What is a nitro beer?
Not so much a beer variety, more a special technique to create a very distinctive drink. A nitro beer differs from a usual brew in that carbon dioxide (which occurs naturally in the brewing process, and is also often added at the bottling stage) is partly replaced with nitrogen gas.
You’ll instantly recognise the results: a cascading effect as bubbles in the liquid appear to be falling rather than rising, a creamy white head and a velvety taste. You can increasingly find both ale and lager nitro beers in many pubs and bars – but the most famous is a stout, and from the brewery that pioneered the technique, Guinness Draught.
Know Your Beer - Nitro
Know Your Beer - Nitro
The history (and science) of nitro beer
Watch a glass of Guinness Draught being poured and it looks like alchemy at work – bubbles seemingly defying gravity, a long mesmeric settle and surge of black and white. What you're witnessing, though, is not magic but pure science, the fruits of many years of brewing innovation.
In the early 1950s, British mathematician and brewer Michael Ash, and his team at St James’s Gate, worked on a pouring system to produce, and easily reproduce, a perfect pint of Guinness on tap. Ash’s solution was to harness the properties of an element born in the stars, in the very heart of a supernova: nitrogen.
At 78% it is the most abundant gas in Earth's atmosphere, and is largely insoluble in water. This property leads to two effects when infused with beer: firstly, its bubbles are smaller and more stable than carbon dioxide, which are interpreted by receptors in our mouth as having a smooth and velvety feel.
Secondly, the stable bubbles, coupled with the type of protein in the barely wheat used by Guinness, make for a longer-lasting beer head. Carbon dioxide, on the other hand, is a much more volatile gas in water, meaning its bubbles pop quicker, and your beer head vanishes quickly as a result.
When Ash and his team revealed the first nitro stout in 1959, to coincide with the 200th anniversary of the signing of the lease at St James’s Gate, they had not only created the perfect draught pint of Guinness, they’d also essentially crafted a new type of beer.
Nitro: frequently asked questions
Why do the bubbles in a Guinness Draught travel downwards?
In nitro beers, such as Guinness Draught, drinkers are often left perplexed by how its bubbles appear to defy the laws of physics by travelling down not up. What they are witnessing though is a actually a flow of tiny stable bubbles of nitrogen around the glass.
When the beer is first poured, some bubbles will stick to the side of the glassware, slowing rising ones around it, while bubbles in the centre of the drink can flow faster, unimpeded. This has the effect of creating a circulating current with a central column of bubbles going up, pulling others, and the ones visible to the drinker, downwards.
Eventually this leads to the forming of the beer head and, as bubbles nitrogen are more stable, one that’s creamy and long-lasting, be that on the top of your drink or on your top lip.


Adults: What is Vision Therapy (Behavioral Vision Care)?
Having a daily routine of eye exercises is becoming more relevant these days with the dramatic onset of computer use, and overall close-up work required daily for students and professionals. Eye exercises help maintain healthy vision, reduce or eliminate the effects of eyestrain, and ultimately help to preserve eyesight (see eye exercise programs further down the page).
In the case of learning disabilities, vision therapy is specifically directed toward resolving visual problems that interfere with reading, learning and educational instruction. Optometrists do not claim that vision therapy is a direct treatment for learning disabilities.
Vision therapy, a type of physical therapy for the eyes and brain, is a highly effective non-surgical treatment for many common visual problems such as lazy eye, crossed eyes, double vision, convergence insufficiency and some reading and learning disabilities. Many patients who have been told, "it's too late," or "you'll have to learn to live with it" have benefited from vision therapy.
Who needs vision therapy?
The simple answer is: "Everybody!" All can benefit from more efficient vision. However, certain individuals will especially benefit from this type of care:
- those who use their near vision a great deal at work or school such as those that spend many hours everyday day on a computer, reading small print, examining small objects, etc.
- students who have been diagnosed as 'learning disabled'
- individuals who have suffered from a brain injury or other cerebral trauma
- people who are myopic (nearsighted), hyperoptic (farsighted), and/or have presbyopia (similar to farsightedness that begins usually after the age of 40.
- anyone interested in improving their vision naturally
History of Vision Therapy
Vision therapy is not new. Physicians in the mid-1800s originally introduced many of the techniques that are used today. Modern Optometric Vision Therapy was introduced in the United States in 1928. Throughout the years, vision therapy has been called various names such as visual training, orthoptics, or eye exercises.
The Bates Method for Improving Eyesight is a method discovered at the beginning of this century by Dr. W. H. Bates, M.D. (1865-1931), a prominent American ophthalmologist, developed the Bates Method for Improving Eyesight which focuses on improving sight and restoring natural habits of seeing, which have been lost through strain, tension and the resulting misuse of the eyes. Dr. Bates had learned to help people see without eyeglasses as early as 1891.
- What is computer vision syndrome (CVS)?
- Is eyestrain from binocular vision problems related to learning disability?
- What is vision therapy?
- Who needs vision therapy?
- Why did this happen to me?
- Will I have to do vision therapy forever?
- Can I wear glasses to avoid vision therapy?
- How much time do I have to spend doing vision therapy?
- What is accommodation?
- What is convergence?
- What is fusion?
- What is stereopsis?
- Research materials: Summary of studies on vision therapy
What is computer vision syndrome (CVS)?
It's a condition, recognized by the American Optometric Association, that affects users of computer monitors and causes eyestrain symptoms, such as blurred vision, dry or burning eyes, delayed focusing, and headaches. It can arise from failure of the eye muscles to work properly.
Is eyestrain from binocular vision problems related to learning disability?
What is vision therapy?
Also called orthoptics or vision training, it's a treatment process for improving visual function including eye movement skills, accommodation and binocular vision. It involves a series of eye exercises of progressive difficulty, performed several times a week, until symptoms are resolved.
Who needs vision therapy?
People who have eye muscle problems that cause eyestrain symptoms - such as blurred vision, headaches, fatigue or concentration difficulty - including computer vision syndrome and vision-related learning problems.
Why did this happen to me?
Our eyes were not made to fixate on two-dimensional written pages or computer screens for hours at a time. Our eyes are more geared for the distance vision primarily used by people in agrarian societies.
Will I have to do vision therapy forever?
Can I wear glasses to avoid vision therapy?
How much time do I have to spend doing vision therapy?
That depends on how quickly your eye muscles learn the needed skills. Most people need to practice computerized HTS Home Vision Therapy Program for 20 minutes a day, five days a week, for about two to three months.
What is accommodation?
What is convergence?
What is fusion?
What is stereopsis?
Vision Therapy Products for Use at Home
For Myopia (near-sightedness), Astigmatism and eye imbalances.
Program for Better Vision The best-selling step-by-step system to improve nearsightedness, astigmatism and eye imbalances in minutes a day. Easy to use eye exercises for better vision. Kit includes:
- 3 audiotapes
- 80-page Program Guide
- 2 Vision Charts
- Fusion String
- Toll-Free Support
For Presbyopia (middle-age vision) and Hyperopia (farsightedness)
Read Without Glasses Method developed by Dr. Ray Gottlieb, is an excellent program targeted to reverse presbyopia, or middle age sight. In as little as six minutes a day you can:
- Keep yourself from needing reading glasses
- See better up close without reading glasses
- See better in dim light
- Reduce the strength of your glasses
- Avoid, eliminate or reduce the need for bifocals.
For General Vision Improvement
The Exercise Your Eyes Total Vision Workout System is an all-around electronic vision trainer that is clinically proven to improve overall visual performance in less than 10 minutes a day. This program is designed for both children and adults.
See a complete list of Vision Therapy Products.
DISCLAIMER: These vision therapy programs should not replace competent optometric or medical care, but should be used as an adjunct to your eye doctors evaluation.


Take the battle to SCN this summer
Soybean cyst nematode has been the buzz since it was first found in our region in 2003. Although the spread has not been rapid and infestation has not reached economically challenging levels, every soybean grower needs to be alert and know how to identify it.
Last fall, North Dakota State University Extension Plant Pathologist Sam Markell led an effort to identify counties where SCN is present. Five new counties in North Dakota, from Emmons in the west to Pembina in the north, are now listed. All Red River Valley counties, with the exception of Walsh (probably just not confirmed), were identified with SCN in soils.
