direction of light is known as refraction of light. In a simple language, light refraction is bending of light. This looks simple and easy but it is unusual.

Supplies Needed

• A piece of paper
• A glass or jar
• Graphic designed papers. You can download the graphic designs from the net and take printouts. (optional)

[*Product links are affiliate links. Your support is highly appreciated]

Experiment  Steps : Refraction of Light

Firstly, ask your kids to fill the glass jar carefully with the clean and clear water. Here I tried to fool my kids saying that I have given them a magical liquid to fill. My younger one (5 year old) is almost in my trap but not my elder one (7 years old). She laughed at me and said ‘hey! Don’t fool me mom, it is just water’! Lollll…

Get a piece of paper and ask your kid to draw an arrow. Let the kids draw the arrow in any direction (any direction of arrow works out) and hold it vertically.

Now place the water filled jar in front of the paper (exactly focusing) just before the arrow. Adjust the glass jar between the glass and the arrow on the paper until the image (arrow) can be seen clearly through the glass.

Ask your kids to come in front of the glass jar and see what happens. Here ask them a few questions like ‘what did they observe’, is there any direction changes’, if the answer is yes (from kids), then ask for any clues or reasons to explain why it happened.

(You can have a debate session by gathering your kid’s friends as well. Having debate sessions improve their communication skills, speaking skills in public as well as builds confidence).

Coming to our activity, in step 4 you can watch the arrow appearing in reverse direction through the glass jar. This is just amazing and pretty surprising right!!

Now, my younger daughter took a paper and showed her drawing skills as well by drawing a small cat to check again through the water. J She is totally in wow mood seeing her cat in reverse direction through the water.

What to explore

• Try different glasses and jars of different shapes
• Repeat the experiments using different distances between the graphics and the glass jar
• Also try the experiment using different liquids like oil, juice etc.

Observations

• You can observe some aberrations or blurriness when you see through the glass. This is due to the imperfections in the glass production.
• Things look like a little funny and different when you see through a glass.

What is the secret behind this activity?

The secret that made the experiment work is “Refraction” . As I already told you, refraction is bending of light as it passes from one medium to another medium. Ex: Air to Water or Water to Air.

In our experiment, the light wave traveled from the image (arrow) into the water crossing the glass cup and finally traveled out of the glass cup and again into the air and reached our eyes. Here, water acts as a magnifying glass which makes light more concentrated towards the focal point (near to the center). When the light ray goes beyond this focal point (to come out of the glass jar in order to reach our eyes) the image looks reversed. That means, the light on the left direction is now changes to right direction.

Hope you all too understand the experiment. Have a try with your kids!! 

Here are some concepts that you can teach to your kids.. 

How is refraction demonstrated in light?

Refraction is manifested as bending of light as it passes from one transparent medium to another. Here, the light wave or ray passes from air to glass and back to air before it reaches our eye lens. The bending is caused due to differences in density between the two mediums. When the light passes from more dense medium to less dense medium, the light refracts in reverse direction and vice versa.

Why does an arrow change direction behind a glass of water?

When the arrow (behind the glass) is observed at a particular distance, it looks like it is reversed itself. Actually what happens is when light travels from one medium to another, it is proved that it can bend or refract. During the experiment, the light initially traveled from the air, through the glass, through the water and comes out to reach our eyes crossing again the same mediums. Anytime that light passes from one medium, or material, into another, it refracts. while crossing the medium the light ray goes beyond the focal point (near to the center) which causes the arrow change its direction.

What is the science behind refraction?

The transmission of light between any two transparent media results in a change in both the speed and wavelength of the wave. The light wave speeds up or slows down and transforms into a wave with a larger or a shorter wavelength along with change in direction when it passes through the boundary of two media. What causes refraction?

The difference in the density of transparent medium that light passes through causes refraction. 

Why does light bend during refraction? Bending of light occurs during refraction because of change in the speed of light wave while crossing the medium.

What is wave refraction?

In oceanography, the wave refraction is manifested as bending of waves while propagating through different depths.

What are some natural examples of refraction? Forming of rainbow when the bending of sun ray’s as they enter rain drops, prism, sunset, etc are some natural examples of refraction.

What are the effects of refraction?

The effects of light refraction are responsible for a variety of familiar phenomena, such as: 1) A straight pencil when dipped in the half filled glass jar always looks bending in the water due to refraction effect. 2) When a coin is dropped in the glass of water, it looks it has raised in its length just because of refraction effect. 3) swimming pool looks shallower than the reality as the light coming from the bottom of the pool bends when it comes out at the surface due to refraction of light. Why is refraction important? Without refraction of light, we could not see any fine details happening or present around us or in the environment since your corneas and lenses enable this, via refraction.

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Optics (Essentials) - Class 12th

Course: optics (essentials) - class 12th   >   unit 3.

• Refraction and Snell's law
• Snell's law example
• Refraction in water
• Snell's law example 2
• Reflection and refraction questions
• Refractive index and the speed of light

Refraction and light bending

• Why do stars twinkle (but planets don't)?

Light changes speed

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Simple Refraction of Light Science Experiment – Why Does the Straw Look Bent?

What do you think, can water bend a straw? What about other clear liquids? In this super simple science experiments, kids will learn about the refraction of light, and why a straw appears to be bent when submerged in a glass of liquid.

Find printable instructions, a video demonstration, and a helpful explanation of how this experiment works, below.

