TIRF Microscopy for Counting Molecules

TIRF Microscopy for Counting Molecules Robert Konstandelos How TIRF microscopy has enhanced the way single molecules are counted in the bacterial flagellar motor Abstract: The counting of individual molecules is important in order to establish how many molecules there are in a particular system. TIRF microscopy is one method to count molecules. The bacterial flagellar motor is a complex system in which motility protein B molecules can be counted using TIRF. Discussion is given for a key research topic based on counting of molecular subunits of this motor. Reviews of the background areas, limitations and confirmations of this research are conducted, and a discussion of the research and its contributions to technological and medical applications. 1. Introduction: The bacterial flagellar motor, TIRF microscopy and associated research Flagellar motors are machines used to drive many bacteria which have to swim in a solution like our bodies. This motor, usually studied in E. coli bacteria, is powered by a flux of H+ or Na+ ions across a cytoplasmic membrane driven by an electrochemical gradient (Sowa and Berry, 2008). The motor itself consists of two components, a rotor and a stator: the rotor spins relative to the cell and is attached by a helical filament known as a hook, whereas the stator is fixed to the cell wall (Francis et al 1994). A method commonly used to visualise the bacterial flagellar motor is Total Internal Reflection Fluorescence (TIRF) microscopy, which is one of the most frequently employed methods in bio-optical research (Leake 2013, P87). TIRF microscopy uses an evanescent field to illuminate the area covered by the specimen in question, which is adjacent to a glass-water interface. Using organic dyes has made it possible to view other properties of bacteria using TIRF (Sako et al 2000). This method is useful in counting the molecular subunits of the bacterial flagellar motor. TIRF microscopy has been used to view single molecules within live bacteria. For viewing the bacterial flagellar motor of E. coli, scientists tagged motility protein B (MotB) cells with Green Fluorescence Protein (GFP) in order to detect them via TIRF. This highlighted the areas within the bacteria where the motor was situated. To visualise the bacteria in a single confined position, the cell was tethered to the slide for viewing on the microscope. This is shown in Fig. 1, where the fixed position of the flagellum limits the bacteria’s movement to rotation. Fig. 1 Tethered cell showing its exposure to the evanescent field used for TIRF (Leake 2006, P355) 2. Background, difficulties and discoveries from the research 2.1 The history behind counting molecules Though the basis of this experiment began in the 60s, initially using the measurement of the activity of single molecules (Rotman 1961), optical detection and spectroscopic methods are now used instead. The counting of complex molecules can now also be achieved, but this area of research also needs TIRF microscopy. TIRF was enhanced in 1984 by Daniel Axelrod after the publishing of a paper on its experimental methods (Axelrod 1984), and those methods remain largely unchanged today. Furthermore, GFP molecules have only been recently understood. Without this research and development in GFP, visuals using TIRF would not be possible (Tsien 1998). 2.2 The difficulties encountered and overcome in counting molecules An estimate of around twenty-two molecules are thought to be present in the flagellar motor, with roughly eleven stator units. The main issue with determining this result explicitly is that there are many MotB molecules not associated with the motor. These molecules cause a problem as they are free to diffuse within the motors of the cell membrane. The fluorescence intensity was estimated from the areas where it was clear that no such molecules would interfere with results. Additionally, an intense laser beam focus for TIRF was required to photobleach GFP molecules. Only an extremely small region of the bacteria was viewed to improve the ability to track a small number of molecules – a significant amount of noise remained in the system, however, meaning that it is not yet possible to count exactly how many molecules are in each motor. Fig. 2 showing bright field (top) and their corresponding TIRF images (bottom) (Leake 2006, P355). The bright areas represent the flagellar motor. Using TIRF, bright spots indicate the centre of the cell rotation of the image shown in Fig. 2. There was a high density of spots centred on the flagellar motor, due to the high density of GFP-MotB molecules around the motor. Short times (between 0-10 seconds) are used because TIRF illumination over the bright spots decreases over longer periods of time, which makes it difficult to detect regions of the flagellar motor. Care was taken to not cause damage to the GFP due to the excitation light on the surrounding water: this means that smaller time steps were required such that the GFP molecules emitted a constant amount of photons. 