I liken these results to saying all counties have yellow houses — perhaps not widespread, but present.
Because we live and farm in the North, we have an advantage that frequently these pathogens and pests spread to our region after a solution has been discovered to fight them. SCN-resistant soybean varieties have been developed — and they are the best defense against SCN. However, don’t press your luck and plant SCN-resistant varieties on non-cyst infected ground, as resistance issues could result.
Crop rotation away from crops that are good hosts (dry beans, soybeans and peas) helps to reduce the SCN numbers but will never rid the soil of the pest. SCN is a microscopic roundworm that penetrates the roots of the soybean. The females remain on the roots and swell into the lemon-shaped cysts that are visible. These cysts contain 250 to 500 eggs each, that either burst, allowing the eggs to infect other roots, or remain in the soil, dormant for many years. SCN harms soybean yields by robbing nutrients and water from the plant. Yield is affected before visual symptoms are noticed. That is why it is so important for growers in regions where SCN is spreading to scout this summer, so the problem can be identified before it robs yield.
Scouting fields is the first step in SCN management. Yield can be reduced before visual symptoms ever appear on the plants. Begin scouting about six to seven weeks after planting and examine high-risk areas, including previously flooded areas, low spots, low-yielding areas and field entrances. Dig up the plants and roots rather than pulling them, as cysts can be lost if plants are pulled from the ground. If possible, rinse the roots prior to inspection. Examine them for yellowish-colored cysts that are less than half the size of the nodules on the roots.
SCN eventually causes visual symptoms that are very similar to iron deficiency chlorosis, soil compaction, water-logged soils and other diseases. Plants will eventually become stunted and yellowed.
With the recent expansion of the SCN infestation in North Dakota and northern Minnesota, I want to get the word out to scout this summer. You’ll be better able to deal with the problem when you know what you have.
Spelhaug is an agronomist at Peterson Farms Seed, Harwood, N.D. Contact him at 701-282-7476.
This article published in the April, 2012 edition of DAKOTA FARMER.


Preparation For Pentecost From The Catechism of the Catholic Church [CCC]
Christ Is Always Present. He Disappears From Sight, But Reappears in the Sacraments
(The Mind of Vatican II as Taught in the Catechism of the Catholic Church)
In this age of the Church Christ now lives and acts in and with his Church, in a new way appropriate to this new age. He acts through the sacraments in what the common Tradition of the East and the West calls "the sacramental economy"; this is the communication (or "dispensation") of the fruits of Christ's Paschal mystery in the celebration of the Church's "sacramental" liturgy. (CCC 1076)
The dual dimension of the Christian liturgy as a response of faith and love to the spiritual blessings the Father bestows on us is thus evident. On the one hand, the Church, united with her Lord and "in the Holy Spirit," [Lk 10:21] blesses the Father "for his inexpressible gift [2 Cor 9:15] in her adoration, praise, and thanksgiving.
On the other hand, until the consummation of God's plan, the Church never ceases to present to the Father the offering of his own gifts and to beg him to send the Holy Spirit upon that offering, upon herself, upon the faithful, and upon the whole world, so that through communion in the death and resurrection of Christ the Priest, and by the power of the Spirit, these divine blessings will bring forth the fruits of life "to the praise of his glorious grace." [Eph 1:6] (CCC 1083)
In the liturgy of the Church, it is principally his own Paschal mystery that Christ signifies and makes present. During his earthly life Jesus announced his Paschal mystery by his teaching and anticipated it by his actions. When his Hour comes, he lives out the unique event of history which does not pass away: Jesus dies, is buried, rises from the dead, and is seated at the right hand of the Father "once for all." [8 Rom 6:10; Heb 7:27; 9:12; cf. Jn 13:1; 17:1]
His Paschal mystery is a real event that occurred in our history, but it is unique: all other historical events happen once, and then they pass away, swallowed up in the past. The Paschal mystery of Christ, by contrast, cannot remain only in the past, because by his death he destroyed death, and all that Christ is ‑ all that he did and suffered for all men ‑ participates in the divine eternity, and so transcends all times while being made present in them all. The event of the Cross and Resurrection abides and draws everything toward life. (CCC 1085) The sacraments, and especially the Eucharist, actualize this continuous presence.
"Accordingly, just as Christ was sent by the Father so also he sent the apostles, filled with the Holy Spirit. This he did so that they might preach the Gospel to every creature and proclaim that the Son of God by his death and resurrection had freed us from the power of Satan and from death and brought us into the Kingdom of his Father. But he also willed that the work of salvation which they preached should be set in train through the sacrifice and sacraments, around which the entire liturgical life revolves." [SC 6] (CCC 1086)
Thus the risen Christ, by giving the Holy Spirit to the apostles, entrusted to them his power of sanctifying: [cf. Jn 20:21-23] they became sacramental signs of Christ. By the power of the same Holy Spirit they entrusted this power to their successors. This "apostolic succession" structures the whole liturgical life of the Church and is itself sacramental, handed on by the sacrament of Holy Orders. (CCC 1087)
"To accomplish so great a work" ‑ the dispensation or communication of his work of salvation ‑ "Christ is always present in his Church, especially in her liturgical celebrations.” [SC 7] (CCC 1088)
The Sacraments as efficacious memorials of salvation: they recall and make present our salvation
The Spirit and the Church cooperate to manifest Christ and his work of salvation in the liturgy. Primarily in the Eucharist, and by analogy in the other sacraments, the liturgy is the memorial of the mystery of salvation. (CCC 1099)
That is why the anamnesis (remembrance) is so important. The liturgical celebration always refers to God’s saving interventions in history... In the Liturgy of the Word the Holy Spirit "recalls" to the assembly all that Christ has done for us… the celebration "makes a remembrance" of the marvellous works of God… The Holy Spirit who thus awakens the memory of the Church then inspires thanksgiving and praise (doxology).
Christian liturgy not only recalls the events that saved us but actualizes them, makes them present. The Paschal mystery of Christ is celebrated, not repeated. It is the celebrations that are repeated, and in each celebration there is an outpouring of the Holy Spirit that makes the unique mystery present. (CCC 1104)
The epiclesis ("invocation upon") is the intercession in which the priest begs the Father to send the Holy Spirit, the Sanctifier, so that the offerings may become the body and blood of Christ and that the faithful by receiving them, may themselves become a living offering to God. (CCC 1105)
Together with the anamnesis, the epiclesis is at the heart of each sacramental celebration, most especially of the Eucharist. (CCC 1106)
Liturgy, Sacraments, Faith and Grace
The whole liturgical life of the Church revolves around the Eucharistic sacrifice and the sacraments. [SC 6] (CCC 1113)
The Sacraments of the Church
The sacraments are "of the Church" in the double sense that they are "by her" and "for her." They are "by the Church," for she is the sacrament of Christ's action at work in her through the mission of the Holy Spirit. They are "for the Church" in the sense that "the sacraments make the Church," [St. Augustine, De civ. Dei, 22, 17: PL 41, 779; cf. St. Thomas Aquinas, STh III, 64,2 ad 3] since they manifest and communicate to men, above all in the Eucharist, the mystery of communion with the God who is love, One in three persons. (CCC 1118)
The Sacraments of Faith
"The purpose of the sacraments is to sanctify men, to build up the Body of Christ and, finally, to give worship to God. Because they are signs they also instruct. They not only presuppose faith, but by words and objects they also nourish, strengthen, and express it. That is why they are called 'sacraments of faith.'” [SC 59] (CCC 1123)


- What are the qualities of a strong person?
- What are the top 10 mental illnesses?
- What causes poor mental health?
- What are the top 5 mental illnesses?
- What is the hardest mental illness to live with?
- What is Anxiety Syndrome?
- What are the 3 characteristics of a mentally healthy person?
- What are the 10 personality traits?
- How can you tell if someone is mentally ill?
- What is needed for good mental health?
- What is the hardest personality disorder to treat?
- What is toxic personality disorder?
- What are the 5 signs of mental illness?
- What are the 7 types of mental disorders?
- What’s a mental breakdown?
- How do I know if Im bipolar?
- What defines good mental health?