JUMP TO SECTION: Instructions | Video Tutorial | How it Works | Purchase Lab Kit

Supplies Needed

• Empty Glass
• Plastic Straw

Refraction of Light Science Lab Kit – Only $5

Use our easy Refraction of Light Science Lab Kit to grab your students’ attention without the stress of planning!

It’s everything you need to  make science easy for teachers and fun for students  — using inexpensive materials you probably already have in your storage closet!

Refraction of Light Science Experiment Instructions

Step 1 – Start with some observations about the straw. Is there anything unusual about the straw? Make sure the kids notice that is it perfectly straight. Next, place the straw in the empty glass. Make a few more observations and point out that the straw is still straight.

Helpful tip: Use a large plastic straw from a water bottle to see the results more clearly.

Step 2 –  Next, pour water into the glass until it is nearly full. Now make a few more observations. What is different about the straw? Make sure to look directly at the side of the glass at the straw. What do you see?

Step 3 –  You will notice that near the top of the water line the straw appears to bend. Remove the straw from the water. Is it still bent? Do you know what caused the straw to appear to be bent? Find out the answer in the how does this experiment work section below.

Video Tutorial

How Does the Science Experiment Work

When you add the water to the glass, the straw appears to bend, but once you remove the straw you see it isn’t really bent at all. This is because the straw is not bending, but the light around the straw is bending due to refraction . Light refracts as it passes from one medium to the next because it travels at different speeds through those mediums. Light travels fastest through air, a little slower through water, and even slower through glass.

WHAT IS REFRACTION OF LIGHT? Refraction is the bending of light and occurs when light travels from one medium to another. For example when the light moves from air to water, or from water to air. 

We need light in order to see. When we look at the straw outside of the glass of water, the light coming from the straw travels through the air straight to your eye. As you look at the straw in the glass of water, the light coming from the straw to your eye bends as it passes through three different mediums (water, glass, and air). As the light passes from one medium to the next, it changes speed and bends.

Other Ideas to Try

Try this experiment with liquids other than water. Liquids to try would be light corn syrup, rubbing alcohol, or clear Gatorade. Does the liquid you use affect how much the straw appears to bend?

More Experiments that Show The Refraction Of Light

Light Refraction Science Experiment – Watch in amazement as the arrow to changes direction.

Ruler Changes Size Science Experiment  – Observe how the size of an object changed when viewed through different liquids. 

I hope you enjoyed the experiment. Here are some printable instructions:

Can Water Bend a Straw Experiment

Instructions.

• Observe the straw and notice that it is perfectly straight. Next, place the straw in the empty glass and take note that the straw is still straight. Helpful tip: Use a large plastic straw from a water bottle to see the results more clearly.
• Pour water into the glass until it is nearly full. Look directly at the side of the glass at the straw. What do you see?
• You will notice that near the top of the water line the straw appears to bent. Remove the straw from the water. Is it still bent?

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10 Easy & Engaging Water Refraction Activities For Kids

August 3, 2023 //  by  Lauren Du Plessis

Are you looking to bring science to life in the most engaging way possible? We’ve curated a list of easy-to-set-up water refraction activities to ignite your student’s curiosity. These experiments, which involve bending light and color spectrums, offer a practical exploration of the laws of physics. While providing endless fun, they will also help you portray fundamental scientific concepts in an easy-to-understand way. So, dive right in and spark some light-bending magic in your classroom!

1. Bending Pencil Experiment

Add a sprinkle of science to a regular day with this hands-on activity. Ask your students to observe as you submerge a pencil halfway into a glass of water. As they watch it appear to look broken, explain how light waves change direction when they pass from one medium to another. This is a fantastic way to introduce the concept of light behavior and optics to your young students!

Learn More: Ingenium

2. Water Prism

For this experiment, expose a glass of water to light rays from the sun. As the light passes through the water-filled prism, it’ll bend and split into a spectrum of colors; showing kids the colorful world hidden in plain light. Be prepared for an array of “oohs” and “aahs”!

Learn More: YouTube

3. Magic Coin Trick

Bring the wonder of magic into the classroom with this simple yet engaging activity. Place a coin at the bottom of an empty container and step back until the coin is no longer visible. Pour water into the container and watch as the coin “magically” reappears. Your students will be amazed at how the path of light can alter what we see!

Learn More: Mombrite

4. Creating Rainbows

Make a sunny day even brighter with this fun and colorful activity. With a garden hose and a good spray of water, demonstrate how sunlight refracts and disperses when passing through water droplets, creating a beautiful rainbow. The best part? No pot of gold is required!

Learn More: Big Bang Education

5. Underwater Color Mixing

Delve into a world of color beneath the water’s surface. Using colored objects submerged in water, ask your students to observe how colors appear to change due to the bending of light waves. It’s a vibrant way to engage the artistic scientist in every child!

Learn More: PBS Learning Media

6. Disappearing Glass Rod

Enthrall your students with this magic trick that makes a glass rod disappear in the water! It’s very easy, too: Pour vegetable oil into a container, add a glass rod, and witness the vanishing act! Explain to your students that when two substances have a similar refractive index, the boundary between them becomes almost invisible, creating the illusion of disappearance.

Learn More: Gr5.org

7. Flipping Arrow Illusion

Who says arrows can’t change direction? Draw an arrow, place it behind a glass of water, and watch as it seemingly flips direction! Your young learners will marvel at this visual twist; understanding how refraction can affect our perception.

Learn More: What Do We Do All Day

8. Water Lens

In this experiment, have your students create a makeshift magnifying lens using only water and clear plastic. By observing how the makeshift water lens magnifies objects, they’ll grasp the refractive properties of water and learn how lenses work in a practical, engaging manner.