2.3 The effects FRAP and FLIP With the noise effects reduced, there was the opportunity for the GFP-MotB molecules to spread into the area which had been bleached beforehand. Focusing the laser beam onto the motor itself resulted in the effects of fluorescence recovery after photobleaching (FRAP) and fluorescence lost in photobleaching (FLIP). Observations of the molecular turnover in the cell found that over a period of five minutes, the intensity of the bright spots around the motor would decrease to nearly zero but then recover to half the initial intensity. This implied that binding and unbinding at the motor and bleaching occurred in the evanescent field (Leake et al 2006, P357), which means that the stator units in the motor only spend half a minute in each flagellar motor. This is demonstrated in Fig. 3, which illustrates the time elapsed after laser focused bleaching and how the intensity decreases but eventually recovers. Fig. 3. Shows the effects of FLIP and FRAP over the period of 5 minutes (Leake et al 2006, P357) 3. Benefits of the research and potential uses for the future An improved type of MotB was used in the research, which enhanced the way in which the molecules in the motor were counted. FLIP and FRAP indicate an alternative means for visualising the motor in motion, confirming that the stator units are dynamic instead of static (Sowa and Berry, 2008, P117). This is one of the first measurements of turnover in a molecular machine, establishing other possible characteristics which could be exploited to gain further understanding of the motor (Leake et al 2006, P357). Scientists are keen to understand more about how such motors work, so that developments in the delivery of medicine or for environmental purposes can be made. It may be possible to replicate the motor (Fukuda et al 2012). Delivery of medicine is one of the key goals: modelling the bacterial flagellar motor such that it could be used for targeted drug delivery would be revolutionary (Leake 2013, P259). Furthermore, through the development of nano-bots, this could be used to visualise diseased tissue or uncover parts of the human body. Summary This area of biophysics is relatively new: from the discoveries in the early 60s through to the 80s, there has not been a clear link between the two subjects. From the late 90s there was an opportunity to visualise biological material using physical optical devices. Over the past two decades, it has now reached to the point where it is possible to count single molecules to a close estimate. The use of GFP molecules combined with TIRF can enhance the visualisation of molecules in bacteria, and there are methods which can significantly improve the estimation of the number of molecules in the motor. This is still a difficult process due to the interference of other, unrelated molecules. FLIP and FRAP methods have proved that the stator is a dynamic rather than a static component of the motor. There are specific parts of this research which may be useful for future technological applications, for example: the delivery of medicine or the bio-sensing of diseased tissue. References Axelrod, D; Ann. Rev. Biophys. Bioeng.; 13; 1984; 247-68 Francis, N, R.; Sosinsky, G,E; Thomas, D; Derosier, D. J; Journal of Molecular Biology 235, 1994; 1261–1270. Fukuda, T; Kojima, M; Zhang, Z; Nakajima, M; Biomed Micro-device; 2012; 1027-32 Leake, M; Single Molecular Cellular Biophysics; 2013 Leake, M C; Chandler, J H; Wadhams, G H; Bai, F; Berry, R M; Armitage, J P; Nature 443; 2006; 355-358 Rotman, B; Biochemistry 47; 1961; 1981-91 Sako, Y; Minoghchi, S; Yanagida, T; Nature Cell Biol. 2; 2000; 1929-1932 Sowa, Y; Berry, R, M; Quarterly Reviews of Biophysics 41, 2008, 103-132 Tsien, R.Y; Annu. Rev. Biochem 67; 1998; 509–44

Lady Liberty

It was 1885 in New York City. A little boy named Frederick Bloomsmen was hurrying down the streets to get to his master's shop. â€Å"Mayhap if I hurry, I'll get to see Lady Liberty,† Frederick thought. But as he ran along, he heard the town clock strike 6:00. â€Å"Oh no! I've got to make haste!† Frederick said desperately. Frederick had been delivering a saddle to the cart builder and had spent too long talking to the cart builder's son. Slam! The door to his master's store opened and shut as Frederick ran in. â€Å"Where have you been† Mr. Ezra (for that was Frederick's master's name) roared. â€Å"Delivering the saddle, sir, just as you asked me to,† Frederick answered timidly. â€Å"Well, you're late!† Mr. Ezra boomed. It was finally Saturday, Frederick's day off. He ran down the streets, as if racing the wind to see who could get to Lady Liberty skeleton first. Suddenly, he stopped still, for in a shop window sat the loveliest carved figure of Lady Liberty herself! His eyebrows slid up. He ran into the shop. â€Å"Excuse me, sir, how much money is that figure of Lady Liberty?† Frederick asked. â€Å"Well laddie, it's worth 15 cents but I'm lowering the price to 10 cents.† â€Å"Ten cents,† Frederick's head rang out as he ran home, â€Å"Where can I get such a fortune?† The next Monday, Frederick had almost forgotten the figurine. â€Å"Mr. Ezra, sir, may I please have my wages?† â€Å"All right, lad, but only because you haven't been sassin' me.† He opened the cash register and dropped a nickle into Frederick's outstretched palm. Halfway there! Frederick's head seemed to yell out with joy. Now he thought about how pretty that figure would look on Mama's mantle. † I'll give it to her for her next birthday,† he said aloud. â€Å"What was that, boy?