Interview

Transcript

Society Is microscopy dangerous or immoral? You might get this impression with respect to the criticism professor Morten Laane got last year. Norway’s leading expert on microscopy (b. 1940) was, in the media, even accused of presenting artifacts and trash science when revealing his research done together with the zoologist Ivar Mysterud in their search for answers to what is causing chronic disease in humans after tick bites. Text Iver Mysterud Photo Geir Holm og Shutterstock Story at-a-glance ✓ The retired biologists Morten Laane and Ivar Mysterud were subject to heated debates in the Norwegian media last year due to their use of microscopy as a tool to examine blood from people who have become chronically ill following tick bites. ✓ In this interview Morten Laane deepens central aspects around microscopy. In the previous interview Ivar Mysterud presents his view of the criticism. ✓ The microscopy methods used have deep historical roots, however, are now considerably improved for detailed observations and digital recordings of bacteria and parasites which reside in red blood cells. ✓ Everyone can easily look through a microscope; however, it demands great and time-consuming work and experience to become decent at microscopy. An understanding of the mechanisms of the microscope is required, and the person executing the microscopy needs to possess interdisciplinary experience when interpreting what is observed in the specimens. ✓ Studying the presence of microorganisms in blood is thus demanding. ✓ In comparison to other test methods, microscopy shows the immediate situation in a specimen. ✓ Laane rejects the criticism that they have not observed real spirochetes (spiral-formed bacteria) and co-infections in the blood of patients sick after tick bites. The structures are not protein fragments from blood cells during decomposition, by some called pseudo spirochetes. He has techniques to distinguish beyond any doubt the difference between spirochetes and pseudo spirochetes and has filmed spirochetes that actively move in and out of red blood cells. Laane also finds it incorrect to call anything pseudo spirochetes and he challenges the argument from opponents suggesting that general basic concepts of blood microbiology may be wrong. ✓ Laane is equally strong in his persuasion that microscopy is an invaluable tool for studying blood of people who have become chronically sick after tick bites. The results from a new, ongoing research project might indeed become useful for this group of patients. 78 l Helsemagasinet VOF l  SEPTEMBER 2014 In spite of professional criticism, Laane is equally strong in his persuasion that microscopy is an invaluable tool for studying blood of chronically sick people. This can provide a good indication of what is really wrong with them. It seems like microscopy as a method is controversial in the eyes of some professionals. What is the reason for this? – Lack of knowledge and loss of microbiological knowledge, Laane answers. He explains that the first bacteria were discovered in the 17th century by Antonie van Leeuwenhoek (1632–1723). Among lots of studies a “tooth spirochete” from a biofilm in the oral cavity is described and illustrated in his book Archana naturae detecta, published in 1695. It is a collection of scientific letters to the Royal Society of London. Laane emphasizes that this meant approval at that time. – Physicians who make statements about microscopy should, at least, be able to repeat what van Leeuwenhoek did, even if it is intricate, Laane thinks. The method you have used to identify the presence of potential Borrelia spirochetes is often presented as new. Why do you disagree with this? – Nothing is basically new. The methods we are currently using are based on insights in microscopy that gradually have been developed throughout history, Laane answers. – However, the techniques that Morten Laane (left) and Ivar Mysterud study both pictures and film from microscopy on the screen. for a long time have been used for observing spirochetes have now been greatly improved. It is important to create conditions with reduced oxygen levels in the living blood specimens. The reduction of oxygen content seems to stimulate inactive, hidden bacteria and parasites to move out of the red blood cells and become more easily observed and imaged, the researcher continues.1 Is it easy to do microscopy? – Everyone can easily look through a microscope. However, it demands a great extent of work and long experience to become good. Only experience will give a good understanding of what can be observed. – Thus it is time consuming, Laane points out. – Modern DNA analyses usually work in a way that the specimen is sent to a special laboratory and the sequences are then returned. Thereafter you search on a computer. This is to a great extent less time consuming than working with a microscope and becoming good at it. Perhaps this is partly the reason why microscopy is not widespread and is less popular today, Laane wonders. You have for decades lectured students in the use of the microscope. What is