9. Floating Flower Fantasy

Enchant your students with a floating flower garden! In this floral variation of the broken pencil experiment, begin by immersing vibrant flowers in a water-filled vase and let your students wonder at the seemingly severed stems! This captivating optical illusion provides a tangible demonstration of water refraction in action.

Learn More: Pinterest

10. Aquatic Amplification – Fish in a Bowl

If you’re looking for a more hands-on activity that indulges your students’ creativity, we have you covered. Have your learners craft their own fish from paper, ask them to pop it into an empty fishbowl, and then fill up the bowl with water. As they observe the amplified and slightly distorted fish, their excitement will rise with the water level. 

Learn More: Frugal Fun 4 Boys

This Old Experiment With Mice Led to Bleak Predictions for Humanity’s Future

From the 1950s to the 1970s, researcher John Calhoun gave rodents unlimited food and studied their behavior in overcrowded conditions

Maris Fessenden ; Updated by Rudy Molinek

What does utopia look like for mice and rats? According to a researcher who did most of his work in the 1950s through 1970s, it might include limitless food, multiple levels and secluded little condos. These were all part of John Calhoun’s experiments to study the effects of population density on behavior. But what looked like rodent paradises at first quickly spiraled into out-of-control overcrowding, eventual population collapse and seemingly sinister behavior patterns.

In other words, the mice were not nice.

Working with rats between 1958 and 1962, and with mice from 1968 to 1972, Calhoun set up experimental rodent enclosures at the National Institute of Mental Health’s Laboratory of Psychology. He hoped to learn more about how humans might behave in a crowded future. His first 24 attempts ended early due to constraints on laboratory space. But his 25th attempt at a utopian habitat, which began in 1968, would become a landmark psychological study. According to Gizmodo ’s Esther Inglis-Arkell, Calhoun’s “Universe 25” started when the researcher dropped four female and four male mice into the enclosure.

By the 560th day, the population peaked with over 2,200 individuals scurrying around, waiting for food and sometimes erupting into open brawls. These mice spent most of their time in the presence of hundreds of other mice. When they became adults, those mice that managed to produce offspring were so stressed out that parenting became an afterthought.

“Few females carried pregnancies to term, and the ones that did seemed to simply forget about their babies,” wrote Inglis-Arkell in 2015. “They’d move half their litter away from danger and forget the rest. Sometimes they’d drop and abandon a baby while they were carrying it.”

A select group of mice, which Calhoun called “the beautiful ones,” secluded themselves in protected places with a guard posted at the entry. They didn’t seek out mates or fight with other mice, wrote Will Wiles in Cabinet magazine in 2011, “they just ate, slept and groomed, wrapped in narcissistic introspection.”

Eventually, several factors combined to doom the experiment. The beautiful ones’ chaste behavior lowered the birth rate. Meanwhile, out in the overcrowded common areas, the few remaining parents’ neglect increased infant mortality. These factors sent the mice society over a demographic cliff. Just over a month after population peaked, around day 600, according to Distillations magazine ’s Sam Kean, no baby mice were surviving more than a few days. The society plummeted toward extinction as the remaining adult mice were just “hiding like hermits or grooming all day” before dying out, writes Kean.

Calhoun launched his experiments with the intent of translating his findings to human behavior. Ideas of a dangerously overcrowded human population were popularized by Thomas Malthus at the end of the 18th century with his book An Essay on the Principle of Population . Malthus theorized that populations would expand far faster than food production, leading to poverty and societal decline. Then, in 1968, the same year Calhoun set his ill-fated utopia in motion, Stanford University entomologist Paul Ehrlich published The Population Bomb . The book sparked widespread fears of an overcrowded and dystopic imminent future, beginning with the line, “The battle to feed all of humanity is over.”

Ehrlich suggested that the impending collapse mirrored the conditions Calhoun would find in his experiments. The cause, wrote Charles C. Mann for Smithsonian magazine in 2018, would be “too many people, packed into too-tight spaces, taking too much from the earth. Unless humanity cut down its numbers—soon—all of us would face ‘mass starvation’ on ‘a dying planet.’”

Calhoun’s experiments were interpreted at the time as evidence of what could happen in an overpopulated world. The unusual behaviors he observed—such as open violence, a lack of interest in sex and poor pup-rearing—he dubbed “behavioral sinks.”

After Calhoun wrote about his findings in a 1962 issue of Scientific American , that term caught on in popular culture, according to a paper published in the Journal of Social History . The work tapped into the era’s feeling of dread that crowded urban areas heralded the risk of moral decay.

Events like the murder of Kitty Genovese in 1964—in which false reports claimed 37 witnesses stood by and did nothing as Genovese was stabbed repeatedly—only served to intensify the worry. Despite the misinformation, media discussed the case widely as emblematic of rampant urban moral decay. A host of science fiction works—films like Soylent Green , comics like 2000 AD —played on Calhoun’s ideas and those of his contemporaries . For example, Soylent Green ’s vision of a dystopic future was set in a world maligned by pollution, poverty and overpopulation.

Now, interpretations of Calhoun’s work have changed. Inglis-Arkell explains that the main problem of the habitats he created wasn’t really a lack of space. Rather, it seems likely that Universe 25’s design enabled aggressive mice to stake out prime territory and guard the pens for a limited number of mice, leading to overcrowding in the rest of the world.

However we interpret Calhoun’s experiments, though, we can take comfort in the fact that humans are not rodents. Follow-up experiments by other researchers, which looked at human subjects, found that crowded conditions didn’t necessarily lead to negative outcomes like stress, aggression or discomfort.