† Mr. Ezra said. â€Å"Nothing,† Frederick said quickly, blushing bright red. The next morning after his chores were done, Frederick ran to the shop where the figure of Lady Liberty sat, to see if she was still there. Phew! There it was, looking as gorgeous as ever. The next week, Frederick got his wages. Another nickle! He finally had 10 cents. But as he walked down the roads he thought, â€Å"What if they need just 10 more cents to finish the Statue of Liberty?† And so silently he headed for the donation box. He listened to the cling as the coins dropped into the box. As the months flew by, Frederick's birthday was drawing nearer. One day, as he was running down the streets to see the Statue of Liberty, a shopkeeper (actually the very one that carried the figurine of Lady Liberty) called out from his shop. â€Å"Frederick! Wait! I've got a birthday present for you!† So Frederick stopped and went inside the shop. The shopkeeper handed him a small box. â€Å"Open it,† he urged. As Frederick did so, his eyebrows slid up as his mouth went down. For there, beautifully arranged in a pile of white tissue paper, sat the figurine of Lady Liberty! Frederick felt dizzy with happiness as he left the shop. Mama's birthday came just as quickly as Frederick's had. Frederick re-wrapped Mama's birthday present. When the time came for her to open it, her mouth dropped in surprise. â€Å"Why Frederick, where did you get this?† she asked in bewilderment, â€Å"It's beautiful! I absolutely love it! I'll put it on the mantle.† Frederick had to tell her the entire story, which was similar to the one I have just finished telling you now.

The Escape – Creative Writing

I couldn't breathe. I could feel a large weight on top of me but when I looked there was nothing there. It was getting heavier and heavier. I tried to scream but every time that I tried I felt a shooting pain in my chest. All that I could do was wait and pray. I thought that it was unlikely that I would be found and I kept going over what had happened and couldn't understand how a life so perfect could go so wrong as easily as it had. It wasn't right. I looked down and released a ear splitting scream†¦ The previous month I had just been a normal teenager, going to school and going shopping at the weekend. Then it all changed. I came home from school to find that a large wagon was parked in front of our house and a man was carrying out our belongings. I saw my TV, stereo and Game cube being carried out of the front door by two large men. At first I thought that we were being robbed but then I saw my mum and dad appear at the front door. My mum was in floods of tears and my dad was stood with another man and signing papers. It never occurred to me what was happening. I ran across the garden and threw my arms around my mum. Although I didn't know what was going on I started to cry. I thought that I should try to be brave as my mum was so upset but it was the only time that I had ever seen her cry. I knew that something must be seriously wrong. I released her gently from my arms and gently asked her what was happening and who the men were. She said that my dad would explain everything when he had signed the papers. It was about five minutes, but seemed like a lifetime, before my dad could come and explain what we were going to do next. As my dad started to talk I became speechless. How could this happen? We hadn't done anything wrong yet we were the ones that were being punished. I couldn't take in what was being explained to me. It was so unfair that because my dad had done the right thing we were going to have to move out of our house and leave our life that my parents had built up for me. What had happened was that my dad had been involved in a robbery at work, the men had been captured but my dad had been asked to give evidence in court in order to get them put in prison. My dad had done this willingly but the thing that he had not realised was that the men who he had helped to send to prison were part of a large gang who were very violent. The other gang members were now out for revenge, with my dad. He had been receiving silent phone calls; evil text messages and he had even been sent death threats through the post. But what had happened the previous day was too much for anyone to cope with. Someone had burst into his office to try to attack him. It could have all gone horribly wrong for my dad if it hadn't been for a meeting being called at the last minute and him leaving his office to join it. I felt awful, it had been the previous day that I had been moaning because I wasn't allowed to go out with my friends. I could now understand that they had done it for my safety and the same men that had wanted to kill my dad could have killed me. I hadn't thought at any point in my life that I would have to be careful where I go and who I go with for the risk of my life. It all seemed so unreal. I had watched TV programmes and horror films about things like this happening but I never thought about what it must be like for people to be put in this situation. I felt disappointed in myself because I remembered when I was at a sleepover and I had joked that this sort of thing would never happen and that if it did the person who was on the run must have done something wrong. Even when I had this explained to me I didn't expect to hear what they told me next. We were being put in the ‘Witness Protection Programme'. I didn't fully understand what this meant. I thought that all it meant was that we had to move away from our home in order to get away from the people that were chasing us. As my parents continued to talk