- What is the rarest mental illness?
- What is the most popular mental illness?
- What are the 8 types of personalities?
- What are 5 qualities of a mentally healthy person?
What are the qualities of a strong person?
What are strong character traits?Tenacious.Confident.Optimistic.Self-aware.Adaptable.Flexible.Drama-free.Reliable.More items…•.
What are the top 10 mental illnesses?
What We TreatDepression.Anxiety Disorders.Schizophrenia & Schizoaffective Disorders.Bipolar Disorder.Autism Spectrum Disorder.
What causes poor mental health?
For example, the following factors could potentially result in a period of poor mental health: childhood abuse, trauma, or neglect. social isolation or loneliness. experiencing discrimination and stigma.
What are the top 5 mental illnesses?
There are five major categories of mental illnesses:Anxiety disorders.Mood disorders.Schizophrenia and psychotic disorders.Dementia.Eating disorders.
What is the hardest mental illness to live with?
Why Borderline Personality Disorder is Considered the Most “Difficult” to Treat. Borderline personality disorder (BPD) is defined by the National Institute of Health (NIH) as a serious mental disorder marked by a pattern of ongoing instability in moods, behavior, self-image, and functioning.
What is Anxiety Syndrome?
However, people with anxiety disorders frequently have intense, excessive and persistent worry and fear about everyday situations. Often, anxiety disorders involve repeated episodes of sudden feelings of intense anxiety and fear or terror that reach a peak within minutes (panic attacks).
What are the 3 characteristics of a mentally healthy person?
Good mental health is characterised by a person’s ability to fulfil a number of key functions and activities, including:the ability to learn.the ability to feel, express and manage a range of positive and negative emotions.the ability to form and maintain good relationships with others.More items…
What are the 10 personality traits?
These 10 Traits Are Key for a “Healthy Personality”—Which Ones Do You Have?Openness to feelings.Straightforwardness (and being “frank, sincere, and ingenuous”)Competence.Warmth (being affectionate and friendly)Positive emotions (experiencing “joy, happiness, love, and excitement”)Low levels of angry hostility.More items…•
How can you tell if someone is mentally ill?
If and when you’re ready to tell others, remember these tips:Pick a day and a time when you’re feeling okay. … Tell the person as much or as little about it as you want. … Rehearse the conversation in your head before you have it – even if it feels a bit strange!More items…
What is needed for good mental health?
Activity and exercise are essential in maintaining good mental health. Being active not only gives you a sense of achievement, but it boosts the chemicals in your brain that help put you in a good mood. Exercising can help eliminate low mood, anxiety, stress and feeling tired and lazy.
What is the hardest personality disorder to treat?
The flamboyant cluster includes people with histrionic, antisocial, borderline, and narcissistic personalities. Except for the borderlines — considered the most difficult personality disorder to treat — these patients enjoyed significantly better lives over time.
What is toxic personality disorder?
Even though most toxic people aren’t criminals, they could have underlying personality disorders, including narcissistic personality disorder, bipolar disorder, or other mental health condition. Or they could just be mean, self-centered, manipulative, egotistical, selfish and calculating.
What are the 5 signs of mental illness?
Five Warning Signs of Mental IllnessLong-lasting sadness or irritability.Extremely high and low moods.Excessive fear, worry, or anxiety.Social withdrawal.Dramatic changes in eating or sleeping habits.
What are the 7 types of mental disorders?
What are some types of mental disorders?Anxiety disorders, including panic disorder, obsessive-compulsive disorder, and phobias.Depression, bipolar disorder, and other mood disorders.Eating disorders.Personality disorders.Post-traumatic stress disorder.Psychotic disorders, including schizophrenia.
What’s a mental breakdown?
A nervous or mental breakdown is a term used to describe a period of intense mental distress. During this period, you’re unable to function in your everyday life. This term was once used to refer to a wide variety of mental illnesses, including: depression.
How do I know if Im bipolar?
Mania can cause other symptoms as well, but seven of the key signs of this phase of bipolar disorder are: feeling overly happy or “high” for long periods of time. having a decreased need for sleep. talking very fast, often with racing thoughts.
What defines good mental health?
But according to the World Health Organization, mental health is “a state of well-being in which every individual realises his or her own potential, can cope with the normal stresses of life, can work productively and fruitfully, and is able to make a contribution to her or his community”.
What is the rarest mental illness?
Also known as Todd syndrome, Alice in Wonderland syndrome (AIWS) is a neurologic condition “in which a patient’s sense of body image, space, and/or time [is] distorted,” according to Medscape. Those experiencing AIWS may have hallucinations, sensory distortion and an altered sense of velocity.
What is the most popular mental illness?
Anxiety disorders are the most common mental illness in the U.S., affecting 40 million adults in the United States age 18 and older, or 18.1% of the population every year. Anxiety disorders are highly treatable, yet only 36.9% of those suffering receive treatment.
What are the 8 types of personalities?
The eight types are:Extraverted Thinking.Introverted Thinking.Extraverted Feeling.Introverted Feeling.Extraverted Sensation.Introverted Sensation.Extraverted Intuition.Introverted Intuition.
What are 5 qualities of a mentally healthy person?
Characteristics of Mental HealthThey feel good about themselves.They do not become overwhelmed by emotions, such as fear, anger, love, jealousy, guilt, or anxiety.They have lasting and satisfying personal relationships.They feel comfortable with other people.They can laugh at themselves and with others.More items…


Speed dial 911, if you suspect stroke
More than 795,000 people in the United States suffer an acute stroke every year, according to the Centers for Disease Control and Prevention (CDC). And the only Federal Drug Administration (FDA) approved medication to treat it is tPA (tissue plasminogen activator), also known as Alteplase.
The drug is used to treat ischemic strokes, which comprise about 80 percent of all strokes, according to Michael Markowski, DO, FAAN, a neurologist at Cape Cod Hospital.
“The relative benefit of receiving Alteplase is reduced disability at three months and beyond,” he said. “One out of three people will have minimal to no disability. If we treat 100 patients with Alteplase, 32 will benefit, and three will have adverse effects. We’re not helping everybody, but this is the only medication we have to treat ischemic stroke, so we want to use it when we can.”
Alteplase works by breaking up the clot that is blocking the vessel and cutting off oxygen to the brain. While it is available to all stroke patients, only 8 percent of patients in the United States receive the treatment, according to Dr. Markowski. The reason is that only about 22 percent of patients come into the emergency room within the three-hour window required for safe administration of the drug, said.
When Alteplase Is Used
“There are very strict criteria for administering the medication, and the four-and-a-half-hour time window is the most stringent of the criteria,” Dr. Markowski said.
The time for giving the medication begins when the symptoms of the stroke start, so getting to the emergency room as soon as possible is crucial.
The window is longer if you are under the age of 80. Those patients have up to four and one-half hours to receive Alteplase from the onset of symptoms. For patients over 80, Alteplase can only be given safely within 3 hours of stroke onset.
The acronym FAST is one way to remember symptoms that could indicate a stroke, according to the American Stroke Association:
- Face drooping
- Arm weakness
- Speech difficulty
- Time to call 911
“An important point to remember is that the symptoms will typically be on one side of the body,” said Dr. Markowski.
After your arrival in the emergency room, many assessments will happen very fast. You will have a neurological assessment by the emergency room physician, lab work will be drawn, a head CT scan will be done, medical history taken and another neurological exam after your CT scan.
“Our goal is to make sure there are no contraindications to giving the Alteplase and to administer it within 60 minutes of the patient’s arrival in the emergency room,” said Dr. Markowski.
Some of the criteria to receiving Alteplase include:
When you can receive Alteplase
- The stroke is an ischemic stroke and not a hemorrhagic stroke (bleeding in the brain)
- Your platelet count is normal
- Your blood pressure is not markedly elevated
- Head CT scan is normal (no bleeding)
- Meet the time and other criteria
When Alteplase cannot be given
- Your stroke is a result of bleeding in your brain (hemorrhagic stroke)
- You have uncontrolled high blood pressure (this may be treated with IV medications in the ER to bring it down so you can receive Alteplase)
- Your platelet count is low, this would increase your risk for bleeding
- You are taking blood thinners such as Warfarin or Coumadin
Other reasons you may not qualify for Alteplase is that you have stroke mimic syndrome, as similar symptoms may rarely occur with migraines or seizures. With this condition, your symptoms are like those of a stroke but may be caused by a migraine, or other neurological disease.