“Rats may suffer from crowding,” medical historian Edmund Ramsden told the NIH Record ’s Carla Garnett in 2008, “human beings can cope.”

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Maris Fessenden | | READ MORE

Maris Fessenden is a freelance science writer and artist who appreciates small things and wide open spaces.

Rudy Molinek | READ MORE

Rudy Molinek is  Smithsonian  magazine's 2024 AAAS Mass Media Fellow.

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The effects of droplet stabilization by surfactants and nanoparticles on leakage, cross-talk, droplet stability, and cell adhesion †

First published on 1st August 2024

Partially fluorinated nanoparticles (FNPs) have been proposed as a promising alternative for stabilising aqueous droplets in fluorinated oils. The exceptional energetic stability of FNPs at the droplet interface holds the potential for minimising leakage, enhancing stability, and promoting improved cell adhesion. However, their lower diffusion coefficient compared to surfactants presents challenges in achieving rapid droplet stabilisation, which is important in microfluidics applications. While several studies have focused on some of these aspects, a comprehensive study and direct comparison with conventional fluorosurfactants is still missing. In this manuscript, we undertake an examination and comparison of four crucial facets of both FNP- and surfactant-stabilised droplets: leakage of compounds, emulsion stability, droplet formation dynamics and cell adhesion. Contrary to what has previously been claimed, our findings demonstrate that FNPs only reduce leakage and cross-talk in very specific cases ( e.g. , resorufin), failing to provide enhanced compartmentalisation for highly hydrophobic dyes ( e.g. , rhodamine dyes). On the other hand, FNP-stabilised droplets indeed exhibit greater long-term stability compared to their surfactant-stabilised counterparts. Regarding the size of droplets generated via a diversity of microfluidic methods, no significant differences were observed between FNP-stabilised and surfactant-stabilised droplets. Finally, the previously reported improvements in cell adhesion and spreading on FNP-stabilised interfaces is limited to flat oil/water (o/w) interfaces and could not be observed within droplets. These comprehensive analyses shed light on the nuanced performance of FNPs and commercial fluorosurfactants as stabilising agents for aqueous droplets in fluorinated oils, contributing valuable insights for choosing the correct formulation for specific droplet-based microfluidics applications.

To effectively utilise such droplets as individual reaction vessels, it is imperative to prevent the transfer of materials from one droplet to another and/or the loss of these materials into the surrounding carrier phase. Achieving this crucial encapsulation relies primarily on the selection of an appropriate system consisting of a carrier phase and a stabilising agent. Fluorinated oils, composed of perfluorocarbon compounds bearing highly polarised C–F bonds, have emerged as a pivotal component in this pursuit. 11–13 These fluorinated oils exhibit notable characteristics that render them exceptionally suited for use in droplet-based microfluidics. Their inherently low hydrogen-bonding capacity 11 and weak London dispersion forces 12 result in minimal solubility of both polar and non-polar substances, effectively preventing loss of encapsulated compounds into the carrier phase. Remarkably, these oils also possess a high capacity for dissolving gases, thereby promoting the survival of enclosed biological entities. 14 Furthermore, unlike certain other oils, fluorinated oils do not induce swelling in polydimethylsiloxane (PDMS) microfluidic devices. 15 To prevent droplets from merging, though, stabilising reagents are typically needed. Traditionally, these have relied on fluorinated surfactants featuring perfluorinated carbon chains linked to hydrophilic groups. Surfactants with longer fluorocarbon tails, such as perfluoropolyethers (PFPE), have been shown to extend droplet stability. 1,16–19

Notably, two prominent PFPE-based surfactants, ‘Krytox’ by DuPont and ‘RAN’ by RAN Biotechnologies (see Fig. 1 ), have gained widespread usage in droplet-based microfluidics. RAN, a PFPE-PEG-PFPE block copolymer, is well-documented for its ability to yield stable droplets with biocompatible interfaces that are also significantly robust against leakage of hydrophilic compounds, 20–22 however for more hydrophobic compounds leakage remains problematic. In contrast, Krytox, a PFPE-carboxylic acid variant, offers cost-effectiveness but is susceptible to increased leakage due to interactions between the carboxylic acid moiety and encapsulated compounds. 1

Despite the remarkable attributes of fluorinated oil-surfactant systems in droplet-based microfluidics, challenges persist in preventing the leakage of certain compounds into the carrier phase or mitigating cross-talk between droplets. 23 It is important to delineate between two distinct phenomena in this context: ‘cross-talk’ and ‘leakage’. ‘Cross-talk’ refers to the transfer of compounds between droplets, whereas ‘leakage’ pertains to the undesired loss of droplet contents into the surrounding oil phase. These two effects arise from a diversity of mechanisms which include diffusion-driven partitioning, micellar-induced transport and surfactant–compound interactions. 4,23,24

In contrast to the conventional approach of modifying molecular surfactants to address leakage concerns, Pan et al. proposed a novel strategy employing fluorinated silica nanoparticles (FNPs) as stabilising agents for droplets ( i.e. , Pickering emulsions). 25–28 In their reports, FNPs are claimed to become more energetically trapped at the droplet interface compared to surfactant molecules, leading to reduced mobility. 29 Crucially, they eliminate the potential for micelle formation, resulting in droplets characterised by lower leakage rates. For instance, in their experiments they provide evidence that resorufin (a slightly hydrophobic dye) remains confined within FNP-stabilised droplets, while leakage occurs in surfactant-stabilised counterparts. 30 Still, a thorough study investigating the behaviour of different dyes in different carrier systems has, so far, been lacking.