I realised that it meant that we were going to get a new identity and that we were not allowed to tell anyone what we were going to do. That was the hardest thing that I had to cope with. I said bye to my friends when I left school that day but it never entered my head that it was the last thing that I would be saying to them. I had always been popular at school and I couldn't understand why something like this would happen to me. Why couldn't it happen to Laura and her family? No one liked her and she didn't have any friends so there wouldn't be anyone who would miss her. I knew that it was a horrible thing to think but I couldn't help myself. What would my friends think if I didn't say anything before I left? I pleaded with my mum to let me phone them, I even said that one would be enough and that they could pass on the message to the others. This wasn't going to happen no matter how much I pleaded. They explained that it was for my safety. If I had told my best friend and she said something to my other friends, someone could overhear what she was saying and this problem could start over again. I did understand what they were saying but it seemed so undeserved. After we had the conversation I decided that I would have one last look inside the house. I was really surprised. The house still had the wallpaper and the carpets but the rest of the house was so bare. I climbed the stairs to where my bedroom was. It was weird knowing that it was the last time that I would be in my room again. It still looked like my room with the carpets and the poster but everything else was bare. I felt a tear fall down my face. I was trying to be strong but there was nothing that I could do, I just couldn't stop the tears from continually falling. I slowly walked down the stairs and quietly sat in the car. The large wagon started to drive off down the street and I knew then that it was the end of what I knew as my life. As my parents got into the car a policewoman came to sit in the back. It all felt so strange and I couldn't take the chance of looking behind me because I didn't know how I would react. When we turned the corner onto the main road my phone started to bleep to tell me that I had a message so I reached into my bag to get it out. It was no sooner that I had the mobile in my hand that the policewoman snatched it off me. She was sympathetic with me but she said that it would be best if I didn't read what it said because it may upset me to know that I was unable to respond to whatever was said. As we pulled onto the motorway it struck me that I did not know where we were going. I had been so wrapped up in my thoughts that we could have been going abroad and I wouldn't have known. I asked my dad but he said that he would tell me when we had arrived. It seemed to me as if I was being kept in the dark about everything that was going on. Did my parents not realise how much this was affecting me? My whole world was being turned upside down and they wouldn't tell me anything about what was happening. It wasn't as though I wouldn't understand; I was a teenager so why wouldn't they trust me. I didn't have any way that I could contact anyone so I couldn't put us in any danger from these people who were chasing us. We slowed down and I noticed a sign that was in welsh and as soon as I saw it I knew where my parents were taking me. We had been on holiday here the previous year and my parents had expressed how much they loved it her. I was really angry now. They were messing up my whole life and they were making me stay in this place. They knew how much I hated it because of how much there was to do. There were fields all around, no shops and no one who was near my age. I couldn't see how I was going to cope in a place like this. My life would revolve around school because there was nothing else to do. If we had to move why couldn't we move to a large town like London or Birmingham? This was going to be my worst nightmare, nothing to do but walk up hills and go to school. I had never realised how great my life was before. It makes me understand how true the saying ‘you never appreciate what you have until its gone' is. The amount of times my parents had said this I hadn't really understood it, I just assumed that if you lost something you would be able to get used to your life without it. I would never get used to this. We drove into the village and there was nobody about even though it was a Friday night. There were always people about when you wanted to go out. I hadn't even seen one person here. We pulled up in front of this tiny cottage. They couldn't seriously think that this was big enough for three people but when I looked round it seemed to be one of the biggest cottages in the area. I hesitantly walked through the front door to find that it seemed bigger on the inside than it looked from the outside. I knew that there was nothing that I could do now to change their mind so I returned outside and took one of the suitcases out of the boot of the car. As I turned around I noticed that there was a boy and a girl walking towards me. They seemed friendly and they looked about my age. I said hello and introduced myself. It seemed weird when I had to introduce myself by a different name. They didn't seem to notice the uneasy tone in my voice because they both introduced themselves to me. They said that they both lived down the road and that they