Modern biomolecular microscopy
Gone are the days when the typical morphologistsought to understand the complex interior of the cellwith his magnifying glass while his colleague, pos-sibly even in the same institution, pursued the samequest with advanced molecular biology techniques.Instead, structural biology and molecular biologyare closely integrated in the modern research envi-ronment thanks to the development of novel imag-ing biomolecular microscopy platforms , whichhave arisen from new, powerful computers, from fast high-resolution cameras, from improved imageprocessing software, and from advances in various
molecular probes specifically designed for labelling fixed and/or living cells . The current boom in thedevelopment of correlative imaging methods illus-trates the point: microscopy is central to the modernbiosciences and is helping to integrate subfieldsin this case, structural biology and molecular biolo-gy . It is no coincidence that high-impact papersdealing with correlative imaging continue to prolifer-ate, even though this avant-garde vision of combin-ing cellular and molecular tools has been aroundsince the early 1970s. Many advances in the clarifi-cation of structurefunction relationships have
Creating next-generation microscopists:structural and molecular biology at the crossroads
F. Braet *, K. Ratinac
Australian Key Centre for Microscopy & Microanalysis, The University of Sydney, Australia
Received: May 6, 2007; Accepted: June 4, 2007
AbstractThis paper highlights the importance of advanced microscopy and microanalysis in the pursuit of qualityresearch in the biological and life sciences. With the growing complexity of modern microscopes, there issubstantial risk of incorrect use or misinterpretation of data by the inexperienced researcher. This paperemphasizes the need for collaboration between biological microscopists and molecular biologists, withinthe context of centralized facilities and supported by first-class training, to fully realize the power of theseunique instruments in modern biology and to create the next generation of molecular microscopists.
Keywords: centralized microscopy facilities correlative imaging molecular microscopy sample preparation
J. Cell. Mol. Med. Vol 11, No 4, 2007 pp. 759-763
*Correspondence to: Filip BRAETAustralian Key Centre for Microscopy and Microanalysis, Electron Microscope Unit, Madsen Building F09, University of Sydney, Sydney,
NSW 2006, Australia.Tel: +61 2 9351.7619.Fax: +61 2 9351.7682.E-mail: firstname.lastname@example.org
2007 The AuthorsJournal compilation 2007 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Modern biomolecular microscopy Complexityally or foe? Centralized microscopy facilities Watch the step
The changing fortunes of microscopy
Merging cellular and molecular biology at the microscope column
760 2007 The AuthorsJournal compilation 2007 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
occurred when molecular and structural biologytechniques were applied concurrently. Classicaloutcomes from the meeting of morphologists andmolecular biologists have included the elucidationof the origin of peroxisomes, the revision of Golgi-transport, the dynamics and structure of protea-somes, the interaction of desmosomal cadherins,and the in-depth revision of the ultrastructure of dif-ferent cellular components .
Complexity ally or foe? Microscopes, like the other machinery of science,are becoming increasingly more complicated andone might wonder how this impacts research out-comes. In principle, more sophisticated tools offerscope for greater insights and understanding, yetthere is also an increased risk of researchers apply-ing these instruments incorrectly. With this in mind,we like the quote: it is important to learn the alpha-bet first, then to learn to write sentences, and if youare lucky you might one day win the Nobel Prize forliterature. The same philosophy is true formicroscopy and microanalysis. Understandably,researchers are attracted to high-end instrumentsbased on the latest vogue in biological research, butthey often make the fundamental mistake of notlearning the alphabet, of not using the most appro-priate instrument for their work or of not fully grasp-ing the inherent limitations of a new microscopytechnique before applying it. This is due, at least inpart, to the buffer provided by advanced softwareinterfaces; though modern instruments are becom-ing more complicated, the commensurate integra-tion of computers and instruments can sometimesmake microscopes seem deceptively easy to use.Without appropriate theoretical insights and practi-cal guidance, researchers, especially molecular biol-ogists, can end up using high-end research plat-forms less than optimally. These issues can be further compounded by the power of modern visual-ization, reconstruction and image analysis software,which can cause essential elements in the data tobe overlooked or, conversely, allow the data to beover interpreted.
