Dear students, in this topic we shall discuss the mechanisms for thermogenesis in endotherms. The endothermic animals respond to temperatures below the lower critical temperature by generating additional heat. This additional heat is produced through three types of mechanisms, shivering thermogenesis, non-shivering thermogenesis and a specialized brown fat oxidation. The shivering and non-shivering thermogenesis both convert chemical energy into heat. These two processes use normal energy-converting metabolic processes, which body primarily uses to produce heat. But the difference is that all the released energy is utilized to produce heat. No energy is used for mechanical or chemical work. Usually, energy generated from the body metabolism process is used for some part-mechanical work and the rest is used to produce heat. But when heat is produced for purposefully thermogenesis, this mechanism works in the same way, but all that is 100% of energy is utilized to generate heat. Dear students, now we shall discuss shivering thermogenesis in detail. Shivering thermogenesis uses the involuntary muscle contractions to generate or liberate heat. This process is controlled by the nervous system, which controls the skeletal muscles. The nervous system at a time activates antagonistic pairs of skeletal muscles for contraction and relaxation. That is why there is no net movement in the shivering. Shivering is just like trembling or vibration. During this process, the muscle activation is hydrolyzed by ATP and energy is released as heat. Dear students, in non-shivering thermogenesis, enzyme systems are activated which metabolize fats. In non-shivering thermogenesis, conventional fats are broken down and oxidized and heat is produced. This process is activated by the sympathetic nervous system, which causes the release of two types of hormones. 1. Norepinephrine and 2. Thyroid. These two hormones activate the breakdown of fats and produce heat through non-shivering thermogenesis. Dear students, third important mechanism is the oxidation of brown fat. This is found in some mammals. A few mammals have specialized type of fat which is called brown fat. This fat contains large number of mitochondria in it. Its color is brown. A enzyme found in mitochondria is caused by cytochrome oxidase. All tissues in which the mitochondria is large appear in brown color. This brown fat, in addition to containing large number of mitochondria, it also has extensive vascularization that is supply of blood vascular system. Dear students, oxidation of brown fat is triggered by the thyroid hormones. This oxidation occurs within the cells which are rich with fat-medabilizing enzymes. The heat produced in this oxidation protein is rapidly dispersed to other body parts where this heat is needed. By the blood, because this brown fat is highly vascularized, there is a lot of blood flow in it, the heat generated by the blood flows to the tissues where the heat is needed.  Ordinary fat deposits are metabolically very inactive because they need to be reduced to fatty acids first and then those fatty acids cannot be oxidized by keeping them in the fat cells. In fact, these fatty acids enter circulation. They are taken up by the tissue cells which oxidize these fatty acids. In contrast, brown fat is oxidized within the containing tissue without delay with massive heat output. That's why brown fat is more efficient in heat generation and thermogenesis.


Y for the prospects of economic growth? I have been very clear about this. This Government will not increase income tax rates at a time of rising inflation, at a time of pressure on household incomes, especially low incomes. That would not be the right thing to do. However, I am equally clear that, given pressure on public services as a result of Tory austerity, it would also be wrong to cut taxes for the top 10 per cent of income earners, so we will not do that either. I am clear about our priorities. I am also pretty clear about the Tory priorities. They prefer tax cuts for the richest at the expense of our NHS, education and those on low incomes. I cannot believe that Ruth Davidson has come to this chamber today to talk about tax cuts for the rich, after the Resolution Foundation said just this week about Tory tax policy. I hope that she is listening to this. They said that Tory tax policy is going to make the poorest quarter of householders up to 15 per cent worse off and the highest quarter 5 per cent better off. They say that it will be the largest increase in inequality since the days of Margaret Thatcher. They also said this about raising the higher-rate threshold. It will do nothing to improve living standards for the majority of households. The Government is on the side of those on low incomes and public services. I will leave Ruth Davidson or Harrison or whatever it is that she has called to argue the case for tax cuts for the rich. Ruth Davidson can just call me the protector of Scottish families pay packets for the answer, but I have here an admiral document that the First Minister signed up to you not so long ago. It is called Let Scotland flourish. In it, the SNP told us that lower taxes would send the message that Scotland is open for business. That same SNP wants to put business taxes up. It then told us that higher rates would send the wrong message for indigenous businesses and businesses coming to Scotland. Now that wrong message is their only message. They then told us that business tax cuts would protect Government revenue because they would drive economic growth. Now they say that the opposite is true. They used to get this. Why not now? I think that Ruth Davidson might have missed something here. I do believe that competitive business taxes are important. That is why we have the most competitive business rates regime of any country in the United Kingdom. It is why the budget that will be debated this afternoon will lift 100,000 small businesses across this country out of business rates altogether. Let me go back to the previous issue of low-income households. The truth of the matter is that the Tories are the destroyers of the living standards of those on low incomes. In case she did not hear me, let me remind her of what the Resolution Foundation had to say about Tory tax policies. They will make the poorest quarter of householders up to 15 per cent worse off and the highest quarter 5 per cent better off. Widening inequality is what the Tories are doing. Will this Government, as we will see in the budget this afternoon, tackle inequality and protect our public services? That is our priorities. The Resolution Foundation also acknowledges that the Conservative tax policy has reduced inequality by the measurement of the Gini coefficient. However, the First Minister has done it already. The First Minister just stood there and said that she understands competitive taxation, but she is about to make us the highest tax part of the entire UK. I will tell her who the First Minister needs to listen to. She needs to listen to our business leaders in Scotland, because my first question this afternoon quoted directly from Liz Cameron, the chief executive of the Scottish Chambers of Commerce. I will quote her more extensively if the First Minister likes quotes so much today. She says that people paying higher income tax in Scotland than the rest of the UK would create a further barrier to Scottish business competitiveness, threatening jobs and damaging Scotland's attractiveness to inward investors. She goes on. She says that the sooner our politicians realise that supporting economic growth rather than hiking up taxes is the route towards increasing revenues and improving investment in key services, the quicker Scotland will prosper. However, we will vote on the budget in five hours' time, and we have been well warned what increasing taxes will mean for families and businesses. Why is the First Minister stitched up a tax-grabbing pact with the Greens rather than protecting Scottish jobs and Scottish pay packets? First Minister, let's take that step by step. Firstly, I'm sure that it's going to be of great comfort to those across this country struggling to make ends meet, those whose welfare protection has been cut by the Tory Government at Westminster, to know that the genie coefficient is all right. The truth, Presiding Officer, is that, as far as the Tories are concerned, the genie is out of the bottle. They are presiding over the biggest increase in inequality since the days of Margaret Thatcher, not my words, the words of the Resolution Foundation. I agree with Liz Cameron about the importance of competitive business taxis. That is why I repeat again that this Government is delivering the most competitive business tax regime in the whole of the UK—100,000 small businesses lifted out of business taxis altogether. Let me finally turn to the impact on householders. The difference between me and Ruth Davidson is that I do not believe that, at a time of Tory austerity, the priority should be cutting taxes for the top 10 per cent of income earners. In terms of our draft budget, the finance secretary will outline any changes to Parliament this afternoon, but in terms of our draft budget, what we are asking the highest earners to do is forgo a tax cut, amounting to £6 per week. That is less than people in England pay for a single prescription. However, in Scotland, of course, taxpayers not only get free prescriptions, they get tuition-free education, free personal care for the elderly, above inflation increases in the NHS, protection of local services, the best deal for taxpayers anywhere in the UK. That is what this Government is delivering. The First Minister seems utterly unconcerned about the impacts that her business policies are having on the ground and the screams