the main emphasis you have exhorted them with when they use the microscope? – A person has to understand how the microscope works, an essential “quality” in order to be able to use it well. Practice and experience in interpreting what you observe in specimens is crucial, and frequent use is necessary to become good, Laane answers. – It is a pity that education in traditional microscopy generally is reduced both in schools, universities and colleges. Highly specialized, so-called confocal microscopes are popular and can be linked to advanced molecular techniques; however, you lose a great deal which could have been detected by much simpler microscopes. We need both English translation of article in Helsemagasinet VOF no. 6/2014 This is a translation of an interview published in September 2014 in Helsemagasinet vitenskap & fornuft (VOF; see www.vof.no) no. 6/2014 on pages 78–81. It directly follows an interview with Ivar Mysterud on pages 74–77 in the same issue. It is translated by Iver Mysterud and slightly edited in cooperation with Morten Laane to better fit an English-speaking audience. types as they complement each other, Laane says. Demanding to study blood The eager researcher explains that it is demanding to study blood. Blood is namely a very complex substance. – First of all around 40 possible cell types exist in blood, all of which you  www.vof.no l 79 Society need to be able to identify. For instance, if you want to detect a bacterium or parasite, you need to know something about “the background conditions” in blood, he emphasizes. Laane tells that one needs insight into what fibrin looks like, i.e. thin threads of protein in the blood, and changes in cell types and other structures in blood that can vary both under preparation and with disease. He further states that for optic reasons red blood cells normally hide both bacteria and parasites. They are small, biconcave lenses, causing optic disturbances. Such problems are solved through a careful swelling so that they become more flat. – The Borrelia bacteria at steep angles may become so long and thin that it is difficult to see them in a light microscope. By using another technique – dark field microscopy – you send the light in from the side, and bacteria and other structures lighten up against a black background. This complements what you see through a light microscope where the light is sent from below. The syphilis bacteria and Borrelia bacteria have morphological similarities of significance for simple detection. In this textbook effective and very simple microscopic techniques are described which later have become more or less abandoned in medicine. Laane’s father used these methods as a military field physician (officer) in the Norwegian army during the Second World War, and later when he resumed his private practice in Tønsberg. At that time Tønsberg was a significant seaport. Many needed to be tested because sailors brought contagion from abroad. In those days microscopic survey methods were common and accepted amongst Norwegian physicians. Laane’s interest for microscopy arose when he observed this at his father’s office in 1947, i.e. when he was around seven years old. At that time he also discovered Bruusgaard’s textbook. This background has since led to Microscopy being a large part of Laane’s professional career, as a biological researcher. Today he is internationally recognized as a highly respected expert in microscopy. Laane is also the author of four textbooks in microscopy, the microscope; however, instead observe What can microscopy contribute with latest from 2007.3 protein fragments from blood cells that other survey methods cannot? – You observe the situation immedi- Unfortunately formal mistakes were during decomposition or other artiately in a specimen. You do not need committed in the research application facts, by some called pseudo spirocomplicated instruments. It is depen- for his criticized research project. This chetes. What is your comment to such dent on understanding how the micro- is something Laane strongly regrets. arguments? scope works and you need the skill to However, there is no resignation to be – Protein fragments from blood cells prepare high quality specimens. In found in Laane. On the contrary, he during decomposition do not move in addition you need broad basic insights is more concerned than ever trying to and out of red blood cells in orderly in biology in order to interpret what get to the core of what is causing these patterns, Laane answers – the moveyou observe, Laane says. He is the first patients to be chronically sick. Laane ments we observe are typical for living to admit that other types of survey is disappointed with the scientific structures. – We study living organisms methods are equally important, for criticism which has emerged. – Why and have filmed spirochetes when they example identifying the species and haven’t any of the most ardent critics move out of and into red blood cells. strains of the bacteria being observed. confronted me directly to lead a fruit- The objection concerning pseudo spiroful