In addition to reducing cross-talk and leakage, FNP-stabilised droplets may offer enhanced stability compared to their surfactant-stabilised counterparts, potentially enabling extended storage periods. 28,30 Such effects, though, have not yet been investigated in depth to date. Furthermore, previous studies have hinted at the potential of FNPs to serve as substrates for cell adhesion and growth within microfluidic droplets, 25,31 but research in this domain has remained limited to illustrative demonstrations.

Despite their postulated advantages, the production of FNP-stabilised droplets presents its own set of intricacies. Due to their larger size and associated lower diffusion rates compared to surfactant molecules, the time required for droplet stabilisation with FNPs is typically extended. Consequently, specialised microfluidic chip designs are needed to facilitate stable droplet formation. Typically, these consist of elongated serpentine channels that afford sufficient time for FNPs to diffuse over the droplet interface before droplets become in contact with each other. 27,32 Alternatively, microfluidic chips featuring additional inlets are operated to enable the deposition of highly concentrated particle solutions in close proximity to the droplet interface. 32 All these approaches, though, focus on droplet generation via flow-focusing strategies and a comparative study for other droplet production strategies ( e.g. , step-emulsification) is still missing.

In view of the significant knowledge gaps on our understanding of the performance of FNPs for droplet-based microfluidic applications, we carried out a comprehensive exploration of several important features of these stabilising agents in these systems. First, leakage and cross-talk phenomena were analysed and compared to two surfactants (Krytox and RAN) typically used to stabilise droplets, which was not yet done in previous research. To do this, we introduced a statistical methodology to rigorously investigate and quantify these phenomena. Notably, this study marks the first systematic comparison of leakage and cross-talk behaviours between droplets stabilised with surfactants and FNPs. To examine this phenomena, the behaviour of 15 different dyes encapsulated within droplets was examined for four different buffer conditions by combining these with empty droplets and monitoring fluorescence on day zero and day one. Additionally, we explored different microfluidic strategies for droplet production and stabilisation, encompassing three distinct flow-focusing configurations, a T-junction, a combination of step-emulsification and flow focusing and two step-emulsification devices. Furthermore, we assessed the validity of the assumption that FNPs are energetically more favourably trapped at the droplet interface than surfactants, comparatively reducing droplet coalescence. To evaluate this aspect, we subjected droplets to various stress conditions, including PCR thermocycling and incubation at room temperature, in a refrigerator and in an incubator for extended periods of time, whereupon droplet size was monitored over time to discern any trends in polydispersity. Finally, we investigated the potential enhancement in adhesion of anchorage-dependant cells on FNP-stabilised interfaces when compared to surfactants.

These comprehensive analyses of four important functionalities in the field of droplet-based microfluidics address some of the most relevant knowledge gaps currently present in the utilisation of FNPs as droplet stabilisers and allow us to provide an assessment of the advantages and disadvantages associated with their use in this field.

1. Materials and methods

15 different dyes were compared in the droplet leakage assay: fluorescein sodium salt (Fl), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Pyr), sulforhodamine 101 (SulfRhod), rhodamine 101 (Rhod101), rhodamine B (RhodB), rhodamine 6G (Rhod6G), resorufin (Res) and alizarin red (AZ) were purchased from Sigma Aldrich. Nile red (NR), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), eosin-5-maleimide (EMA), Alexa fluor 350 (AF350), Alexa Fluor 488 (AF488), Alexa Fluor 568 (AF568) were obtained from Thermofisher. Sulfo-cy5 NHS ester (Cy5) was acquired from fluoroProbes and sulfo-cyanine 7 NHS ester (Cy7) from LumiProbe. 10 mM stock solutions were prepared in DMSO which were diluted 100× upon use for droplet production in the correct aqueous solution. Chemical structures of each dye are depicted in Fig. S2 and S3. †

The distribution coefficients were calculated with Marvin Sketch 23.8 by using ChemAxon and can be retrieved in Table 2 . The distribution coefficient is defined as the expected ratio of the sum of concentrations of all forms of the fluorophore (ionized plus unionized) in water and in a non polar solvent (in this case octanol) and gives a good indication of the hydrophobicity of a molecule. 23 All tautomers and resonance structures of the dyes were taken into account during the calculations. The electrolyte concentrations and pH values were based on the solutions as shown in Table 1 .

1.1 FNP production and characterization

Dynamic light scattering (DLS) was performed with the zetasizer Nano ZSP (Malvern Panalytical, UK) for analysis of (F)NP size distribution. DLS samples were transferred to a quartz cuvette (ZEN 2112) at 173° (25 °C). Each sample was measured three times with the following material properties: EtOH (refractive index: 1.287, dynamic viscosity: 1.074 cP, dielectric cte: 25.3), HFE 7500 (refractive index: 1.290, dynamic viscosity: 1.240 cP, dielectric cte: 5.8), silica particles (refractive index: 1.540).

Transmission electron microscopy (TEM) was utilised as an additional tool for particle size analysis. For the TEM a 300 mesh copper grid was glow discharged for 15 s. Next 3.5 μL sample was transferred to the grid and incubated for 5 min. After blotting samples were imaged.

Energy dispersive X-ray (EDX) composition analysis allowed for analysing successful fluorination of the silica NPs. Here the samples were oven dried at 70 °C overnight and then transferred to carbon tape. Afterwards samples were imaged with the Quanta 200 ESEM FEG (FEI, USA) with EDX system.