of pain from companies across Scotland. We have not, and we have been speaking to staff at businesses effective. One of them is called the Banff Springs hotel. On 1 April, its rates are going up by £50,000, so it has been faced with a choice to either reluctantly put up its charges or to go bust. It has been forced to pass those charges on, and it has had its first complaint from a customer who is now having to pay £80 to hire a room. Let me read it out. The increase in the higher fee is excessive, to say the least. Should this fee of £80 apply to future meetings, I can confirm that there will be no further bookings and our business will be taken elsewhere. The name of the customer is the Banff branch of the Scottish National Party. First Minister's own party cannot support our policy. Isn't it time that she did something about it? What Ruth Davidson is talking about is an independent revaluation of business rates. As we have outlined, two weeks in a row, the final valuations will be issued later this year, and all businesses have the opportunity to appeal if they think that their valuation is wrong. Let's get back to the core issue here. We have the most competitive business rates regime in the whole of the UK. 100,000 small businesses lifted out of business rates altogether. We have a tourism sector that, thanks to the good work of those in it, is booming, employment rising much faster than it is in the rest of the UK. We are the best-performing part of the UK outside of the south-east of England for inward investment. These are the success stories of the Scottish economy, and we will continue to make sure that we invest in the success of our economy. We will also make sure that we protect our public services and we protect those on low incomes. That is what the budget will deliver, and I will be proud to put it forward to this Parliament later today. The member seems a little excitable ahead of the budget. Can I just ask members to please show respect to each other and to the proceedings? Kezia Dugdale. To ask the First Minister what engagement she has planned for the rest of the week. First Minister. At engagements to take forward the Government's programme for Scotland. Kezia Dugdale. In September, I asked the First Minister about the number of young people being referred for mental health treatment only to have that referral rejected. At the time, the First Minister expressed concern and said that she was determined to act. Can the First Minister now tell us how many more cases of young people with a referral have been rejected for mental health treatment since I last brought this crisis to the chamber? First Minister. We continue to invest in improving our mental health services. I have made it very clear to voices across this chamber the priority we attach to that as a Government. We do have rising demand for mental health services, which I have said before is something that we should welcome because it means that the stigma is reducing and more people are coming forward. We are seeing waiting times reduce. We are seeing more people treated. We are seeing not just rising investment but rising numbers of staff. I do accept, though, absolutely readily accept that we have much more work still to do. We are not unique in that sense in Scotland. Many countries are experiencing the same challenges, but we are absolutely determined through our investment and through our new mental health strategy that we will meet those challenges head on. Members will have noticed that the First Minister was unable to answer that question, so let me share the reality with her. Since the First Minister promised to act, there have been another 1,600 cases of young people who were rejected for mental health treatment. That takes the total now to 10,500 cases overall. That is thousands and thousands of children and young adults in crisis who have turned to professionals for help only to be turned away. We could reduce the number of young people needing clinical treatment in the first place. School-based counselling is key to that. Five months ago, I came to this chamber with a published plan for every secondary school to have access to a qualified counsellor. The First Minister said that she would look at it. We were not asking for any new money, just a fraction of the £150 million that the Government is already spending on mental health. We have had the warm words, First Minister. When will we get the action? As Kezia Dugdale knows, the plans that she brought forward are being looked at in the context of developing the mental health strategy. That is work that is on-going, and I would have thought that Kezia Dugdale would have welcomed that work and the consideration that has been given to those plans. In terms of numbers, let me point out to Kezia Dugdale that, in recent statistics, the number of patients seen by CAMHS services has increased by 21 per cent. We have seen long waits reducing, and we have seen the number of patients seen within the waiting times also improving. That is progress, but it is progress on an issue where I have readily accepted and continue to do so that we need to do more work. That is why the £150 million of extra investment backing the mental health strategy is so important. On an issue that is so important, I think that we all agree about its importance. I would hope that all of us in the chamber would get behind it. Kezia Dugdale said just there that her Government is looking at it, and she is considering it as part of the mental health strategy. That is interesting, because that is not what Maureen Watt told the health committee back in January. In fact, she told the health committee that provision of councillors in schools was a matter for local authorities. How on earth does the First Minister think that local authorities can do this when they are faced with millions of pounds worth of cuts? The cuts that we are faced with voting on this afternoon will make it all the harder for schools and other local services to provide the help that young people need. Those cuts will punish kids already in crisis. It does not have to be this way, so will the First Minister do the right thing to scrap the cuts and invest in Scotland's public services instead? Any mental health strategy that is going to be successful has to involve the Scottish Government working not just with the national health service but with local authorities. The fact that councillors are a matter for local authorities does not mean that it is not something that we will look at in terms of the mental health strategy. I thought that that point would have been very obvious. Kezia Dugdale stands up here and talks about extra funding for mental health services. Kezia Dugdale and her colleagues are intending to vote against a budget today that includes extra commitment to mental health services. Kezia Dugdale's approach to this budget, which has involved her as the leader of the third party, coming to the party that won the election and saying that they will only talk to us if, effectively, we rip up our own manifesto and implement theirs—not so much student politics as the politics of the playground. It is that lack of any constructive approach to the budget that has meant that Labour has rendered itself irrelevant, had no influence and delivered absolutely nothing on behalf of the people that it is supposed to represent. When it comes to the budget discussions, Labour should be deeply ashamed of itself. Two constituency questions at this stage. The first is from Claire Baker. Thank you, Presiding Officer. It is almost two years since Shekou Bayou died on the streets of Cercode while in police custody. Despite an investigation by Park and a report sitting with the Lord Advocate since August, the family still does not know the facts of what happened that morning in May 2015. There is now the potential for civil action as the family search for answers. Can the First Minister today assure the chamber and Shekou's family that the Crown Office will reach a decision on the report as a matter of urgency, and will the Scottish Government now commit to a wider inquiry into death in custody, as is the case in England, to ensure that no other family has to go through the same experience that Shekou's had for the past two years? The First Minister will be aware that this is a live, independent investigation, so it would not be appropriate for me to comment on the specific circumstances of the case. However, it is a complex investigation, and the Crown Office has already indicated that a decision will be made as soon as possible. Indeed, I understand that the Lord Advocate is meeting Shekou Bayou's family next week to discuss the case. The previous Lord Advocate, of course, made it clear that, regardless of the perk investigation, a fatal accident inquiry will be heard, and that will provide public scrutiny into the circumstances of the incident. I personally made it clear to the family when I met them that I am not ruling anything out in terms of a wider inquiry at an appropriate point in the future if that is required. I hope that the member will agree with me that it is important to allow those processes to take their course and to conclude. The First Minister will be aware of the report from NHS Greater Glasgow and Clyde that sets out cuts of £333 million and the sweeping centralisation of services. What is clear is that talk about shifting the balance of care is being used by that health board as a cover for cuts. Specifically, the report talks about cutting unscheduled care assessment and admission points. On that basis, will she today guarantee what she promised in the VEL vision that the medical assessment unit will remain in place? Jackie Baillie is being slightly misleading in her question because she is referring