discussion? Had they done that, chetes is misunderstood and irrelevant, they could have had the opportunity to he continues. Early influence Laane himself was exposed to micros- see for themselves what we have found copy early in life. His father was a through studies by microscope, he says How do you distinguish between spiphysician and used microscopy in his with slight resignation. – A scientific rochetes and pseudo spirochetes in the practice. Laane’s father studied medi- debate rarely benefits from extensive microscope? cine at the University of Oslo in the verbal abuse and criticism in the media, – It is actually incorrect calling something pseudo spirochetes. If you, by 1930s, followed the lectures of, among Laane adds. pseudo spirochetes, mean protein others, Professor E. Bruusgaard in fragments from blood cells during microbiology. One of the textbooks was False bacteria Bruusgaard’s Forelesninger over syfilis Some of your critics have objected decomposition, you should call them [=Lectures on syphilis] from 1930.2 that you do not see spirochetes in the that, Laane reasons. – The structures 80 l Helsemagasinet VOF l  SEPTEMBER 2014 actually demonstrate that they are bacteria, not protein fragments from red blood cells. Human red blood cells normally lack DNA (they do not have a cell nucleus), and green spots usually identify living bacteria, he says. Laane has never doubted their observations in the blood of chronically sick patients after tick bites: – It is without doubt spirochetes, and they are probably Borrelia bacteria since the patients have been bitten by ticks. However, the species we cannot know with certainty without using other survey techniques, because microscopy is not sufficient for this, he stresses. ”Old guys” can contribute are of varied nature and are connected to infectious processes and cell membrane events. The concept creates confusion and lack of understanding for very complex processes linked to red blood cells. Some “pseudo spirochetes” emerge because the bacteria have two membranes, and are able to shed the outer membrane. In the microscope this will be observed as a thin residual, and it often has chromosomes inside, detectable with specific stains. Thus, this has nothing to do with protein fragments from red blood cells, however, it involves parts of a spirochete and sometimes also the red blood cells, the experienced researcher explains. – In a microscope you can thus observe products from the cell membranes of the red blood cells. They often contain some DNA from bacteria that were inside the blood cell. Such products conduct so-called Brownian motions. It is possible to film what you observe in the microscope and filtrate Brownian motions. On the film you will then Laane is retired, however, still actively involved in research. After their previous project was closed by the order of the Norwegian Board of Health Supervision – due to the regrettable, formal mistakes in the project application – he and Ivar Mysterud have now started a new research project proposed and directed by the Norwegian Institute of Public Health – this time with all formalities in order. Their microscopic method is to be tested and compared see slow, clear spirochete movements, with other survey techniques. – I hope Laane says. that this research can become useful for the chronic sick patients struggling, as – Real spirochetes cannot just move out soon as possible, Laane says. of and into red blood cells, they can also move against the blood stream. One He adds that major medical advances can prepare such stream on a micro throughout history have been done by level in a blood specimen studied under non-physicians. One example is the microscope, and only living bacteria chemist and bacteriologist Louis Pasteur can move targeted, Laane emphasizes. (1822–1895), who still did important discoveries after he had suffered a stroke. Additionally, the researcher focuses – Also old guys can contribute, says on a certain simple staining technique, Morten Laane, and hope the critics will which is important for clarifying what calm down and look forward to seeing ”pseudo spirochetes” in reality are. – If the results of the new research project. you use the dye “acridine orange” correctly, it binds to nucleic acids (DNA, RNA), yet not to proteins. Single- Sources: Laane MM, Mysterud I. A simple method for stranded nucleic acid, i.e. RNA, emits 1. the detection of live Borrelia spirochetes in human orange-red light. A double-stranded blood using classical microscopy techniques. nucleic acid, DNA, emits green light, Biological and Biomedical Reports 2013; 3: 15-28. Laane explains. – Green dots are the http://www.biomedicalreports.org/index.php?jo bacteria’s (simplified) cell nucleus. urnal=bbr&page=article&op=view&path%5B% 5D=98 You can clearly observe this in a fluo- 2. Bruusgaard E. Forelesninger over syfilis. Oslo: rescence microscope. ”Pseudo spiro- Aeskulap forlag, 1930. chetes” turn out, by this technique, to 3. Laane MM, Lie T. Moderne mikroskopi med contain DNA within themselves, which enkle metoder. Oslo: Unipub, 2007.  www.vof.no l 81