1.2 Microwell plate partitioning

1.3 microfluidic chip production, 1.4 materials and imaging conditions for droplets.

A volume of 10 μL of droplets containing only buffer was combined with another 10 μL of dye-encapsulating droplets. Droplet populations were imaged directly after combination and after one day of incubation by transferring 4 μL of droplet solution inside a cell-counting chamber from which brightfield and epifluorescence micrographs were captured with a Nikon Ti2 microscope.

For the on-chip leakage fluorescence imaging three dyes were selected: AF488 (a well retained dye), sulford (a moderately leaky dye), Rhod6G (a strongly leaky dye). These were imaged on the Nikon Ti2. Chip design 1 (20 μM thick, see Fig. S1 † ) with 1 μL min −1 (aqueous phase) and 2 μL min −1 oil flow rate utilised for droplet generation.

1.5 Determination of fluorophore leakage and cross-talk

With this fitting, the medians and standard deviations of two different normal distributions and the mixing parameter were calculated. If the mixing parameter was close to zero or one, the bimodal normal distribution approaches a normal distribution with only one bell-shaped curve. That indicates that there is only one droplet population instead of two indicating transfer of dye from the droplets encapsulating dye to the empty droplets. However, this parameter is not a perfect indicator of cross-talk on its own: if the medians of two bell-shaped curves are close to each other (in the case of cross-talk with two overlapping populations), the mixing parameter can still be close to 0.5. For this reason a calculation of the intersection area was used, since it provides a more accurate indication of the occurrence of cross-talk ( e.g. , the intersection area corresponds to the percentage of droplets that cannot be differentiated into two distinct populations). In the absence of cross-talk, the intersection area was close to zero. Based on our analysis and our experimental observations, an intersection of 10% was chosen for identifying cross-talk. For a few experiments, though, the bimodal distribution fitting was unreliable ( e.g. , R 2 < 70), likely due to some variability in the experimental conditions at different time points. In these rare cases, the final classification was made based on the visual inspection of the data and the observations made during the experiments.

To obtain the data for the fitting images were analysed with a custom Phyton script. The location and size of the droplets were detected on the brightfield images through a Hough circle transform. The fluorescence intensities at these location were measured and averaged across each droplet. The background was detected by measuring the intensities at three different locations outside of the droplets (image background) and in pure buffer solution (buffer background). The droplet size distribution and fluorescence data were extracted from the Phyton pipeline. Then, this data was fed to a MATLAB R2023a (statistics and machine learning toolbox) to form the histograms and do further statistical analysis by fitting the data to probability distributions as described above.

To check for the leakage of fluorophores from the droplets, the percentage of droplets with an average intensity value below the background value (image background) was detected. If more then 10% of the droplets had an intensity below the background the sample was categorised as very leaky (category 4, see below). To calculate the cross-talk between the droplets, buffer background was subtracted from the fluorescence data of droplets and the resulting data were normalised between 0 and 100. This scaling provided a more suitable way for comparing different populations. The data was fitted to a bimodal normal distribution with the function “fitgmdist” with two components to represent two normal distributions. Then, a probability density between 0 and 100 was calculated based on the predicted median, standard deviation and mixing parameter, and drawn as a linear curve. In order to check for cross-talk, the intersection area of the two normal distributions was calculated by “cumtrapz” function. This function calculates the integral of the minimum values of the two probability densities by trapezoidal numerical integration. Then, histograms were generated with a hundred bins based on the raw normalised data, finally the intersection area was calculated. Based on the calculation above dye leakage and cross-talk was divided into four categories (see Fig. 4 for a visual overview).

• Category 1: more than 10% of the droplets show a lower average fluorescence intensity than the background value on day 0 indicating severe leakage of the dye into the oil phase.

• Category 2: less than 10% of the droplets show a lower average fluorescence intensity than the background value on day 0 and the bimodal distribution shows a minimal overlap of 10% on day 0 and 1. These droplets show limited leakage and severe cross-talk.

• Category 3: less than 10% of the droplets show a lower average fluorescence intensity than the background value on day 0 and the bimodal distribution shows a minimal overlap of 10% only on day 1. These droplets show limited leakage and mild cross-talk.

• Category 4: less than 10% of the droplets show a lower average fluorescence intensity than the background value on day 0 and the bimodal distribution shows an overlap of less than 10% on day 0 and 1. The droplets show limited leakage and cross-talk in this category.

1.6 Droplet storage and stability

1.7 cell adhesion and encapsulation, 2. results and discussion, 2.1 fnp characterisation.

2.2 Fluorophore retention and leakage

For NR no significant enhancement of dye leakage was measured compared to the HFE samples, this results from the strong partitioning of the dye into the oil phase. 42 If the majority of the dye already leaks in the pure HFE case, additional surfactant will not be able to induce any additional leakage.

A colour-coded overview of the leakage categories in which all the different conditions tested were classified is presented in Fig. 5 . A first important observation that can be made from these results is that cross-talk was minimally influenced by the buffer solutions in which the dyes were dissolved, in line with previous observations by Janiesch et al. 23 Another clear trend that can be inferred is that, for all these conditions, the more hydrophobic a dye was, the more leakage observed.