to a report that was never discussed by Greater Glasgow and Clyde health board because the chair of the board said that he did not agree with it and did not think that she would go forward for discussion. The health secretary has also made it clear that, had such a report gone forward for discussion, she would not have approved any of the proposals in it. For Jackie Baillie to stand up here today and try to somehow give the impression that this is the policy of Greater Glasgow and Clyde health board or the Scottish Government, I think, is misleading, terms of the veil of leaving, this Government will continue to do what this Government has done since day 1 in 2007. Continue to do what the previous Labour administration failed to do over many years and that protects services at the veil of leaving hospital. Thank you to ask the First Minister when the cabinet will next meet. We will next meet on Monday. I am delighted to say that we will meet in Piltlockry, which will be the first of the travelling cabinets this year. Everyone in this chamber and I think everyone in this country understands the value of public local services to all of our quality of life. Over recent weeks, councils in this country have been forced to contemplate cuts to a wide range of services, unacceptable cuts, from bigger class sizes to scrapping public transport and active travel, from ignoring late night noise and vandalism complaints to scaling back recycling, from removing librarians and specialist support staff from schools to increasing charges for people burying their relatives. That is not a position that any Government should leave our councils in. Late last year, under pressure from the Greens and others, the Government gave ground on the centralised control of additional council tax revenue and that will now be available for councils to allocate as they see fit in their local circumstances. Is not it clear that, even if we see this afternoon a budget concession restoring significant funding to protect local services in every part of Scotland, that that was not only essential but should also mark the beginning of a new approach, one in which we invest not only resources but local control back into the hands of our communities? As the chamber has heard me say before, the draft budget that the finance secretary outlined to the chamber at the end of last year involved additional funding, potential funding if councils used council tax powers of £240 million in additional funding for local services. I think that that was a strong draft budget for the protection of local services. The finance secretary also said that he wanted to listen to parties across the chamber and to enter into constructive discussion about how we could take forward some of their priorities, as well as the priorities that we have already identified. It is fair to say that the Conservatives and Labour refused to take part in any meaningful way in that constructive discussion whatsoever. The Lib Dems, at least, made the pretence of doing so—whether it was serious, I am not sure—but what the budget this afternoon I think will outline is the continued priority of this Government to protect local services, but it also makes clear that those in the chamber who are prepared to take part in constructive discussions actually do manage to achieve something on behalf of those that they represent. Perhaps other parties across the chamber could learn something from that. I am sure that all our colleagues look forward to hearing the detail of what is announced this afternoon. I can hear how eager they are to hear the detail of that even now, but can I make a further point? Is it not clear that, despite the progress that we hope to see this afternoon, tax policy with the new powers that are devolved to the Scottish Parliament can no longer be based for the long term for the duration of a Parliament on manifestos written years in advance? That was the approach in the first era of devolution when this was just a spending Parliament. We now, to some extent, make fiscal policy in Scotland and it is essential to respond not only to the balance of views across the Parliament but also to events. The events that we have seen since manifestos were written for last year's election include the Brexit vote, the fall in the value of the pound, a new UK Government, changes to UK fiscal policy. Is it not clear that tax policy throughout this Parliament cannot be based on manifestos written in previous years and has to be a living debate in which we take new directions forward with the new powers that we have available? I agree that any responsible Government has to take account of developments and things that are happening in the economy and in wider society when it comes to its budget decisions. The manifesto in which the Government was elected was not written years ago, but it was written less than one year ago. It is reasonable for the Government to say to the Scottish people that we want to seek to implement the promises that we made to them. In terms of Patrick Harvie's legitimate comments about the impact of Brexit, those impacts cut both ways. On one hand, one of the implications of the Brexit vote, partly because of the fall in the value of the pound, is rising inflation, putting greater pressure on household incomes, which I think underlines the commitment of this Government not to increase income tax rates in this budget. It also puts pressure on public services, which I think underlines the commitment of this Government at this time not to give a tax cut to those who earn the most in this country—the top 10 per cent of income earners. I think that the budget that will be outlined this afternoon by the finance secretary strikes the right balance. However, as I said before, it demonstrates that, where other parties in this chamber are willing to come forward with constructive suggestions, they will find a Government that is willing to listen. There will be one Opposition Party in this chamber this afternoon that can say to the people that they represent that they have managed to achieve something. The other Opposition parties, and this is a serious point, have achieved not one single brass penny in this budget for the people that they are supposed to represent, and I think that they should be ashamed of that. Willie Rennie, question 4. To ask the First Minister what issues will be discussed at the next meeting of the cabinet. To the people of Scotland. Once upon a time, the First Minister said that the police were safe in her hands. Now she says the same about Highlands and Islands Enterprise, but this week we discovered, through freedom of information, that the economy secretary had to be educated about what it does after he had made a decision to abolish the board. Instead of carrying on regardless in the dark, can the First Minister announce today that the board of Highlands and Islands Enterprise will not be abolished? I thank Willie Rennie for reminding us at the start of the question that the Lib Dems occupy a fairytale world. In terms of the police, an important priority for this Government, the draft budget that was outlined before Christmas, of course, delivered real terms protection for the resource budget of the police, which will see an additional £100 million going into front-line policing. Who knows? I am only speculating. The finance secretary may have more to say on matters such as this later this afternoon. On the question of Highlands and Islands Enterprise, I and indeed the economy secretary are on record on many occasions praising the great work that Highlands and Islands Enterprise does and the determination of this Government to support it to continue to do that work. The review that is under way right now is about looking at how we make sure that all of our enterprises, all of our agencies that work in the area of economic development and skills provision are working together in a co-ordinated way to deliver maximum impact on our economy. We will continue to allow that process to take its course. The economy secretary just before First Minister's questions was answering here about the work that Lorne Creerar is doing on our behalf in this. Of course, we will report back to Parliament on those matters in due course. Willie Rennie. I think that she needs a new joke writer. Another review by one of our Quango bosses is no substitute for a vote in this Parliament to reject her plans. The former chairman of Highlands and Islands Enterprise, Professor Jim Hunter, denounced the move as ministerial control freakery and centralism run riot, and he is a member of the SNP. Highlands and Islands Enterprise did not ask for this change. Local people do not want this change. The democratically elected Parliament of this country voted against the change, yet this Government is hell bent on taking control of running everything from the centre, of ignoring the needs of the Highlands and Islands. Why is it that, despite all the experience, she is so determined that she knows better than everybody else? I am not sure if Willie Rennie was in the chamber just a few moments before First Minister's questions when Keith Brown was answering questions on this very issue. Maybe he was too busy loving himself outside the chamber for him to have managed to find his way into it. If he had been in the chamber, he would actually have heard Keith Brown quote Jim Hunter and indeed quote some of the representations that have been made not just by Jim Hunter but by council leaders and by MSPs on my own benches or doing a good job on behalf of the people they represent. We will continue to listen to those representations. We are in the second phase of a review, and we will allow that review to conclude in due course. Then we will come back to Parliament and report