Another Tool to Prevent Disability
“Mechanical thrombectomy is a new tool in the toolbox of treating stroke in addition to Alteplase,” said Dr. Markowski. It is a procedure to remove blockages of larger blood vessels, and is done by a physician who is a specialist in interventional procedures in an academic hospital setting.
The physician removes the clot by going through a catheter inserted into the groin to the brain. It must be done within six to eight hours of onset of symptoms of your stroke.
The benefit of this procedure is even better than intravenous Alteplase, said Dr. Markowski.
“The absolute benefit of Alteplase is 12 percent; the addition of the mechanical thrombectomy raises it to 14 to 30 percent.”


- Address people’s needs holistically through a healthcare package that includes contraception, maternal and child health, HIV prevention and treatment, and other services
- Strengthen health centers, satellite clinics, and community health workers so more people in more places have access to high-quality services
- Pioneer the use of digital technology to collect and use data in real time, help community health workers improve care, and monitor clients
- Focus on the unique sexual and reproductive health needs of adolescents—the largest generation of young people in history—as they come of age
- Strengthen partner organizations that best understand local needs and can continue to deliver services long-term
Without Fear or Boundary in Haiti
When the earthquake struck Haiti in 2010, it displaced more than 1.5 million Haitians. For much of Haiti’s recent history, people have faced significant challenges accessing the health services they need. 69% of married women aren’t using a modern method of contraception. 15,000 people become infected with HIV each year.
We believe every person has the right to sexual and reproductive health care. 20 years ago, Pathfinder helped prevent maternal deaths in Haiti by expanding access to contraception and lifesaving postabortion care. Today we’re partnering with the government to provide millions of Haitians with the services they need and the quality of care they deserve.


The candidates for ‘oldest avenue of trees in England’ include:
- The yew trees in Westbourne said to have been planted in 1544.
- The Bucklebury Oaks, also known as The Queens’ Avenues, which may have been planted to commemorate a visit by Queen Elizabeth I as well as a later visit by Queen Anne
- Joris Hoefnagel’s drawing of Nonsuch Palace makes it look as though a line of trees leads to the entrance and there was a similar feature is shown on reconstructions of the Palace of Beaulieu
But the ‘correct’ answer depends in the interpretation of the question:
- the processional route at Stonehenge is often described as an avenue and probably passed through trees for some or all of its length. Other stone circles (eg Callanish) also had what are assumed to be processional routes, as did Egyptian and Mesopotamian temples
- the word ‘avenue’ (from the French avenir) was not used in English until the mid-seventeenth century. A similar feature made before this date would probably have been called an alley (from the French aller)
So on a strict interpretation of the word ‘avenue’, the oldest avenue in England may be in Greenwich Park. The chestnut trees, which survive, were planted c1660 and John Evelyn, who is recorded in the OED as the first English author to use the word ‘avenue’, may have advised on the layout. He had an avenue, which does not survive, in his nearby garden (at Sayes Court in Deptford). The best-looking avenue of old trees in Greenwich Park runs north from a point near the intersection of the Great Cross Avenue with Blackheath Avenue. The view along this avenue was blocked a few years ago by the erection of a ‘hit-and-miss’ fence round an outdoor rubbish dump for the park cafe. It makes one think that the Royal Parks, who manage Greenwich, either have no knowledge of garden history or no interest in garden history. So one does not know whether to conclude ‘forgive them, Oh Lord, for they know not what they do’ or whether to conclude ‘forgive them, Oh Lord, for they know exactly what they do’. But I regard the positioning of this rubbish dump as unforgivable. When an airplane crashes, an accident investigation team is established. There is a need for a similar investigation of the Royal Parks Agency. The Commission of Enquiry should have plenipotentary powers to call for papers, to summon witnesses, to take evidence under oath and to make binding recommendations, if necessary for the future involvement of expert garden historians and landscape architects in decision making for the Royal Parks. How many managers of Royal Parks have qualifications in garden history? Are there any qualified garden historians on the agency’s payroll? Several excellent landscape architecture firms have given advice on Greenwich Park but, so far as I know, no trained designers or historians have had a role in the Greenwich Park management hierarchy. And it shows. Greenwich Park is to be closed for a month in 2012 for the Olympic Equestrian Events. Wouldn’t it be wonderful if the rubbish dump were removed as part of the Olympic legacy to Greenwich Park?


Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics.
Citation: Kawano Y, Otsuka C, Sanzo J, Higgins C, Nirei T, Schilling T, et al. (2015) Expanding Imaging Capabilities for Microfluidics: Applicability of Darkfield Internal Reflection Illumination (DIRI) to Observations in Microfluidics. PLoS ONE 10(3): e0116925. doi:10.1371/journal.pone.0116925
Academic Editor: David T. Eddington, University of Illinois at Chicago, UNITED STATES
Received: July 20, 2014; Accepted: December 16, 2014; Published: March 6, 2015
Copyright: © 2015 Kawano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors used the following grant for their study: Grants-in-Aid for Scientific Research (A), No. 26242039, http://www.jsps.go.jp/english/e-grants/index.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Olympus America Inc. and Olympus Corporation provided microscope equipment but was not a funder of, and did not influence the outcome of, the study. The VS120 WSI scanning system was used. Yoshihiro Kawano and Chino Otsuka are employed by Olympus Corporation, James Sanzo and Christopher Higgins are employed by Olympus America, Tatsuo Nirei by Olympus Engineering and Tobias Schilling by Olympus Soft Imaging Solutions GmbH. This particular microscope setup (VS120 with BX-DSU) is not available in the market. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Micro total analysis systems (μTASs) have become popular over the last two decades . Microscale tools to control fluid flow, such as micro valves, pumps, and flow sensors have been developed . Microfluidics technologies are now capable of manipulating nanoliters of fluid, molecules, bubbles, particles, and cells , and these have been used for micro-fabrication in engineering settings as well as diagnosis in clinical settings . Recently, techniques for producing microbubbles using microfluidic devices have been developed, and the two-phase flow in microchannels has been measured [5–12]. Some groups have succeeded in fabricating monodispersed microbubbles for drug delivery, and in clinical diagnosis they have been used as contrast agents. Droplets and vesicles can also be fabricated by microfluidic devices, which can then be used in synthesizing molecules, such as proteins and DNA . To design such microfluidic devices appropriately requires an accurate dynamic analysis of the motion of bubbles and droplets, with high resolution in time and space. In particular, interactions between bubbles and channel walls are important, because microbubbles may block microchannels, which alters the flow compared to what was expected (i.e., clogging). Moreover, bubble shape is important in the breakup and coalescing of microbubbles in microfluidics. Thus, visualization techniques that enable detection of the surfaces of bubbles and walls are important [13–15].
In microfluidics research, the velocity field is often measured using microparticle image velocimetry (μPIV) [16–19]. Recently, confocal laser scanning microscopes (CLSM), such as disk scanning confocal systems, have been adapted to μPIV systems to improve image contrast and reduce the depth of field of the image [20, 21]. Three-dimensional optical sectioning has become possible and semi-3D velocity measurements have been performed [22–30]. To enable highly accurate velocity measurements, high-contrast images of tracer particles are required. Moreover, the wall configuration must be observed clearly to evaluate the wall shear stress, which can be calculated from the velocity gradient observed at the wall. Thus, it is necessary to simultaneously and clearly observe the wall configuration within channels and fluorescent tracer particles flowing through the channels.
Microfluidics technologies have also been used in cancer diagnosis. For example, an antibody-based microfluidics platform can capture cervical cancer cells using immunocytochemistry techniques . Oral squamous cell carcinoma (OSCC) cells were detected with a microfluidic device using magnetic beads conjugated with antibodies plus real-time polymerase chain reaction (RT-PCR) . Microfluidic devices have been used to identify circulating tumor cells (CTCs) in the peripheral blood of patients with metastatic lung, prostate, pancreatic, breast, colon, and ovarian cancers [33–50]. In the design of such microfluidic devices, it is important to observe the behavior of cells within channels with complex geometry. Moreover, cancer cell adhesion and invasion have been investigated using microfluidics to assess the mechanisms of cancer metastasis [51–57]. As the interactions between cancer cells and vessel walls are important, visualization techniques to detect cancer cells and their relationships to channel walls are critical to understanding cell behavior [13–15]. Such techniques are investigated in this study.