Perhaps the most striking result from our experiments is that, while it was previously reported that FNPs in fluorinated Pickering emulsions create a protective shell around droplets that effectively deters leakage, 25 our observations clearly contradict these claims: our FNP-stabilised droplets indeed exhibited significant degrees of leakage. This phenomenon was particularly pronounced for highly hydrophobic dyes (typically also neutrally charged), which in all cases surpassed the leakage levels observed with commercially-available RAN surfactant. There was a single, notable exception to this trend, namely for the Res dye, which leaked out from RAN- and Krytox-stabilised droplets but not from FNP-stabilised samples. This exceptional behaviour might be the result of the neutral charge of resorufin, in combination with its intermediate distribution coefficient and small size. As explained in the previous section, leakage from FNP-stabilised droplets can be explained by a combination of dye-particle interactions with a consequent change in the surface energy of these FNPs.

The leakage of DAPI from Krytox- and FNP-stabilised droplets possibly results from the electrostatic interaction between the Krytox and FNPs charged groups and the many amine groups present in the DAPI molecules, providing it with a positive charge. Additionally, 23 showed that low molecular dyes with planar structures can cross the droplet barrier more easily, which could potentially further explain the higher leakage observed for the DAPI, Fl and Res dyes. The UV-vis plate experiments and the observation of leakage in droplets do show some difference. This can be attributed due to the higher sensitivity of the plate assay compared to the droplet experiments.

Finally, it also needs to be noted that the low leakage of RAN-stabilised droplets could be partially explained by the use of BSA in our inner aqueous media, which was previously shown to reduce leakage by adsorbing to the o/w interface. 42 We chose to perform these experiments with BSA since BSA was essential for the rapid stabilisation of droplets with FNPs. To allow comparison the same buffer conditions were used for Krytox and RAN, although here BSA was not necessary for droplet stabilisation.

2.3 On chip leakage

2.4 Droplet stability

The assessment of droplet stability upon PCR-like thermocycling is presented in terms of the evolution of the coefficient of variance (CV) values of the droplets' diameter before and after this treatment, and is depicted in Fig. 7 . The most striking outcome of this process occurred in droplets stabilised with Krytox, which acquired irregular, non-circular shapes (and were, therefore, not analysed for their diameter and CV after the thermocycling process). Remarkably, these droplets were still quite stable: no fusion events were observed during the imaging process when droplets collided with each other. The effects of temperature on surfactant solutions were previously studied with molecular dynamics simulations, which concluded that as the temperature increases, thermophoresis of water molecules causes the hydrogen bonding between water and surfactant molecules to fracture and reconnect. 43,44 As mentioned previously, Krytox is composed of a PFPE chain functionalized with a carboxylic acid group. 1,20–22 To explain these results, we hypothesise that the carboxylic acid groups of Krytox molecules are fracturing their hydrogen bonds with water molecules and reforming them with carboxylic acids of neighbouring surfactant molecules, a phenomenon that has previously been observed in carboxylated PFPEs. 45 In these interfacial conditions, this reaction possibly leads to the formation of some form of hydrogen-bonded (fluoro)organic framework 46 at the droplet interface that behaves more like a gel and, therefore, can sustain deformations and support the non-circular shape of such droplets.

For the other two stabilisation systems exposed to a PCR-like thermocycling, the original droplet size was around 60 μM with a CV below 5%. After thermocycling, the CV of RAN-stabilised droplets became three times larger, indicating that a significant portion of the droplets stabilised with this reagent fused under these conditions. On the other hand, FNP-stabilised droplets remained relatively stable with only a limited increase in their CV as was shown in previous research. 47 The lower stability of RAN droplets can be attributed to the thermoresponsive nature of the PEG molecules in this triblock copolymer, which potentially destabilises droplets during temperature changes. 48–50 Additionally, the production of RAN does not result in a perfectly pure product, with traces of precursors, ionically-coupled surfactant and diblock copolymers still present. As previously suggested, this small amount of contaminating species might decrease the stability of RAN droplets. 17,50

With regards to the long-term stability upon storage in different conditions, as illustrated by the CV values of the diameters of droplets (see Fig. 8 ), these always increased over time for RAN-stabilised droplets but for Krytox-stabilised droplets a CV increase was only seen after storage in the incubator. Conversely, for FNP-stabilised droplets, CV values remained constant in all storage conditions.

From the results of the above treatment and storage conditions, we can conclude that FNP-stabilised droplets have a superior stability compared to surfactant-stabilised droplets. This can be explained by the higher energetic stability of the particles at the droplet interface. 41

2.5 Droplet production

For all cases the aqueous phase consisted of DPBS with 5 mg mL −1 BSA. As it was shown in previous papers, BSA promotes a more rapid stabilisation of droplets. 30 Alternatively, a long serpentine channel could have been added to the end of the design to specifically allow FNPs sufficient time to adsorb at the droplet interface. We did not make use of this strategy since for the creation of small droplets this results in a large increase in hydraulic resistance and the consequent increase in pressure to drive flow usually leads to the delamination of the PDMS chip from its sealing glass substrate and failure of the device.

Resulting graphs for the three different flow focusing designs are plotted in Fig. 9 . For the 20 μM flow focusing designs, Krytox- and RAN-stabilised droplet production behaved in a similar fashion. Similar flow rate ratios resulted in a similar droplet size. The FNP-stabilised droplets tended to be smaller for design 1 but in case of the other two designs droplet size was similar to that of the droplets stabilised by surfactants. In all of the above cases, the standard deviations are comparable, indicating a robust production of monodisperse and stable droplets in all systems.

For the 10 μM designs, production of RAN- and Krytox-stabilised droplets remained again relatively similar. For FNP stabiliser, though, droplets were slightly larger for the second and the third designs. In all cases the standard deviation are slightly higher compared to the 20 μM design, which could arise as a result of a higher sensitivity to pressure fluctuations of the pumps for devices with smaller channel sizes.