the findings of that review. That is the appropriate and right way to go about things. However, as we do that, we will continue to protect Highlands and Islands Enterprise's ability to do the fantastic job that they do on behalf of people in the Highlands and Islands of our country. Does the First Minister agree that if this Parliament chooses to have a referendum on Scotland's future, then no Westminster Tory should try to stand in the way? If this Parliament voted to have a referendum on independence, then absolutely I agree that no Westminster Tory should stand in the way of the voice of this Parliament. The mandate of this Government in relation to this is unequivocal. It was the Tories, after all, who put us in the position of being taken out of the European Union against our will. With the support of only one of the 59 MPs in this country. Strang is it not that a Tory party that proclaims that it would be so confident of winning a referendum on independence now talks about trying to block it? Is it not the case that the Tories are running a wee bit feared? Question 5, Rob Doris. To ask the First Minister what representations the Scottish Government has made to the UK Government regarding the announcement of further job centre closures in Scotland. I am very concerned at the announcement by DWP to close up to 23 job centre plus sites in Scotland. The lack of impact assessments and consultation with the communities affected is totally unacceptable. Those closures will see people who rely on job centre services travelling further, incurring increased transport costs and phasing increased risk of benefit sanctions. It is essential that the UK Government reconsider that approach. The minister for employability and training has raised those concerns directly with the UK Government, seeking urgent clarification of the impact on people who use job centres and the staff who work in those vital services. Bob Doris. I thank the First Minister for that answer. In a recent debate that I brought to this session, there was strong cross-party support to save my local job centre in Mary Hill and others right across Glasgow. The Conservatives, however, refused to condone or condemn the eight closures. Given that there are now 23 closures nationwide with one of them in Ruth Davidson's own Edinburgh constituency, does the First Minister agree that it is time for all MSPs in this place, including Ruth Davidson, to put their constituents first and demand that the DWP halt the closure of all 23 job centre plus offices until there is full and meaningful consultation with the communities and staff-affected, as well as ensuring that the quality impact assessments are carried out such as to defend our constituents? Yes, I agree that it is important for all MSPs across the chamber to unite, to urge the UK Government to reconsider its approach to the proposed programme of closures, and to consult all of the Scottish communities affected by the closure of what are essential local services. The Scottish Government is taking a lead on that. I know that Bob Doris and others have played a key role in opposing the closures in Glasgow, for which there is now significant cross-party support against those closures. It is unfortunate that Tory MSPs are declining to stand up and be counted on the issue that is so important to Glasgow and for other parts of Scotland. It is equally unfortunate that Ruth Davidson herself is declined to stand up for vulnerable people in her constituency who might be affected by those closures. It is time for all of us across the chamber to say to the UK Government that those closures are wrong, that they will harm vulnerable people and that those proposals must be urgently reconsidered. To ask the First Minister what the Scottish Government's response is to the auditor general's comments regarding Police Scotland and that the lack of progress that has been made in demonstrating financial leadership is unacceptable for any public body. The auditor general has signed off the Scottish Police Authority's 2015-16 accounts unqualified. As I set out in December, I agree with the auditor general's conclusion on the 15-16 audit that the Scottish Police Authority and Police Scotland have begun to take steps to improve both financial leadership and management and governance arrangements, but those have not yet had a chance to have an impact. Those steps, of course, include appointing a director of corporate services, strategy and change and an interim chief financial officer to provide strategic leadership and direction on all financial matters, and this interim post will soon be filled permanently. Douglas Ross. I thank the First Minister for that response. Just five months ago, she said and I quote, I don't think that single force is in crisis. In response to the auditor general, seven days ago, senior SNP MSP Alec Neill said and I quote, the organisation is in crisis. Which SNP politician should we believe? First Minister. Well, our police service is not in crisis on the contrary. Our police service is doing a fantastic job, the length and breadth of this country, and I think that it is incumbent on all of us to get behind them. It is a sign of the importance this Government attaches to the work of the police, that the draft budget did protect in real terms the police budget, and over the life of this Parliament that will mean £100 million more going into front-line services. Of course, Derek Mackay will present any changes to that later this afternoon. We will continue to support the fantastic work of our police officers, because they do an essential job in keeping each and every one of us safe. Question 7, Anas Sarwar. To ask the First Minister how many refugees the Scottish Government expects to welcome in 2017. Scotland has already received around 1,300 refugees under the Syrian Resettlement programme since October 2015, and refugees continue to arrive. The arrival of refugees is dependent on many factors, including assessment and screening by the UN Refugee Agency and the Home Office, the matching of refugees with accommodation and services that meet their needs, and other logistical matters such as the arrangement of flights, travel documents and visas. It is for those reasons that I cannot give a figure for the exact number of people who will arrive this year. However, I can say that Scotland will continue to be a country that welcomes those seeking refuge from war and persecution. We are committed to welcoming as many as we can of those who arrive in the UK in 2017. Anas Sarwar. Presiding Officer, in a little under two weeks, President Trump has defended torture. He has banned US aid to health providers, providing care for women in developing countries. He has insulted the Jewish community on Holocaust Memorial Day. He has imposed a ban on Muslims from seven countries from entering the US and has imposed an outright ban on Syrian refugees and, at the same time, has held hands with Theresa May. Many of those actions are designed to incite hatred and create division. Will the First Minister draw me in saying that, while we cannot be complacent about acts of hatred and prejudice, we should recognise the 1.8 million people across the UK who have signed a petition to withdraw the red carpet from President Trump and paid tribute to all those people right across the world, irrespective of their faith, their colour or their nationality, who have joined together to say that we accept hatred and recognise that we support humanity in all its forms? I do, in Dorsal's comments. I disagree deeply and profoundly with the executive orders issued by President Trump last week, banning Syrian refugees and imposing a travel ban on people from seven Muslim-majority countries across the world. Banning people or even giving the perception that people are being banned on the basis of their faith, religion or origin is profoundly and, in my view, morally wrong, and I think that all of us should stand up and say that. I have already made my views clear about how inappropriate it would be to allow a state visit to proceed while those bans are in place, and I hope that the UK Government will think again on that. Indeed, I had the opportunity to express those views directly to the Prime Minister when I met her in Cardiff on Monday. The last thing that I would say is that people around the world have expressed horror at those policies, and we all have a duty on matters as fundamentally important as those to speak out, to speak up and to oppose where that is necessary. However, I think that we are under a duty to do more than that. All of us have to lead by example around the kind of world that we want to live in. Scotland is a relatively small country, but I think that in the action that we have already taken on welcoming Syria refugees, the action that we are determined to continue to take to give refuge to those fleeing from war or persecution, we can demonstrate through our actions the kind of world that we want to live in. So, yes, let's oppose, but let's also lead by example, and I want Scotland always to do that. Question 8, Donald Cameron. To ask the First Minister what action the Scottish Government is taking to support people who have been newly diagnosed with dementia. First Minister. We will soon publish our new dementia strategy, which will outline a range of actions that we will be taking to help to further improve the planning and delivery of dementia care services. We are also working with health boards to continue to improve access for people with a new diagnosis of dementia to post-diagnostic support from an appropriately qualified link worker. The First Minister may be aware that recent figures show that out of all those newly diagnosed with dementia in 2014-15, only two in five patients received 