went to the school that I would be going to. I told them that I had to take my things inside but I would hopefully see them later. They said bye and walked off. Things looked much better than I had originally thought that it could be. I slowly got used to living in a quiet village and by the end of the second week I had lots of new friends and I had told then why I had moved here-The made up version obviously. Everyone seemed nice but because it was so secluded there was only six people in my class. It was strange to begin with and I would go home upset because of how few people there are but after I thought about it I realised that it was much better for my education. It was as though I was having one to one tuition, which meant that I was learning much more than I did at my previous school. I still missed all my friends and in a strange way some of the teachers. After we had been at our new home for a month things started to go wrong for us. Someone had found out my mums new mobile number and she was getting prank phone calls and silent phone calls. We notified the police but they assumed that it was my fault and that I had been in contact with someone from my old school. Nobody believed me when I said that I hadn't done anything that I wasn't supposed to do and I was kept off school to be questioned and to ‘learn a lesson'. I hadn't realised that it was so serious if I had spoke to someone from my old school. I hadn't and that was what upset me, no one believed me. I stormed out of the house my eyes full of tears and a lump in my throat†¦ †¦ My scream rang through the dark lane and my chest hurt even more. I couldn't believe what I was seeing. My white T-shirt had turned red with the blood that was pouring out of my chest. I tried to think what had happened to me. I didn't know how long I had been here for. I didn't know how much longer I would be here. I heard a car engine in the distance. I tried to move but I found that no matter how hard I tried my legs would not move. I couldn't believe that I was going to be rescued but my luck changed. The car turned down a small lane. If only I had waited with my parents I wouldn't have been in this situation. Why didn't I wait at home and try to explain further? I didn't know what to do. I had no way of phoning home and I didn't know where I was. In the very far distance I saw a small yellow dot that was becoming larger with every second. I realised that it was someone on a bike. I wanted to scream for help but each time I tried there was only a small noise that escaped. I could only wait and hope that the bike wouldn't turn. It was coming straight towards me. My eyes closed and everything went dark. As I opened my eyes I could hear lots of noise and see lots of people hurrying around me. I was in a total daze. I could hear lots of people saying my name and asking if I could hear them. I could, I just couldn't respond. It was as though I was watching these people with someone else and there was nothing that I could do. I felt someone grab hold of my hand so I squeezed the hand as much as I could. When I had done this I heard the reassuring voice of my mum. It was saying â€Å"Everything will be okay. I wont let anything happen to you ever again. There is no-one that can harm you now. † I believed every word that she was saying to me. I knew that I would be okay from now on.

Myths About Spanish and the People Who Speak It

When many people, especially those in the United States, think of Spanish, they tend to think of mariachis, their favorite Mexican actor and Mexican immigrants. But the Spanish language and its people are far more diverse than the stereotypes suggest. Here we debunk 10 myths about Spanish and the people who speak it: More People Grow up Speaking English Than Speaking Spanish Because English has become a worldwide lingua franca for science, tourism, and business, its easy to forget that English is far surpassed by two other languages in terms of numbers of native speakers. Easily ranking No. 1 is Mandarin Chinese with 897 million native speakers, according to the Ethnologue database. Spanish comes in a distant second with 427 million, but thats well ahead of English with 339 million. One reason English seems more prominent is that its regularly spoken in 106 countries, compared with just 31 countries for Spanish. And English does rank ahead of Spanish when non-native speakers are counted as it is the worlds most common second language. Spanish Is the Language of Latin America The term Latin America traditionally is applied to any of the countries of the Americas where a Romance language is the dominant language. So the most populous country of Latin America — Brazil with more than 200 million residents — has Portuguese, not Spanish, as its official language. Even French-and Creole-speaking Haiti is considered part of Latin American, as is French Guiana. But countries such as Belize (formerly British Honduras, where English is the national language) and Suriname (Dutch) are not. Neither is French-speaking Canada. Even in countries where Spanish is the official language, other languages are common. Indigenous languages such as Quechua and Guarani are widely used in large swaths of South America, and the latter is co-official in Paraguay, where it is spoken even by many who arent of Amerindian heritage. Nearly two dozen languages are spoken in Guatemala, and in Mexico, about 