The most common illumination systems used for cell and microfluidics observation in previous studies were brightfield, darkfield, fluorescence, and confocal imaging. Regular darkfield illumination is typically used for particle observations . An example is its use in a blood cell counting application . Fluorescence and confocal techniques are useful for observing specific cell markers. However, simultaneous detection of the channel wall and the cell marker is difficult. Lima et al. combined transmitted halogen illumination and confocal imaging to increase the visibility of fluorescently labeled cells and walls . The combined imaging methods were effective, although the fluorescent signal was partly masked by the halogen illumination. In the present study, we overcame this problem by using a Darkfield Internal Reflection Illumination (DIRI) system combined with a spinning disk confocal microscope. Our group recently developed a modification of the darkfield microscopy system . In the previous study, the applicability of using a DIRI for a fixed brain slice sample was investigated . In the present study, we investigated the applicability of fluorescence and DIRI for microfluidic systems containing microbubbles, fluorescent particles, and/or cells. We show that the combined illumination techniques can simultaneously detect the channel wall and the microbubbles, fluorescent particles, and/or cells with a good apparent signal to noise ratio.
Microfluidic devices with multiple reactor areas and/or multiple measurement sites are often larger than the field of view of a typical 20× or 40× microscope objective lens. Thus, whole-slide imaging (WSI) is useful to observe the entirety of these areas. WSI technology is well suited for observing areas larger than one field of view under a microscope, and is used for tile imaging, remote viewing, archiving, and analyzing images for neuroscience and pathology fields. In this study, we adapted WSI technology to microfluidics observation. The proposed methodology should be useful in future for controlling microfluidics by monitoring multiple locations.
The paper is organized as follows. In the Materials and Methods section, we first describe our DIRI system and compare it with conventional darkfield illumination. We then explain the details of experiments using brightfield, DIRI, and fluorescent illumination. In the Results and Discussion section, we first investigate the combination of brightfield and DIRI imaging that enables visualization of both microbubbles and channel walls simultaneously. We show that the methodology is useful for dynamic phenomena, such as the detection of bubbles flowing in a channel. Second, we investigate the combination of fluorescence and DIRI imaging. We show that the present system can detect both the channel wall and fluorescent tracer beads. Third, we investigate the combination of multi-channel fluorescence imaging and DIRI imaging using two types of cells, fluorescently stained red or green. We show that the present system can detect both the channel wall and the target cell marker. Last, we discuss the advantage of our DIRI system. In the Conclusion section, we summarize the study and provide final thoughts.
Materials and Methods
1. Darkfield Internal Reflection Illumination (DIRI)
Fig. 1A shows the newly developed DIRI (Darkfield Internal Reflection Illumination) unit. A multiple LED light assembly is installed on the stage plate and used for side illumination of a microfluidics cell or a glass slide. The DIRI system can be used alone, or may be used with a fluorescence microscope. Acquired fluorescent images are used to show fluorescent markers of target objects. The DIRI system creates a broad scattering of illumination from the edges of the PMDS channel walls. The refractive index mismatch between PMDS and buffer (liquid) produces good image contrast, resulting in high-quality images of the microchannel walls. Superimposition of the two images (fluorescence and DIRI) allows observation of both fluorescent markers and the edges of the channel walls. There are two ways to produce such superimposed images: (a) fluorescence and DIRI images can be acquired simultaneously, or (b) fluorescence and DIRI images can be acquired sequentially as two or more channels (depending on the number of fluorescent channels to be captured separately), and the channels can be superimposed later. Method (a) can be applied to dynamic phenomena as well as static phenomena, whereas method (b) can be applied only to static phenomena.
(1a) The photograph shows the microfluidic device used with the DIRI illuminator. The illuminated area is almost the entire microfluidic device. (1b) The photograph shows the microfluidic device used with a regular darkfield illuminator. The condenser lens is a DCD condenser, NA 0.8–0.92 (Olympus). The illuminated area is shown by the blue arrow. Note the small area properly covered by the illuminator. (1c) The experimental setup for the system. The left side camera is an Orca R2. The microfluidic device consists of a special microfluidics module placed upon a plastic adapter plate. The size of the microfluidics module in the photograph is 25 × 75 mm.
In the case of conventional darkfield microscopy, light enters the specimen at an oblique angle and is blocked at the periphery of the optical components. As the scattering of light based on the refractive mismatch passes through the optical path and is observed by the darkfield microscope, the background image is dark, and the specimen appears bright. If the image background is bright, the detection sensitivity is considerably reduced, resulting in conditions similar to brightfield microscopy. Darkfield microscopy can often provide enhanced detection sensitivity and contrast for specimens that cannot be effectively imaged using brightfield illumination.
Conventional darkfield units are commercially available for standard microscopes. There are two types of darkfield condenser: dry and oil darkfield condensers. Some phase turret condensers include the required darkfield annulus within the condenser turret. In Fig. 1B, a dry darkfield DCD condenser (NA 0.8–0.92; Olympus, Tokyo, Japan) is shown below the microchannel slide. The illuminated area is limited to a spot (see arrow). Normally, most dry darkfield condensers are capable of illuminating fields of view of 10× to 20× objectives with NA < 0.5. A dry darkfield condenser cannot cover objective lenses with NA > 0.8, such as a 40×, NA 0.95 lens because the illumination cone of light passes directly into the objective lens and eliminates the dark background of the image. Use of the 40×, NA 0.95 objective lens requires optical coverage that can be provided by an oil darkfield condenser. A common practice during imaging sessions is to find the required field of view using a 10× or 20× lens, then to switch to a higher magnification and higher NA objective (such as the 40×, NA 0.95 lens) for image acquisition. However, if the user later needs to switch back to the 20×, NA 0.75 dry lens, the condenser also has to be switched back to the dry darkfield condenser. This necessitates the disruptive and somewhat laborious process of removing oil from the slide, and requiring that the investigator begin again with dry optics. The procedure of switching back and forth between dry and oil systems poses considerable inconvenience to investigators and may introduce unacceptable delay into the experimental protocol, rendering some experiments impossible to perform. In fact, this is the case for many microchannel setups with multiple delicate attachments to pumps, drains, sensors, etc.
As described here, the newly developed DIRI system eliminates the inconvenience and delay inherent in the conventional approach. Thus, experiments can be performed that require frequent or rapid switching of magnification to locate fine details within larger fields of view before acquisition is appropriate. In addition, the area of view that is illuminated for DIRI imaging is larger than that which a conventional darkfield condenser is capable of illuminating (compare Fig. 1A to Fig. 1B). Fig. 1A clearly shows that the DIRI system can illuminate most of the area of a glass slide. The illumination intensity has also been enhanced in this version of the device. The latest design for DIRI is 1.5-fold brighter than the previous design . Moreover, the DIRI can be used with 10× to 100× objective lenses, including both dry and oil objectives. Thus, the DIRI system has important advantages in usability compared to the conventional darkfield condenser.
The DIRI system also has an advantage in terms of the small space required for implementation. The DIRI illuminator is thin and built into the stage insert plate, as shown in Fig. 1A. Therefore, it does not interfere with tubes connected to the microfluidics equipment for liquid handling. Our DIRI is optimized to work with a standard microscope slide measuring 25 × 75 mm. As the size of the microfluidic device used in this study is 25 × 50 mm, we implemented a plastic support plate to adapt the microfluidic device size to 25 × 75 mm. The simplicity of this adaptation is another indication of the robustness and flexibility of our DIRI system as applied to microfluidics.
Our DIRI system is easily installed by simply attaching the LED array to the side of the microfluidics observation location. When we constructed the first prototype, we started from such a simple design. By adding automatic control of the confocal illumination, the widefield fluorescence excitation and emission, DIRI, transmitted (brightfield) illumination, stage movement, etc., a more sophisticated system can be constructed (cf. Fig. 1C).