12 months of post-diagnostic support. Given that the target set out in the local delivery plan states that all people newly diagnosed with dementia should receive such support by 2015-16, does she accept that her Government hasn't done nearly enough to ensure that this crucial target has been met? First Minister. Well, I do agree that we have much more to do in terms of some of the commitments we give around diagnosis generally but also post-diagnostic support. We are well ahead of most other countries anywhere in the world and it is important to recognise that. Those who are now getting guaranteed post-diagnostic support would not have been getting it at all unless we had set a very clear commitment around that. I was health secretary when we set that commitment, so I know exactly how important that is. The figures that the member has cited do underline the fact that we have more to do. It made similar comments earlier on in relation to mental health. We know that, with the changing demographics in our society, more and more people are going to be living longer, which is a good thing, but that means more and more people are going to be living with dementia. That has implications for all aspects of our society and it is absolutely vital that we get it right and that is exactly what this Government is determined to do. Linda Fabiani. I ask the First Minister if she recognises the very particular issues that come to a family where there is a diagnosis of early onset dementia. Sometimes there are still children in the home and people can be of working age still working. It has very particular difficulties. Can she let me know whether there are plans to improve data collection on this so that provision can be made and a recognition across all departments of government that, in those particular cases, very special attention has to be paid? First Minister. I think that Linda Fabiani is absolutely right to raise the issue of early onset dementia. The diagnosis of dementia for anybody at any stage in their life is a devastating one, but there are particular issues that are raised around those who are diagnosed with dementia at a younger age implications, or even greater implications for family, for example. Data is important here. Last year, Health Scotland published dementia and equality with recommendations on improving services for under-65s, including increasing workforce knowledge, better information for employers and more age-appropriate services. We will continue to consider the report's recommendations as part of the next dementia strategy. We are also taking action for people under-65. The post-diagnostic services focus on key areas such as ensuring that social networks are sustained as far as possible, signposting to age-appropriate peer support and helping with some of the financial issues that can impact on this particular care group. All of that is important, but Linda Fabiani is right also to talk about the importance of data so that we know exactly the challenge that we are dealing with and how best to do that. Last week's figures on post-diagnostic support for those newly diagnosed with dementia show that there is a huge gap between the Scottish Government's pledge of support and the real experiences of people living with dementia. First Minister, given that health and social care partnerships are already struggling to meet the Government's guarantee and partnerships will have to make tens of millions of pounds of further cuts if the Government's draft budget is agreed, where exactly will the additional funding come from to deliver the guarantee that everyone with a new diagnosis of dementia in Scotland will receive a minimum of one-year post-diagnostic support? As well as the above-inflation increase that we are committed to delivering for the national health services, the member will be aware. We are also committed to ensuring that money goes from the health service into social care, given the importance of the integrated service that he talks about. Last year, that was £250 million. We are adding an additional £107 million to that this year. That is part of the funding commitment that will help to ensure that services such as those services can be delivered. I said that in part in a previous answer. That commitment is really important. It is a commitment that does not exist in many other countries. We were one of the first countries to give that commitment to post-diagnostic support. We have made progress in delivering that, but we have more progress to make. I think that it is important that we do not shy away from giving ground-breaking commitments because it may be difficult to deliver them. Rather, we were working towards that, as we are doing with the funding and the strategy that is necessary to deliver it, and that is what we will keep very focused on. That concludes First Minister's questions. We will now turn to the member's business in the name of Liam Kerr. We will take a few seconds to change seats.


Hello students. In the last lecture, we have had a lab demonstration on how to screen the presence of autoantibodies in the bio fluids of cancer patients using microarray. In today's lecture, we will discuss about various parameter that should be considered during image scanning and processing in microarray workflow. Detection probes have been briefly discussed in our previous lectures. These probes are major determinants of finding obtained from any microarray experiment. Detection strategies for quantifying signals from these probes in microarrays have been categorized into two basic categories, the label-based and label-free methods. The advancement in confocal laser scanner technology has led to the extensive development of protein microarray technology. As a result, protein microarray detection system widely uses label-based methods. Scanners capture the fluorescent label signal using cold charge couple device or CCTV cameras. Some scanners also employ lasers for excitation and a photo multiplier tube, BMT detector. In other cases, LED is used for a uniform light source and is detected using a cold CCD in combination. Today, we have with us Mr. Pankaj Khanna, who will be talking to us about GenePix microarray scanner. He will first discuss about microarray scanner as there is a software which controls this and will further discuss about the data acquisition parameters that should be implemented to acquire the data from the process microarray chips. So, let us welcome Mr. Pankaj Khanna for today's lecture. In the form of hardware, as we have discussed, there are a number of different hardware possibilities. In the form of GenePix right from 4000B to 4003-4400, these are actually doing the hardware part which allows the lasers to scan the chip. So, once we are ready with the chip, so it will be an interface between the CPU and the system which is allowing the data acquisition to happen as GenePix is being attached with a software called GenePix Pro software, which helps in understanding how a data is being acquired with the hardware usage. So, basically another software which is also used is Acuity, which is actually a third level statistical analysis software. So, in brief the hardware is being attached with a data acquiring software called GenePix Pro and this GenePix Pro software is now give the results to Acuity which can be further analyzed for statistical analysis. GenePix microarray as such is based on two special designs. One of them is a non-confocal design of hardware and second is inverted chemistry. Non-confocal design is like where you have spots in the same plane where the scanning is done is called as a 2D dimensional. Say when you go for a tissue arrays or analysis where it is very very higher, so in the form of spot size especially at the level of imaging of scanning images. So, there at the level of microscopy people use a technology of confocal microscopy so that they focus the laser at different wavelengths. So, at the end of the day they build a 3D image, but usually for all microarrays which is the highest is the tissue microarrays it is shown that all the slides are in the same plane which is why it is non-confocal chemistry which just resides in the same plane of the slide. So, that is why we choose non-confocal chemistry attached with the inverted chemistry of scanning which helps in the best results in the form of signal to noise ratio. So, as we discussed non-confocal coupled with the inverted scanning how does it help ease? Usually we see in the glass slides there are lot of small deformities which our eyes cannot see. So, what we do is we invert the slide so that it goes to the level of the same scanning and they are supported at the level of the edges with the controllers which see the deformities at the glasses. So, what it helps is it helps in looking at the deformities and directly allow the scanning to happen on the phase of the scanning area. So, what happens is because of the confocal design attached with this inverted chemistry we get best signal to noise ratio so that your data is more valid for any analysis. So, classically microarrays are being used for the differential gene expression which usually people categorize in the gene expression format. So, later as the development has happened people went for SNP arrays as well as a CGS which is called as comparative genome hybridizations. They are used to look at the chromosomes apart from that protein arrays chip on chip to look for the control genes they are also being focused and the protein arrays are now really catching up in many sense because the protein is actually the biological functions what many scientists believe and I am also a strong believer. So, now people are moving from the DNA to the real functional part how they are directly affected. The major