The risk of researchers collecting poor-quality oreven artefactual data can be minimized throughcareful management of high-end microscopes and
provision of quality user support and training. In anideal world, therefore, every Biomolecular ResearchInstitute would be affiliated with the centralmicroscopy facility of their institution, rather than run-ning their own Molecular Imaging Unit. In that way,they would avoid problems in sample preparation,imaging and data processing, and the many possibleartefacts that can arise during this multi-stepprocess. Moreover, the expert staff and the differenttraining programs offered by a centralizedmicroscopy facility (see, vide infra) can eliminatemost misinterpretation of data and/or the publicationof artefact-rich images. Mistakes in microscopy are,unfortunately, still frequent and have major budget-ary implications on many fronts, such as wasted con-sumables, instrument downtime, unnecessary staffload and, most importantly, potential ethical issues.Although one can only speculate as to the overallimpact and cost of this worldwide, it is clear that thecareful collected research equipment and researchfunds should be nurtured with more care to respectthe investments made by tax payers and grant fund-ing bodies.
Centralized microscopy facilities The ideal approach for supporting researchers intheir use of high-end microscopes is through a cen-tralized microscopy facility, in which users needs arecarefully monitored by facility staff throughout thelifetime of the project. For biological projects, it isadvisable to start out with overall dynamic observa-tion in time; i.e. time-lapse live cell imaging. Thisallows facility users to first understand what is goingon in the live context and then to define the particu-lar point in time where the relevant (sub)cellular phe-nomena occur. From that moment onwards, the high-end instruments come into play as appropriate,thereby avoiding the incorrect use of advancedmachines. Increasingly, this approach will be facilitat-ed by new sample preparation devices that are onthe way to bridge the time-resolution gap betweenlight optical microscopes and the high-end electronmicroscopes by using rapid-sample-transfer prepara-tion systems. Another core element in managing theuse of high-end microscopes is the availability of acomprehensive staff profile, blending academics,research associates and technical staff, all with
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different training and backgrounds. This unique poolof know-how and skills helps solve the vast majorityof issues that arise in the use of advancedmicroscopy. In addition, quality-training programstypically offered as short, field-dedicated trainingcourses that blend both theory and hands-on prac-ticehelp to ensure that researchers have sufficientexpertise to use instruments correctly. These alsoserve as the first training ground for the next genera-tion of biomolecular structural biologists .
As part of the user experience in a centralizedmicroscopy and microanalysis facility, projectsshould start with a new user meeting in which theresearch project is discussed in depth with skilledmicroscopists and technicians. Subsequent atten-dance at one or more microscopy training courses isusually of value before the (molecular) cell biologistgets hands-on with the microscopes. This approachhas proven to be highly successful and productivehere in the Australian Key Centre for Microscopy &Microanalysis and guarantees successful outcomesas shown in Ph.D. theses, reports, peer-reviewedjournal papers, and so on.
Of course, core microscopy or imaging facilitiesoffer many more advantages than the users experi-ence alone. For example, they have the large user-base necessary to justify substantial governmentinvestment in increasingly costly instruments, com-bined with the staff capital required to take full advan-tage of such equipment. Centralized facilities alsohave the capacity to ensure that suites of micro-scopes work correctly, and that problems are quicklyidentified and rectified with minimal downtime for thelifetime of the instruments. Furthermore, placingexpensive microscopes within such facilities reducesthe precious time spent by individual academics andresearchers in independently exploring, often in vain,the mechanisms to attract such infrastructure.