6 percent of people dont speak Spanish as their first language. Native Spanish Speakers Talk Like Speedy Gonzales The Spanish of the cartoon character Speedy Gonzales is an exaggeration of Mexican Spanish, of course, but the truth is that a minority of Spanish speakers have a Mexican accent. The Spanish of Spain and Argentina, to take two examples, doesnt sound like Mexican Spanish—just as U.S. English speakers dont sound like their counterparts in Great Britain or South Africa. Although much of the regional variations in English tend to be with the vowels, in Spanish the variation is in the consonants: In the Caribbean, for example, speakers may tend to distinguish little between the r and the l. In Spain, most people pronounce the soft c with the tongue against the upper teeth rather than the front of the palate. There are substantial variations as well in the rhythm of speech from region to region. The Spanish R Is Difficult to Pronounce Yes, it does take practice to get the trilled r to come naturally, but millions learn it every year. But not all Rs are trilled: You can pronounce the common word pero close to correctly just by sounding out peddo, and mero sounds very much like meadow. In any case, its undoubtedly easier for native English speakers to pronounce the Spanish r than for native Spanish speakers to pronounce the English r. People Who Speak Spanish Are Spanish As a nationality, Spanish refers to people from Spain and only Spain. People who are from Mexico are, well, Mexican; people from Guatemala are Guatemalan; and so on. I wont try to settle here any controversy over how to use terms such as Hispanic and Latino. Suffice it to say that traditionally in Spanish, hispano is used to refer to someone from the Iberian Peninsula, while latino can refer to anyone from a country that speaks a Latin-derived language — and sometimes specifically to people from the Lazio region of Italy. Native Spanish Speakers Have Brown Skin, Brown Eyes and Black Hair In their totality, Spain and the Spanish-speaking countries of Latin America are every bit the melting pot of races and ethnicities that the United States is. The societies of Spanish-speaking Latin America descend not only from Spaniards and indigenous Amerindians but also from peoples of Africa, Asia, and non-Spanish Europe. Most of the Spanish-speaking countries of the Americas have a population that is majority mestizo (mixed race). Four countries (Argentina, Chile, Cuba, and Paraguay) are majority white. In Central America, many blacks, usually descendants of slaves, live along the Atlantic coast. Cuba, Venezuela, Colombia, and Nicaragua each have a black population of around 10 percent. Peru especially has a large population of Asian ancestry. About 1 million are of Chinese heritage, and thus the abundance of chifas, as Chinese restaurants are known there. One of the former presidents of Peru, Alberto Fujimori, is of Japanese heritage. You Can Form Spanish Nouns Just by Adding O to the English Word This works sometimes: A car in much of Latin America is a carro, a telephone is a telà ©fono, an insect is an insecto, and a secret is a secreto. But try this often and most of the time youll just end up with gibberish. Besides, an a works sometimes too: A jar is a jarra, music is mà ºsica, a family is a familia, and a pirate is a pirata. And, please, dont say No problemo for No problem. Its No hay problema. People Who Speak Spanish Eat Tacos (or Maybe Paella) Yes, tacos are common in Mexico, although it should tell you something that Taco Bell markets itself as U.S.-style fast food in Mexico, not as a Mexican-style chain. And paella is indeed eaten in Spain, although even there its considered something of a regional dish. But these foods arent found everywhere that Spanish is spoken. The fact is every region of the Spanish-speaking world has its own culinary favorites, and not all have crossed international boundaries. Not even the names are the same: Ask for a tortilla in Mexico or Central America, and youre likely to get a sort of pancake or bread made from cornmeal, while in Spain youre likelier to receive an egg omelet, possibly prepared with potatoes and onions. Go to Costa Rica and ask for a casado, and youll get a simple if tasty four-course meal. Ask for the same in Chile, and theyll just wonder why youd want a married man. Spanish Will Take Over English in the United States While the number of native Spanish speakers in the United States is projected to increase to around 40 million by 2020 — up from 10 million in 1980 — studies consistently show that their children will grow up bilingual and that their grandchildren are likely to speak English exclusively. In other words, the level of Spanish speaking is tied more closely to current immigration rates than it is to use of Spanish by those born in the U.S. The descendants of Spanish speakers switch to English as they assimilate just as did those who came to America speaking German, Italian and Chinese. Spanish Is an Official Language in Just Spain and Latin America Of the African territories that were once part of the Spanish Empire, one independent country still uses Spanish. Thats Equatorial Guinea, which gained independence in 1968. One of the smallest countries in Africa, it has around 750,000 residents. About two-thirds of them speak Spanish, while French, Portuguese and indigenous languages also are used.