bottleneck was looking at the antigen antibody reactions like the proteins are all being seen there is a no general formula, but now the field is catching up. So, even at the level of protein arrays this is compatible the incoming tissue arrays which is really in infancy is also now being compatible with gene peaks problem. So, our scanners ranges from 5 micron resolution to 2.5 micron resolution. The basic thumb rule for all the resolution is that amount of your size of your spot be it protein or RNA that should be 10 times that of the resolution what you are scanning with. So, interestingly most of the DNA is less than 50 microns and most of the protein usually stands at 200 microns. So, essentially the rule goes that 10 is to 1 if you say 10 is a resolution and 1 is the spot size. So, that is a major rule. So, just to give you a brief out say for example, if your spot of protein is 100 microns in size. So, what best if you do at 10 microns basically you are getting 78 pixels. So, this is actually 1 by 10 if you do by 5 micron resolution all of the pixelation will increase to 314, but the size of the image actually also increases. So, apart from making more than 10 times the size of spot it does not really help at the level of resolution if you increase the only thing is you are increasing the size, but the data quality is just increased by few very very minor changes. So, that is why the best rule is 10 is to 1 which 5 to 2.5 micron usually is most suitable where the people are making the slides. Essentially because they are all not present. So, the data could be right from the 0 intensity which will be equivalent to the background to the very highest intensity. So, best way to control the variations is looking at a photomultiplier tube actually that is nothing, but once you do a fluorescence. So, the intensity is coming out of that. So, the very low expressing genes or the proteins will give you very low fluorescence intensity whereas, the high will give very high. Yes, I will just interrupt you here. When people scan these micro-race slides it always ask for the PMT gain. So, I think that is what you are talking here photomultiplier tube and the PMT gain. Yes. So, just maybe you can clarify here like how one can adjust that parameter of PMT gain. So, the basic thumb rule there is in any scanning you do not want to see a wide spot. The wide spot essentially means the saturation and to avoid saturation exceptional cases for few controls people avoid that happening. So, the best way is to look at the photomultiplier tube condition. So, it ranges actually from something like 300 to 700 which helps one control how much photon multiplication can be done. So, the lowest can also be seen and highest does not become saturated. So, this is how the photomultiplier tube is very very essential to control the different kind of variability within the chip which happens due to the biology. So, the major variations actually come owing to the different sources one could be technical one could be biological and another one is owing to the essay or the chemistry. So, the technical ones in the form of hardware can be controlled at the level of PMTs and when you are looking at signal to noise ratio the small play of PMT and very less with the help of laser power and then with our own design of inverted scanning as well as the non-colonial chemistry we are able to achieve the best. So, in short if by visibility you may not be able to look like what is happening to the images. So, in the form there here in the image which is being shown the which which looks brighter to the eye may not be a true sense. So, when you look at even the del there the background is very low without looking at numbers if I see them I said this is del that is bright, but that is not true always. Because when you calculate reality based on what you have controlled at the level of PMTs and others you quickly come to a conclusion that signal to noise ratio can be seen by eyes at the level of PMT which the laser balance can be seen. So, in this fashion while you are scanning a slide you can always look at that. So, as as you go for the signal corrections. So, basically what you are trying to see same tunnels of not looking saturations and then you expect the image is going to be with least background. So, the valid information which is coming in the form of intensities become a true signal. Basically if you scan once. So, image is being collected. So, if you want to have multiple scannings being done. So, that that all intensities can be averaged upon and get to a signal value it is being seen that when you increase the number of scan usually people prefer 2 to 3. So, then average them. So, this helps in reducing the signal to noise ratio in a fashion that you get a real good intensity coming up. So, the basic concepts in the form of hardware design the basic one is non-confocal optics and another one is the focal depth. The meaning of focal depth is where our laser can focus in the form where it can get or acquire the data and the depth is 64 microns which allows a wide variety of applications possible. So, if you want to use some slide based slides that is having a cover slips. So, that the depth is little bit you know kind of lower. So, you want to focus at a different range. So, in this fashion the non-confocal optics with a focal depth of 64 is our major feature apart from that up down upside down that is inverted chemistry is actually what we are going to use in the form of printed slides being scanned directly and controlled for the variability I have discussed earlier and the resolution at 2.5 or 5 microns based on the system which is available can be also used. And real time PMT adjustment PMT we have already elaborated the photo multiplier tube and the real time is at the level of scanning we can look at the data when the scanning is live. So, in this form we can control the PMT live in between the scan and then auto PMT adjustment for those who are not real burst of looking at how to control them we have given auto PMT option. This auto PMT option allows acuity to get the best output, but GenePix Pro allows the multiple scan by itself. So, the best PMT suitable for one application can then be seen immediately. GenePix Classic 4000B is having 6.5 minutes of simultaneous scan. The meaning of simultaneous scan is both laser scan at the same time. So, you have very less time for scanning apart from that it has got two lasers actually 635 and 532 nanometers which is classically used for sci-3 and sci-fi and they are on compatible dice. So, in view of this we have standard green and standard red emission filters to accommodate all sci-3 and sci-fi applications. The laser power can also be adjusted in the form of 100 percent to 33 percent to 10 percent. So, based on what application and what intensity you want best the laser power can be adjusted as well. So, adjusted focal position is from minus 50 to 200 microns. So, this allows you to focus in a different ways. So, we can have a different slice compatibility coming up with the scanners 5 micron resolution maximum it allows to go for and it can go up to 100 microns of resolution. So, non-confocal again it is using the non-confocal optical design and any standard slide can be used for the scan. So, it gives a wide applications possible in that. As we discussed in brief like the GenePix Pro software we are going to use for data acquisition. So, basically they are different steps involved what in the usual process of bioinformatics the first one comes is imaging. So, imaging is being done in the form of multi image or single image TIF format. So, we use TIF format to create the images form and export these color images in 24 bit JPEG formats. So, these JPEG formats allow one to only see, but the basic data processing will be done on TIF format. So, once GenePix Pro is being allowed in a different sense of hardware controls then image acquisition can be done using the number of lasers availability it ranges from 2 to 4 and many times your application use one laser as well. So, based on the channel type laser type you select how many to select for again on your chemistry basis and this third party image alignment and manual automatically being done. So, what this mean is each particular array has got different blocks which in turn contains the features. So, these features are actual genes or the proteins or the representative of the biological material what you are checking now these have to be aligned with the annotation information. So, this can be done by a third party and the GenePix Pro is doing by itself in the form of automation to do the job. So, automated block and feature alignment is possible even the different sizes and shapes of the spot can be handled right from circular square to irregular features which can be handled at the level of image alignments. So, in any micro array after the image first thing comes is the alignment which we have just covered in the form of data acquisition because there is a very important part to cover and apart from that you have a background subtraction so that you get a true signal coming in. So, GenePix Pro does help in the background subtractions in a different format and also in the normalization of the features. So, if you see the background subtraction can be done in the form of local global and negative or morphological control. So, this negative and morphological controls are subjected to the design of the slide type say negative controls in your slide there should be some spots we should not bind to anything and leave blank and whereas the local and global can be calculated in a general space where there are no spots available and the area which is not being spotted nearby your particular feature. So, in this fashion background