In the T-junction device, droplets could be produced for all three droplet stabilisers. The trend shows that the higher the flow rate ratio was, the smaller the droplets were, starting at around 35 μM for a flow rate ratio of 1 decreasing to around 20 μM for a flow rate ratio of 8, this can be expected upon inspection of the literature. 51 No significant differences between droplet size and monodispersity, as well as stability, could be observed for the three different stabilisers after these experiments.

With the two different step-emulsification designs droplets could be produced with 45 and 15 μM in diameter (see Fig. 10 ). Interestingly, when BSA was added to DPBS, no droplets could be produced with FNPs as stabilising agents. In these conditions, the droplets did not pinch-off as expected and only very large, polydisperse droplets were formed. When utilising MiliQ water without BSA, though, droplet pinch-off did effectively happen. This indicates that the compounds present in the aqueous solution that one attempts to encapsulate in droplets can critically influence droplet formation when FNPs are used as droplet stabilisers.

The diverse influence that BSA exerts on the production of droplets in these different droplet-generation systems can be explained due to the attraction between the positively-charged amino acids in BSA and the negative charges on these silica-based FNPs. For the flow focusing designs (if no long serpentine channel is present) BSA may play an essential role in promoting a rapid particle accumulation ( i.e. beyond their diffusion-limited rate) at the droplet interface. Additionally, hydrophobic–hydrophobic interaction between the fluorocarbon chains and the BSA molecules could also facilitate this more rapid stabilisation. 30 In the case of the step emulsification devices, since droplet formation is a relatively slower process as it is not dependant on high shear energy as in flow focusing devices, 52 there appears to be no need for the use of a stabilisation enhancer. In these circumstances, BSA seemed to actually negatively impact droplet formation, possibly by affecting the droplet necking and pinch-off processes due to its exertion of dynamic, localised changes in surface tension, which is a critical parameter regulating droplet formation in these systems. 52–54

Finally, in the device combining flow focusing with step emulsification, droplet production resulted in droplets of about 10 μM in size for all three different droplet stabilisers. Here, solutions both with and without BSA could be utilised without any detrimental effects in droplet monodispersity and stability.

In general droplet size and polydispersity was very similar for all the different stabilisers. For the use of FNPs for droplet stabilisation, though, it is important to highlight some important differences with respect to surfactants. During our experiments, we noticed that a good modification of the surface of the devices ( i.e. , with fluorinated silanes) is of paramount importance for all designs except for the step emulsification ones. In flow focusing chips that were silanised more than two days before droplet production, some of the droplets readily wetted parts of the channel where, presumable, the fluorosilane layer had become deteriorated. These wetting issues resulted in droplet fusion and splitting events that led to a final polydisperse droplet population. Secondly, FNPs aggregated when in direct contact with the silanising agent (probably due to the high reactivity of the silanising agent with silanol groups), resulting in channel obstruction. This could be avoided by immediately rinsing the devices with pure HFE 7500 upon finalisation of the silanisation treatment.

2.6 Cell adhesion

In another study by Lin et al. 31 it was observed that MCF-7 cells could adhere to the droplet border when encapsulated in droplets stabilised by FNPs. Given the observed adhesion in the plate-based experiment, both cell types were encapsulated in droplets of approximately 100 μM in diameter, which were stabilised by 2% Krytox, 2% RAN and FNPs (see Fig. 12 ). Once more, since there was an interaction between the cell medium and Krytox (ESI S4 † ), only results of RAN and FNP stabilised droplets are discussed. After an incubation of 24 h and 48 h, it was observed that when imaging droplets stabilised with RAN, all cells were in the same focal plane, while when imaging droplets stabilised with FNPs, cells were in different focal planes. Although these results indicate adhesion of the cells to the droplet edge, the typical morphology of spreading cells ( i.e. when cells flatten and extend their membranes after adhering to a surface), could not be observed at the droplet interface, which might indicate that the adhesion is non-specific or that the adhesion does not result in spreading due to the high curvature of the droplets. Given the different results obtained here and in the work of Lin et al. 31 where spreading was observed, future research is needed to investigate different parameters that might influence cellular adhesion to the FNP stabilised droplets, such as droplet size, FNP concentration, FNP fabrication, FNP characteristics, etc.

3. Conclusion

The process of droplet production exhibits strong similarity between FNPs, RAN, and Krytox for droplets generated by flow focusing. Initially we hypothesised that the slow diffusion of the particles to the droplet interface might prove problematic for droplet formation. However, this can be effectively addressed by the addition of BSA to the inner buffer solution, with the exception of step-emulsification devices, where BSA appears to impede the droplet pinch-off process. Encouragingly, FNPs demonstrate superior droplet stability under all examined storage conditions when compared to RAN and Krytox-stabilised droplets. Finally the adhesion of anchorage dependant cell is enhanced in a plate-based assay but could not be observed inside the droplets.

Looking ahead, further modifications of silica particles through silane chemistry hold the promise of providing droplets with more functionally tailored surfaces that can interact in specific manners with the droplet contents. Currently, the absence of commercially available fluorinated particles presents an opportunity for future advancements in the field, potentially leading to greater reproducibility and facilitating broader applications within microfluidics.

This study provides a comparison between FNPs, Krytox and RAN, comparing droplet leakage, cross-talk, stability, production and the adhesion of anchorage dependant cells. The findings shown in this manuscript can help in the choice of the most suitable surfactant for the right applications, driving further microfluidic innovations.

Conflicts of interest

Acknowledgements, notes and references.

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