2. Experimental Setup and Procedures
In this study, we conducted three types of experiment: (1) observation of microbubbles by combination of brightfield and DIRI imaging, (2) observation of fluorescent tracer particles by combination of fluorescence and DIRI imaging, and (3) observation of two types of stained cell by combination of multiple fluorescence imaging and DIRI imaging.
In all of these experiments, we used the Virtual Slide System VS120 (Olympus) including VS-ASW WSI software (ver. 2.7) as a base system, with the cellSens software (ver. 1.9; Olympus Soft Imaging Solutions, Münster, Germany) for video and 3D imaging. We attached a BX-DSU spinning disk confocal system (Olympus) to the VS120 system for confocal observations. We also attached the DIRI illuminator as in our previous study  for WSI. Before use, this integrated system was calibrated using the VS-ASW software and the recommended calibration slide from the manufacturer. The system described here is a prototype and is not yet commercially available.
A photograph of the system is shown in Fig. 1C, and the optical layout is shown schematically in Fig. 2. The DIRI system consists of LEDs attached to the microscope stage , which enables visualization of the sample by refractive index mismatch. The DIRI system can be used with widefield fluorescence or confocal imaging. A microfluidic device on an appropriate stage adapter is placed on the stage, and the condenser lens for the transmitted illumination is set up about 10 mm below the Köhler illumination position. The condenser lens is located between the stage and mirror unit below the stage (not shown in Fig. 2). The halogen lamp is used for transmitted illumination. The spinning disk confocal system is set between two dichromatic mirrors (cf. Fig. 2). Mercury vapor short-arc lamps are used for confocal and fluorescence illumination. The observed images are recorded with a CCD camera (Orca R2; Hamamatsu Photonics, Hamamatsu, Japan). We typically use the Orca R2 camera for all modes of image capture, including DIRI, fluorescence, confocal, and brightfield imaging.
This system is capable of brightfield, DIRI (Darkfield Internal Reflection Illumination), conventional fluorescence, and confocal imaging. The DIRI image was obtained by illumination using the LEDs at the side of the slide. The system can also provide whole-slide images (WSI).
Fig. 3 shows the microfluidic device used in this study (SynVivo Bio-chip Microvascular Network; CFD Research Corporation, Huntsville, AL). The original device measures 25 × 50 mm with a thickness of 2 mm (Fig. 3A), and the top surface is covered with a coverslip 0.17 mm thick. The microchannel network is molded into the polydimethylsiloxane (PDMS) block that composes the microchannel device. For this device, the channel width is about 100 μm. Ports A and D in Fig. 3B are the inlet and outlet for fluid flow, respectively. Ports B and C are closed, but are used to remove bubbles in the channel before the experiments. The fluid flow is generated by manipulating a syringe (Norm-Ject, 1 mL; Henke Sass Wolff, Tuttlingen, Germany) with a programmable syringe pump (Model Fusion 200; Chemyx Inc., Stafford, TX). The refractive index of PDMS is approximately 1.41, the buffer has a refractive index similar to water (n = 1. 33), and that of cells is in the range 1.35–1.40. When we used DIRI with buffer-filled channels, scattered light was observed at the interface of the two materials (channel walls) due to their differing refractive indexes.
(3a) Schematic of the entire plate. The microfluidic module itself was 25 × 50 mm, which was extended to 25 × 75 mm by the addition of a plastic adapter plate. (3b) Channel geometry within the microfluidic device. The image was taken using DIRI (Darkfield Internal Reflection Illumination). Port A is the inflow port, and port D is the outflow port. Ports B and C are closed during the experiment.
In the microbubble experiment, we added a diluted surfactant (Triton X-100; Sigma-Aldrich, St. Louis, MO) to phosphate-buffered saline (PBS). The bubbles were generated manually by pushing the syringe plunger back and forth before the experiment. Then, the two-phase fluid was injected into the channel manually using the syringe.
For our fluorescent bead experiments, we added diluted surfactant (Triton X-100; Sigma-Aldrich) to phosphate-buffered saline (PBS) to avoid adhesion of the beads to the channel walls. We used fluorescent beads 1 μm in diameter (F8823 FluoSpheres fluorescent beads; Invitrogen, Carlsbad, CA) diluted with PBS.
In the cell experiments, we used two types of cell emitting two different colors under fluorescence illumination: human umbilical cord blood endothelial cells at the fourth passage (HUVECs, #KE-4109; Kurabo, Osaka, Japan) and fibrosarcoma cells (HT1080; American Type Culture Collection, Manassas, VA). All cells were incubated at 37°C in 5% CO2. The endothelial cells were stained with 10 μg/mL rhodamine 123 (Wako, Osaka, Japan) and emitted green fluorescence when illuminated with blue light. Fibrosarcoma cells were stained using Mito Tracker Red CMXRos (Life Technologies, Gaithersburg, MD) and emitted red fluorescence when illuminated with green light. The numbers of endothelial cells and fibrosarcoma cells were adjusted to 6×105/mL. Before introduction of cells into the microchannel device, the channels were coated with fibronectin to promote adhesion to the channel walls.
In all of these experiments, we used DIRI from only one side of the microfluidic device. Therefore, the DIRI induced asymmetric illumination. More isotropic illumination could be created by the attachment of additional DIRI illuminators located around the periphery, creating concurrent illumination from multiple directions. For example, DIRI illuminators could be attached to both sides of the sample or surrounding the sample.
Results and Discussion
1. Combination of Brightfield and DIRI Imaging
When using a microfluidic device, air bubbles may adhere to the inside of the microchannel, and thus make flow control difficult. Microbubbles are small in size and difficult to find within the entire microfluidic device, so it is important to develop a methodology to detect microbubbles throughout the device. In addition, microbubbles are difficult to detect using standard brightfield illumination because both the bubble signal and the background signal produce images of similar brightness with minimal contrast. In most cases, liquid and air bubbles are both transparent, only differing in refractive index. Darkfield illumination, however, allows the microbubbles to be detected because of the image contrast created by the refractive index differences. Under darkfield illumination, microbubbles appear as bright objects against a dark background. Here, we investigated the utility of our DIRI system for detecting microbubbles.
Fig. 4 shows a DIRI image of the whole microfluidic device, in which bubbles were observed as bright spots, as indicated in yellow. The four large bright circles indicated by the blue arrows are the inlets and outlets. The VS-ASW software was used to construct a whole-slide image by stitching 1088 individually acquired images. Our system facilitated detection of bubbles in the entire microfluidic device with high contrast.
Bubbles trapped in the microfluidic channels are indicated by the yellow arrows. The blue arrows indicate outlet ports. Photograph was taken using DIRI (Darkfield Internal Reflection Illumination) with WSI (whole-slide imaging).
To clarify the advantages of the system, we compared microbubble images using brightfield illumination alone (Fig. 5A), simultaneously used DIRI and brightfield illumination (Fig. 5B), and DIRI alone (Fig. 5C). The images were obtained with a 2× objective lens (NA 0.08), and no flow was induced (i.e., the bubbles were stationary). The brightfield image (Fig. 5A) shows the darker edge of the channel and bubbles on top of a brighter background. The DIRI image (Fig. 5C), however, shows the bright edge of the bubbles on a darker background, although the edge of the channel is unclear. This bright edge of the bubble image is produced by total reflected light from the LEDs of the DIRI. When DIRI and brightfield illumination were used simultaneously (Fig. 5B), the edges of both the channel and bubbles themselves could be observed clearly.
This image was acquired using a 2× objective lens (NA 0.08) and an exposure time of 10 ms. (5a) Photograph obtained with brightfield illumination alone. (5b) Photograph obtained by simultaneous using brightfield illumination and DIRI (Darkfield Internal Reflection Illumination). (5c) Photograph obtained with DIRI alone.
To quantitatively discuss the edge detection sensitivity with different illumination, we measured the intensity profile along a line crossing a bubble and the channel walls, as shown in Fig. 5D. The measured line is indicated as a white line in Fig. 5A—C, which has a length of about 220 μm. In this figure, the intensity varies from 0 to 65535, given that the camera acquired a 16-bit image. As can be seen in the figure, the curve of the line (labeled “Simultaneous”) from the simultaneous illuminated image (5B) shows the greatest variation in brightness at the channel wall and the bubble surface, indicating that the dual illumination technique of brightfield and DIRI is advantageous for simultaneous detection of bubbles and channel edges.