subtraction can happen and then feature viewer and feature pixel plot. So, basically the major thing comes in the acquisition is after acquisition is visualization of the data and this visualization comes in the form of pixels and the plots the graphs. So, graphs help us in understanding globally what is happening in short. So, it gives you a real image how the things are happening. So, this can also be done and there are multiple ways of calculating in the form of ratio calculations after normalization or during the normalization of the data. So, analysis immediately after those involve few of the normalizations process which GenePix Pro can very well handle and other important feature is the flagging of the spot in a biology we see some spots are really not good or because of some artifact they are not supposed to be taken for analysis. So, essentially we need to make that spot as flag as good bad or absent. So, these can also be done with GenePix Pro software lastly normalization of the images and the different formats is also allowed to happen in GenePix Pro. So, once the image is being acquired. So, immediately scientist wants how my data has per. Especially few people use even the bacterial controls which is non related to the biology as such totally they want to see just the essay control. So, first they say whether my essay has worked or not. So, best way to do for them is looking at the graphs which are being given differentiation in the level of backgrounds and though also can be done at the level of different histograms and the scatter plots possible. And these scatter plots can be plotted once against the channel type say laser 1 versus laser 2 or wavelength versus wavelength 2 in classical say psi 3 versus psi 5. So, how these two things have behaved for me. So, again different kind of grabs and so also the images can also be exported to PDF as well as being visualized in GenePix Pro for your further screening for different QC applications. The basic GUI which we are going to see in few minutes actually contains three different areas. The first one is image laser controls. So, there you wanted to see which kind of laser I am going to use and second one is a different features which are used for the controlling of the image and towards the right hand side we have a pane which is allowed or helpful in hardware controls. So, the basic one is in the hardware control is first as we said that we can look at auto PMT and other adjustments which is being done for that we use preview scan. So, preview scan and that they are different tabs which allows you to go for the true scan and the preview scan and based on the laser which particular scan you are using. So, like for example, if you start with the preview scan you decide on which best pixelation suits me which different power of laser suits me. Once you are able to do these decisions being made you can go for your own data scan people prefer that because they are sometimes a bleaching effect on the flow of flows. So, they want to avoid the exposure for a longer time. So, multiple scanning is avoided. So, once you see even a single wavelength scan. So, if your application requires only one wavelength to be scanned there you can select in the tab only a single wavelength scan and then. So, also a preview scan with the data scan followed by you can also do an automation that once you do a preview scan you see that it is all good then immediately it can go for that, but very rarely people use that because you see once and then only you want. There is a button towards the side where you have a having a control for the scanning time. So, as it is a dual channel that means the two lasers are present in that it is allowed to select whether you want to use one and two. And then based on one or the user application you select both the lasers and then at the level of fly scan you control for the PMT and the MIGFAR resolution you want to use for. So, these are within the same software towards the right hand side you see the pain where you have a hardware control button there you can also look for all these different images which is being now suited for your own biological application. So, basically in the preview scan when you are scanning your live data you can just switch on to the histogram graph right there what it gives is how much red and green channels are contributing towards the intensities. So, you really want that they are overlapping. So, they are really balancing there could be a small variability in the beginning going to the fact that they are just a background and then the spots coming in. So, there you want that they are really overlapping after little bit of lag that is few seconds of lag that is it. So, once you are able to do them you can see and select whether yes this PMT is being good for me. So, this is a way you check which PMT is more suitable. So, you select the PMT look at them see the overlap wherever there is a best overlap without saturation you want to go over those settings. So, what you see now is a GUI or graphical user interface of GenePix Pro software. On top it is in the form of different tab buttons which allows you in a different work group. Say for example image allows you in different ways and controlling of the image acquisition and histogram looks at how that image has performed. So, this is what we were speaking about in the earlier slide where you can see a life kind of term which is happening. And then lab book actually gives what all you have done in a different step wise. So, what is being every movement of yours in this particular software is being logged in and analysis can be done in the form of batch form which allows multiple slides. So, that you can do alignment and the analysis which can be performed with the batch analysis. And once the analysis is over results can be seen and scatter plot can be now plotted at the level of this graphical user interface. Once you are through you can look at the reports as well. So, let us look at the major function of imaging. So, what and how one controls for the best image acquisition. Let us quickly go through like different kind of buttons here. So, now the imaging can be done at a different wavelengths and the like preview can be done at 635 and 532. In a single laser based the wavelength can be done at 635 or wavelength at 532. So, even the ratio of the imaging how the both has performed together can be looked at looking at a button of ratio of imaging. So, this one allows you to see how the image is being done after the scan. You can look at one channel preview channel or different channels. And now let us look at different tools which are available to you while or after the scanning. So, the major ones are here where you can move across the chips in the form of this hand tool. The plus indicates the zoom tool and the other tools are actually this is one to unzoom and you can also look at the whole image button. So, once you have the image these two becomes activated. So, these two are actually for the blocks and looking and the controlling the blocks and these ones are the features. So, many a times what happens usually you get the GAL file which is actually the feature information file. So, GAL stands for gene array list. So, actually it gives the x and y coordinates where each array usually is being presented in the form of blocks which blocks in turn are in the form of features. So, these blocks and feature positions are being recorded in GAL file and then the information or annotation is given to each part. So, GAL GL that is gene array list file essentially contains the x, y and the number of columns and so also the information of each part how they are being annotated and placed on the chip. So, if by chance if you do not know or you are prepared by yourself these buttons here allow to make your own blocks and we create your own GAL file with the help of the tool which is called as gene array list generator. So, now let us look at the control button which is towards your right side. So, the first one is a preview scan and then you can also have a data scan one stands for one wavelength. So, it allows you to take image from only one wavelength and then you also have a multiple scan. So, you do a preview scan then you do a scan with this button. You also have other buttons which will light up as and when you acquire the image and this is for the analysis. So, the once the analysis is being done if you click this button the analysis will be performed after the alignment. So, this is actually open button. So, this is like normally your file where you want to open or save your images and this one is actually a flag. As we discussed the different features can be flagged you can look at once the image is available to you you can look at good bad or absent and you can give them a different ratings. Here again is looking at different zoom buttons. So, which allows you that which we want to focus on feature names or the feature IDs where you want to go for a one particular one. The major one here is particularly this which allows a different workflow controls right. So, this allows either for a different ways can be had. Now, let us quickly go through a one particular scan which is a simultaneous simultaneous scan. So, both users will be acquired at the same time. So, if I press on a data scan button the image the after putting your inverted slides in the hardware it is scanning. So, you just see on the top which is very less visible. So, let us try to zoom inside. So, if I just put this button and allow you to zoom you can see particular how the scanning is happening. So, you are looking at different image type. So, if I click on only one wavelength this because it is live after the scanning you can see it is going for the ratio image