Watch the step Biologists encounter various pitfalls during the collec-tion of microscopy images, though these vary fromproject to project, user to user, and sample to sam-ple. One should start at the beginning, with the art ofsample preparation. The relevant quote here isgarbage in, garbage out. Good microscopy starts
with proper sample preparation according to thehighest international standards, which can be foundin relevant literature sources. Sample preparationhas been an intriguing and ongoing research topicfor decades, but still continues to grow and evolvewith the microscopy techniques, illustrating its impor-tance. Often, inexperienced microscopists find it diffi-cult to adapt complex preparatory procedures to theneeds of a typical experiment at hand. This is naturalgiven the plethora of factors that can play a key rolein the meticulous processing of specimens from aspecific tissue or organ: surgical skills, route of per-fusion, gravity or pump perfusion, pressure, flow,composition and sequence of perfusion fluids,saline washing, osmotic value, ion composition,quality of chemicals, prelevation techniques, cuttingor fracturing methods, the frequency of changingfluids, temperature, and so on. Fortunately, moststructural biologists are aware of the importance ofgood sample preparation and many make it theirmission to guide molecular biologists in the fine artof sample preparation.
Another common pitfall occurs at the other end ofthe process; after acquiring the end product,researchers face the challenge of interpretation andof retrieving the relevant information from thoseimages. It is essential that the molecular biologistand morphologist meet with each other well beforethis point, and on an ongoing basis, to help avoidsubsequent problems in the collection, analysis andinterpretation of microscopy data. From our perspec-tive, such meetings are most productive interactionsas both parties educate each other about the avail-ability of techniques to dissect cells molecularly andthe complementary methods to image and character-ize cells at nanometre resolution. This crossover is akey-indicator of high-quality and, ultimately, high-impact research.
The changing fortunes of microscopyHistorically, microscopy and microanalysis wereexpected to become obsolete once molecular biologytools became readily available for every laboratory.There was even a time when senior academic man-agement looked askance at you if you made the brave
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claim of being a microscopist. Of course, these viewswere just heralds of changing scientific fashions:microscopy was in style in the 1960s and 1970s, butdefinitely was not popular in the late 1980s and early1990s . Fortunately, this situation has changed inrecent years with the introduction of cryo-electrontomography techniques and rapid, live-imaging tech-niques. A simple scan of websites for academic andscientific jobs shows just how many positions now are available for trained molecular microscopists.Certainly, the Australian experience confirms theessential role of microscopy in modern biologicalresearch; Australias bio-scientists clearly recognizethe importance of more advanced molecular image-analysis methods, such as fine-structure immunogoldtechnology and (cryo-) electron tomography . TheFederal Governments recent funding of an AustralianMicroscopy and Microanalysis Research Facility,headquartered here at the University of Sydney,illustrates the high value that policy makers placeon this key scientific infrastructure. Ultimately, com-bining advanced biological microscopy with molec-ular cell biology techniques is the only way toensure that research is highly competitive at theinternational level  and, perhaps more important-ly, to avoid being limited to molecular biology dataalone .
It was suggested once at a conference that micro-scopists have become an endangered species andthat it is high time to invest proper funding in preser-vation programs for them and in the training of thenew generation. This is true; quality training is essen-tial to meet the growing demand for molecular micro-scopists. An important aspect in the integration ofstructural and molecular biologists is offering themthe possibility of doing dedicated training programsin microscopy. At present, however, there are only afew dedicated training programmes on offer on theinternational scene. There is no doubt that Australiais a global leader in this area. For example, theUniversity of Western Australia runs a unit in electronand optical microscopy for undergraduate students inbiology, engineering, and materials and earth sci-ences. The University of Sydney offers full postgrad-uate courses in microscopy and microanalysis, aswell as doctoral research programs. This trainingcapacity goes some way to meeting the localdemand for skilled molecular microscopists in
Australasia, and some of the fresh graduates end uptaking jobs in the United States and Europe.
Merging cellular and molecularbiology at the microscope column Modern advanced biomolecular microscopy compris-es the full range of instruments that explore speci-mens with light, lasers, soft x-rays, electrons orscanned probes to generate images and spectra atresolutions from the microscale towards the atomicscale. Based on the increasing availability of diffe...