Next, we induced fluid flow, to put the bubbles in motion. Although the local velocity fluctuated due to breakup and coalescing of bubbles, the velocity of bubbles in the observed region was on the order of about 10 mm/s. Fig. 6 shows a comparison of microbubble images using three kinds of illumination. Images were acquired with a 10× objective lens (NA 0.4). Fig. 6A—C were acquired with different exposures although the velocity of fluid flow remained constant, so the bubble sizes appear different in each image. The brightfield image (Fig. 6A) clearly shows the edges of the channel walls and the edges of the bubbles. The DIRI image (Fig. 6C) shows total reflected light from the top side at the edges of the bubbles. Note that the edges of the channel walls are difficult to observe. Fig. 6B was taken using brightfield illumination and DIRI simultaneously.
This image was acquired using a 10× objective lens and an exposure time of 60 ms. (6a) Photograph obtained with brightfield illumination alone. (6b) Photograph obtained by simultaneous using brightfield illumination and DIRI (Darkfield Internal Reflection Illumination). (6c) Photograph obtained with DIRI alone.
To quantitatively discuss the edge detection sensitivity with different illumination, we again measured the intensity profile along a line crossing the bubbles and the channel walls. The measured line is indicated as a white line in Fig. 6A—C, which has a length of about 130 μm. In this figure, the intensity ranges from 0 to 255, given that the video mode camera setup stored 8 bits of intensity depth. The simultaneous brightfield and DIRI illumination (Fig. 6B) has high contrast at the bubble surfaces as well as the channel wall. This system has the advantage that it can provide a high-contrast image by simultaneously using two kinds of illumination, rather than requiring the superimposition of two images taken sequentially using the two different kinds of illumination. Therefore, this method can be applied to dynamic phenomena, such as gas-liquid two-phase flow.
2. Combination of Fluorescence and DIRI Imaging
To assess the applicability of the system to fluorescent illumination, we observed fluorescent beads using DIRI and fluorescent illumination. We first observed the channel wall using DIRI with WSI mode (Fig. 7A) using a 10×, NA 0.4 objective lens. The software was programmed to construct a whole-slide image by acquiring and stitching together 108 individually acquired images. Next, we observed the trajectories of fluorescent beads by confocal microscopy in WSI mode. Fig. 7B shows the trajectories of fluorescent beads over 0.81 s with a flow rate of 1.2 μL/min. The image was obtained with fluorescent illumination (confocal) alone in this case. Note that the channel walls are difficult to identify. Fig. 7C shows superimposed DIRI and fluorescence images that were acquired sequentially. By superimposing the two images, tracer trajectories and channel geometry can be observed clearly. The long trajectories (visible as long green streaks) indicate beads moving at high velocity, whereas short trajectories (shorter green streaks) indicate low bead velocity. Locations that appear to have many lines indicate those with many moving beads. Thus, there are several advantages to this imaging technique: 1) the superimposed image provides information on the flow field in the whole microfluidic device, making measurements possible using channel architecture as fiduciary points; 2) this imaging modality does not require an expensive camera, such as those used frequently in high-speed imaging; and 3) high quality images can be obtained with a regular cooled CCD or sCMOS camera designed for fluorescence imaging.
The exposure time for darkfield was 100 ms and that for confocal fluorescence was 810 ms. In total, 108 images were captured using a 10× objective lens, and were used to construct the WSI (whole-slide image). (7a) DIRI (Darkfield Internal Reflection Illumination) image. (7b) Confocal fluorescence image. (7c) Superimposed image of the fluorescence and DIRI images.
The velocity field in the microfluidic device can be roughly estimated by taking images with various exposure times, as shown in Fig. 8. The exposure times used in these images were 10 ms (Fig. 8A), 30 ms (8B), 90 ms (8C), 269.9 ms (8D), 810 ms (8E), and 2.4 s (8F). The velocity at each location can be roughly estimated by measuring the length of each bead trajectory and dividing it by the exposure time. This methodology is useful for determining whether the flow field of a microfluidic device is induced as designed.
Flow rate 1.2 μL/min. In total, 108 images were captured using a 10× objective lens, and were used to construct the WSI (whole-slide image). Images are superimposed the fluorescence and DIRI images. (8a) Exposure time 10 ms. (8b) Exposure time 30 ms. (8c) Exposure time 90 ms. (8d) Exposure time 269.9 ms. (8e) Exposure time 810 ms. (8f) Exposure time 2.4 s.
The images shown in Fig. 9 were obtained by simultaneously using DIRI and fluorescent illumination. No flow was induced in this case, and the three-dimensional confocal image was obtained using a 30× silicone oil immersion objective lens (NA 1.05). In the case of fluorescent illumination alone (Fig. 9A), fluorescent beads could be observed clearly, but the edges of the channel were difficult to identify. When fluorescent and brightfield illumination were used simultaneously (Fig. 9C), the shape of the channel became clear, but the contrast of the fluorescent beads was lost because the background image was brighter. The bright background considerably reduced the contrast of the image. When DIRI and fluorescent illumination were used simultaneously (Fig. 9B), the shape of the channel could be observed clearly, and the contrast of the fluorescent beads was maintained. This technique can also be applied to dynamic phenomena, such as fluorescent beads flowing through the microchannel. A movie taken using simultaneous fluorescent and DIRI is available as supplementary data (S1 Video).
The image was taken with a 30× objective lens (NA 1.05, silicone oil immersion). (9a) Confocal fluorescence image. (9b) Image obtained by simultaneous using a confocal fluorescence excitation and DIRI (Darkfield Internal Reflection Illumination). (9c) Image obtained by simultaneous using confocal fluorescence excitation, and brightfield illumination.
3. Combination of Multiple Fluorescence Imaging and DIRI Imaging
Finally, we investigated the applicability of the present system for imaging cells within microfluidic devices. Endothelial cells (0.5 mL, 6×105 cells/mL) were injected into the microfluidic device, followed by incubation for 5 h at 37°C in a 5% CO2 atmosphere to allow the cells to attach to the walls of the microfluidic channel. Immediately before the experiment, fibrosarcoma cells (HT1080; 0.5 mL, 6×105 cells/mL) were injected into the microfluidic device. The endothelial cells were labeled with rhodamine 123, which was visualized in the green fluorescence emission channel, whereas the fibrosarcoma cells were labeled with MitoTracker Red CMXRos for visualization in the red fluorescence emission channel. A superimposed whole-slide image of green fluorescence, and red fluorescence images is shown in Fig. 10 (20× objective lens, NA 0.75). The system is capable of providing a high-contrast whole-slide image of two fluorescent colors.
Endothelial cells (emitting green fluorescence) and fibrosarcoma cells (emitting red fluorescence) in the whole microfluidic device. The image was generated by superimposing two-color fluorescence images. The 20× objective lens (NA 0.75) was used.
Fig. 11 shows the advantages of multiple channel fluorescence imaging plus DIRI imaging in certain types of experiment. These images show a magnified image of two types of stained cell acquired with a 30× silicone oil immersion objective lens (NA 1.05). In the brightfield image (Fig. 11A), high contrast can be seen at the edges of the channel walls, and the cells stand out well from the background. However, the difference between endothelial cells (green arrow) and fibrosarcoma cells (red arrow) is not obvious. Use of appropriate fluorescent illumination (Fig. 11B) enabled observation of one or the other population of cells, but rendered the walls of the microchannel difficult to see. In this image, the endothelial cells are fluorescing under blue excitation (green arrow) and can be seen clearly, but the edges of walls and the fibrosarcoma cells were unclear or not visible. Simultaneous observation of the green fluorescence emission and DIRI (Fig. 11C) facilitated observation of the endothelial cells (green arrow) and the edge of the channel walls. The high-contrast images were obtained using two types of illumination simultaneously, not by superimposing two independent images in post-processing. Thus, the present system is ideal for observing cell adhesion and invasion, in which dynamic interactions between multiple cell types and blood vessel walls can be visualized in a simplified in vitro model of a complicated in vivo situation.