scan. So, now quickly at the histogram you see it is start coming up because it is scanning is going on live. So, it is start reducing the basically as we discussed it should be overlapping. So, my settings are usually looking very nice in this particular one. So, in this fashion the image acquisition is being happening right and you also have you we give you a power that in between the slides usually people keep barcodes. And our system or GenePix Pro is compatible with reading the internal barcodes which is being done. So, so that you can have multiple scans also being possible. So, nowadays each slide is coming with multiple arrays because of the variable densities people are focusing on the custom type. So, this can also be done with the new software upgrade developments. So, now as the scanning is being performed let us look like I will save the image and once I save the image I would like to see how the different processes is being done. So, say for example I have saved this image in the form of say this is the east. So, I just want to open an image which I have just saved. So, so basically as we discussed each particular array can be divided into the blocks. So, this particular array of east contains four blocks and each block is having features. So, number of feature information is given in GAL. So, basically terminology is array block and features. So, I need to align that GAL information of positions on top of this. So, I have to put a GAL file and do by further analysis. So, what I am going to do now is open a GAL or GAL file which allows me for alignments. So, best feature of GenePix Pro is its capability of identifying feature by itself it is literally comes in. So, let us just see how the zoom button looks like. So, only thing you have to do here is just take your block and allow it to move to the first alignment and then what you can do is click the button over here which is for the align. Align can be done in different ways I recommend to use the first which finds all features all blocks and do automated fashion. So, if you click once you see software automatically finds all its features wherever by chance the features are absent or there are some physical deformity it say it is not present or it flag it as bad. So, as you see you can actually move it, but it does not affect life as long as you have just kept once and the data is being stored. But usually people ask me is it good idea if I by chance move to do once again. So, it is not a bad idea because it takes few seconds to do it. So, once you have done this particular alignment let us look these two slide which I said you can zoom out. So, you can see whole particular slide now is being scanned and aligned as well. So, it is a very quick process which software performs very easily for doing the job and once you have done this you can always hit a button of results. Now, if I go to results actually this is empty. So, if I click on results results are being calculated and there are some 40 different columns which will be output in a form which GenePix Pro understands different ways. So, just quickly looking at the major ones the major ones here are looking at this F means the intensity from different channels 635 or 532. And this background calculation is being done accordingly in the same laser range. So, once you do a corrections what happens is you want to correct your intensity mean values with the values of the background. So, this is what is the most important which usually people use for the further calculation apart from a ratio of means or ratio of medians which can be calculated again and being presented to you in a different column formats. So, each column signifies different ones like for example, SD standard deviation, CV coefficient of variations and then different channels coming up. So, in this fashion the results will be outputted if you are image acquisition first controlling the part then allowing you to align and then do the analysis. So, this is a basic steps which anybody or everybody want to do in micro steps. So, once you see the images that the people end up in the form of results and you have different columns available to you. So, there are different ways people want to visualize how my column because number make very less sense. So, the best way to look is a scatter plot. Scatter plot allows you in a different ways what you are plotting at x and y accesses and here if you see I am just plotting actually towards f 60 35 median over the f 6 35 median. So, you are comparing two different channels how they have behaved. So, essential rule is they should mostly the micro assumes all the chips are having the spots and which are genes which are not varying too much. So, you expect most of them to stand nearby to the origin of the center. So, this is what you want to look at. How close is their align? Yes, they should be not too far away from each other. So, that they are not naive actually to each other. Because you expect there are few differences, but not very significant which can be seen at very large scales. So, GenePix Pro as we discussed in acquisition software and the molecular devices recommend activity software for further data analysis which can be at the level of secondary or tertiary based on that. So, you do statistics as well as visualizations on a single or multiple data to handle that. Molecular devices introduces the world simplest most reliable automatic microarray slide scanner. Now you can walk away from scanning while the GenePix auto loader 4200 AL automatically loads, scans, analyzes and saves results for up to 36 slides. The auto loader accommodates microarrays on standard glass microscope slides labeled with up to four fluorescent dyes. These microarrays can contain just a few hundred spots or tens of thousands of spots representing an entire genome. As many as 36 slides can be loaded into the convenient slide carrier. As the carrier is inserted into the scanner sensors detect the location of each slide indicated by a blue bar on the slide carrier map. On the batch scan tab in GenePix Pro you have complete flexibility to define the most appropriate settings and analysis parameters for each slide or for groups of slides. You can also choose to automate scanning analysis and file saving steps. Enter an email address and GenePix Pro will notify you remotely when your batch is complete. Using the defined scanning parameters the precision robot arm leaps into action and moves to the first slide. Our unique never let go whippers securely clamp the slide and carry it to the scanning area. A barcode reader records the barcode and then the slide is positioned for scanning. The GenePix auto loader 4200 AL can be configured with up to four lasers. A neutral density filter wheel can be used to attenuate the laser power if necessary for especially bright samples. The laser excitation beam is delivered to the surface of the microarray slide. The beam scans rapidly across the short axis of the slide as the robot arm moves the slide more slowly down the long axis. Fluorescent signal emitted from the sample is collected by a photomultiplier tube. As the scan proceeds sensors detect any non- uniformity in the slide surface and the robotic arm adjusts the slide position accordingly to ensure the array surface is always in perfect focus. Each channel is scanned sequentially and the developing images are displayed on the monitor. The multi-channel TIF images are saved automatically according to file naming conventions specified by the user. After the slide has been scanned the precision robot arm replaces it safely in the slide carrier before picking up the next slide. As each slide is scanned a list of each saved image with its associated settings and analysis files accumulates in the batch analysis tab until the batch is complete. GenePix Pro automatically finds the spots and calculates up to 108 different measurements for each spot. The results are saved as a GenePix results or GPR file. GPR files can be saved automatically to the acuity database for statistical analysis, clustering and other advanced investigations. So, I hope in today's lecture and demonstration for various parameters you are now familiar with the basics of scanning parameters, image processing and data acquisition. In a protein micro experiment intensity of a spot is a representative of the interaction between the sample and analyte. To achieve this target proficiency in image processing and data acquisition is required. As discussed the artifacts due to the contaminants such as dust particle or even very high background on the chip these issues make automation in image processing and data acquisition very challenging. Researchers have devised several segmentation algorithms to reduce the manual interventions. However you have seen there is a degree manual flagging is necessary to mark the low quality spots. This data would now be pre-processed for background correction and normalization. You have also seen the demonstration of one such software GenePix Pro micro array image analysis software for data acquisition and you will further see how to play used for data analysis in subsequent lecture. There are several commercial softwares like proto array prospective software and many others as well as there are several open source software. I recommend you to play with many softwares and try to explore the features. While the basic steps remains the same many softwares offer some very specialized features which also includes sometime the data visualization, data plotting and a better and effective ways of data representation. In our next lecture we will continue our discussion on micro array experiment workflow and how to analyze the micro array data obtained from the images generated from today's lecture. Thank you.