The images were captured using a 30× silicone oil immersion objective lens (NA 1.05). (11a) Brightfield image with an exposure time of 250 ms. (11b) Confocal fluorescence image with an exposure time of 200 ms. (11c) Image obtained by simultaneous using a confocal fluorescence excitation and DIRI (Darkfield Internal Reflection Illumination). The exposure time for fluorescence was 250 ms. The green arrow indicates an endothelial cell, and the red arrow a fibrosarcoma cell.
In this study, we investigated the applicability of using DIRI for observing a microfluidic device containing microbubbles, fluorescent particles, or fluorescently labeled cells. Whole-slide imaging was combined with these modalities to extend the capabilities of the system to allow multi-modal imaging of an entire microchannel device. Multi-modal imaging allowed clear visualization of both microbubbles and channel walls by utilizing DIRI and brightfield illumination simultaneously. The imaging methodology is useful not only for examination of static phenomena, such as clogging, but also for observation of dynamic phenomena, such as the detection of bubbles, fluorescent tracer particles, or labeled cells flowing through a channel. Observation of these dynamics makes possible a whole range of physical and physiological measurements of systems used for modeling more complex in vivo situations. This new multi-modal observation technique will make possible many previously difficult analyses, including observation of phenomena such as vessel wall adhesion of circulating cells under the moderation of pharmacological or physical agents, clot formation and inhibition, blood vessel obstruction, certain chemotaxis studies, the influence of turbulence or vessel walls, the influence of pharmacological moderators on circulating cell behavior, etc. The system developed here and the model validated in this study will be useful for a wide variety of engineering and biomedical applications using microfluidic devices.
S1 Video. S1 Video is a movie of bubbles in the microfluidics system using brightfield and DIRI.
This image was acquired using a 10× objective lens and an exposure time of 60 ms. Video obtained by simultaneous using brightfield illumination and DIRI (Darkfield Internal Reflection Illumination).
We would like to thank Robert Schmalzigaug of Olympus America for his advice on the figures.
Conceived and designed the experiments: YK CO JS CH TN TS. Performed the experiments: YK CO JS CH. Analyzed the data: YK CO JS CH TS TI. Contributed reagents/materials/analysis tools: YK CO JS. Wrote the paper: YK CO JS CH TS TI.
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(Medical Xpress)—Popular computer-based training programs designed to improve behavior or academic performance in children with attention deficit hyperactivity disorder, or ADHD, do not deliver on their intent, according to research findings published this month in Clinical Psychology Review.
Michael Kofler, assistant professor and director of the University of Virginia Curry School of Education's Children's Learning Clinic, and colleagues at the University of Central Florida took a critical look at 25 studies evaluating the effectiveness of cognitive training programs. These programs use computer-aided activities that are designed to help children improve brain functions thought to be underdeveloped in children with ADHD.
"We found that these treatments are not effective for treating children with ADHD," Kofler said. "They don't improve ADHD symptoms or behavior, they don't improve academic achievement, and in many cases do not improve the cognitive functions they claim to target."
According to Kofler, many of these programs are intended to target working memory, which is an area of difficulty for many children with ADHD. Working memory plays a key role in children's learning, and affects reading, math performance, writing and note-taking, interpreting social cues and preventing minds from wandering. Previous research by Kofler and colleagues also indicates that working memory plays a key role in core ADHD symptoms including the lack of attentive behavior, excess gross motor activity (hyperactivity) and impulsivity.
Interestingly, although many of the interventions claim to improve working memory, these programs actually trained short-term memory.
There are two primary parts to working memory: the memory component, which involves holding information in our head for a few seconds, and the working component, which involves processing or doing something with that information.
"Our research shows that it's the working part of working memory that is related to ADHD symptoms," Kofler said, "but unfortunately the current cognitive training protocols are only targeting the memory part. So it's not surprising that they're not working."
Kofler says this is a particularly important topic because parents are currently spending thousands of dollars in hopes of getting help for their children.
"Right now, the only effective treatments for ADHD are medication and behavioral interventions," Kofler said. "We're working on changing that, but none of the cognitive training programs out there right now are helpful for treating ADHD."
The Children's Learning Clinic research program was established as the result of a gift from the Galant family this past spring. As a result, the center has been working with a nationwide group of experts to develop a new cognitive training that focuses on working memory. The clinic is affiliated with the Curry School research center Youth-Nex as well as the school's doctoral program in clinical and school psychology. The clinic takes a strengths-based approach to ADHD.
Kofler says that cognitive training is a promising new direction for helping children with ADHD, but emphasizes that what's currently out there misses the mark.
"We need to develop better interventions for children with ADHD that focus on their strengths and not just their difficulties," he said. "If we do not consider all aspects of ADHD – including strengths and weaknesses – we are unlikely to provide the best possible services for these children."
Youth-Nex director Patrick Tolan said Kofler's work is breaking new ground. "Michael is taking a new and potentially game-changing approach to ADHD by looking at how to produce effective development, not just how to treat symptoms and lessen impairment," he said.
"Our ultimate goal is to do more than stop ADHD from impairing these children's academic, peer and family functioning," Kofler said. "We believe these children can go from struggling to thriving."
More information: Read the complete study: psychology.cos.ucf.edu/childre… hed-Version-2013.pdf


Everywhere the metaverse is expanding. It will become a major trend in the technology sector in the upcoming years. Although the metaverse itself is nothing, its technologies give it life.
The metaverse is a persistent, open, and shared virtual space in which users can exist as digital avatars. The metaverse is built on the idea that users can perform any action in the virtual world that they can in the physical one. However, the metaverse is still in its early stages, though. While B2C businesses continue to establish a presence in the Metaverse, a fully functional virtual reality is still a few stoplights away.
Top 5 technologies that power the Metaverse
Virtual and augmented reality
Immersive gaming has long made use of augmented reality (AR) and virtual reality (VR). It is clear how these technologies are used in the Metaverse, and both the Metaverse and immersive technology will develop further. With advancements in wearable technology, you can add full-body sensors to AR and VR technology to create an immersive experience.
In the past few years, VR has become more crucial due to the demand for a safe distance. In order to allow attendees to attend and interact with others just like they would in real life, several in-person events were VR-enabled. Despite still being in its infancy, augmented reality is a more user-friendly option for immersive experiences. Augmented reality adds virtual elements to the user’s real-world surroundings rather than creating an entirely virtual environment.
Blockchain technology will be used to power the Metaverse. The technology that underpins cryptocurrencies like Bitcoin and Ethereum is called a blockchain. Blockchain is a distributed, decentralized ledger that publicly and chronologically records transactions. This renders it secure and tamper-proof. In the Metaverse, assets will be purchased, sold, and traded using cryptocurrencies. They are it already.
Hybrid and distributed cloud environments
The adoption of cloud computing makes complete sense given that hosting 3D environments requires significant computing and storage resources. Cloud computing companies like AWS, Azure, Oracle Cloud, and Google Cloud Platforms may offer a more optimized cloud environment for the Metaverse, depending on how widely used the Metaverse and its offerings are.
Making sure that users’ experiences are not harmed by bandwidth restrictions and internet connectivity outages is one of the major challenges facing Metaverse cloud providers. Furthermore, it is a given that the requirements of the Metaverse will extend to the secure storage of personally identifiable information, which is a requirement of nearly every nation.
Introducing artificial intelligence is unnecessary. For many years, it has been the focus of our culture. Interest in and investments in this technology have significantly increased over the past few years. This year, the AI market will generate $62 billion, as predicted by Gartner. AI will be used in the Metaverse for many different purposes, including making digital avatars, handling payments, and managing resources. For instance, you can create a digital avatar in Decentraland using the Ava app. The app creates a 3D model of your face using AI.
It is the next iteration of the internet, to put it simply. The web is now smarter, easier to use, and safer. Web 2.0, the current web, is built on centralized programs like Facebook and Google. It will be decentralized in Web 3.0. There won’t be a single point of control or failure as a result. On Web 3.0, the Metaverse will be constructed. Instead of being kept on a single server, the data will be spread out among a network of computers.
Also Read: Top 5 Metaverse Predictions For 2023