4x Objective Lens for Shared Multiphoton Microscope
The Departments of Chemical Engineering and Bioengineering, the School of Medicine, and the Institute for Stem Cell and Regenerative Medicine, jointly propose the purchase of a shared 4x objective lens for the existing Olympus FV1000 MPE BX61 Multiphoton Microscope housed in the Garvey Imaging Center user facility at the South Lake Union Medical Research Campus. The multiphoton microscope is a crucial and popular tool used to visualize both cellular and synthetic materials generated by stem cell biologists and engineers alike. Moreover, the microscopy technique is uniquely powerful at imaging deep within thick tissue samples and even living animals. As the current configuration includes only a single objective (25x), the addition of the proposed 4x objective would enable students and researchers to image samples over a significantly larger field of view, simultaneously increasing the area that can be studied and decreasing image acquisition times for large tissues.
The Garvey Imaging Center is one of the most heavy utilized facilities at the South Lake Union Campus and is fully staffed and maintained by Dr. Ron Seifert. While the purchase of the Multiphoton microscope and its service contract have both already been very large investments for the ISCRM facility, the multiphoton microscope is not currently utilized at its full potential; the single existing non-adjustable objective lens has made the microscope less appealing than its confocal counterparts. Though several labs have suggested the purchase of a lower magnification lens, the high cost has dissuaded any single lab to make the investment necessary to buy, install, and share such a piece with the entire facility. The proposed objective purchase would enable new science to be performed, expand capacities, and decrease the work burden on similar but oversubscribed microscopes within the facility.
Prof. Cole DeForest in Chemical Engineering is prepared to contribute up to $1,000 towards the purchase of this objective.
The 4x objective lens is a crucial and state of the art instrument necessary in cellular biology, stem cell biology, tissue and biopsy analysis, biomaterials, and tissue engineering research. User training and maintenance costs will remain largely unchanged after installation of the 4x objective. Moreover, this objective can be interchanged with the current 25x objective with relative ease. There should be no additional charges after purchase of objective lens and nosepiece fitting. The lens will represent a key accessory for the existing $500k+ Multiphoton microscope.
The Institute for Stem Cells and Regenerative Medicine (ISCRM) is an interdisciplinary consortium of over 40 core and almost 100 affiliate clinicians and researchers dedicated to the advancement of our understanding of stem cells and the development of novel medical therapies and cures to diseases spanning from diabetes to heart failure. The breath of expertise within ISCRM extends from clinical specialties like the Department of Vascular Surgery to technical specialties like the Department of Chemical Engineering. Together through collaboration and innovative new approaches to medicine, UW researchers have been able to begin to understand the molecular mechanisms of disease and have become one of the world’s leaders in regenerative medicine.
At the cornerstone of ISCRM research exists the Lynn and Mike Garvey Cell Imaging lab. Through the generous donation of Lynn and Mike Garvey, ISCRM purchased and maintains state-of-the-art light microscopy systems supporting nearly all imaging work conducted by the ISCRM associated labs. Access to this facility is currently either free to any ISCRM member lab or in the case of the confocal system subject to a small nominal hourly fee. For labs outside the ISCRM a case-by-case fee and basis is applied. Housed in the imaging lab is a Nikon A1R confocal microscope, two Nikon TiE Inverted Widefield Fluorescence Microscopes, and an Olympus FV1000 MPE BX61 Multiphoton Microscope. Each microscope enables researchers to image within samples, probing cell function in real time, and penetrate deep into samples to understand the structural organization and function of complex tissues.
The multiphoton microscope, in particular, is a unique instrument capable of capturing high definition fluorescent images of living tissue up to a high depth of 1-2mm. It utilizes higher wavelength infrared light that exhibits little scattering through biological samples. For this reason, multiphoton microscopy is often regarded as a superior alternative to confocal microscopy as it is capable of the same features of a confocal while in addition can penetrate deeper into samples, exhibit greater light detection and signal resolution while eliciting minimal phototoxicity. Utilizing this technology, researchers and students are able to observe complex structural architectures of tissues, probe intricate intracellular processes, and generate 3D reconstructions of either individual cells with great resolution or image whole tissues either living or fixed.
Recently, the utility of a multiphoton microscope has been greatly expanded by groups here at UW as well as abroad, as not only a tool for imaging biological samples, but also as a mean to synthesize and generate biomimetic structures. In multiphoton microscopy, a single laser pixel is rastered throughout a 3D space exciting and recording signals from a small volume. These measurements taken at specific locations are then utilized to reconstruct 2D and 3D images of a sample. Using this individual laser pixel and software in conjunction with novel light sensitive biomaterials developed in the Bioengineering and Chemical Engineering departments, researchers have been able to build and degrade materials with full 3D control (most recently described in a forthcoming accepted manuscript appearing in the May issue of Nature Materials). In an expanded use of these technologies, UW researchers have developed novel ways to essentially 3D print complex tissues using light with greater resolution than ever previously explored. Purchase of the 4x dry objective lens and nosepiece would greatly expand the ability of researchers to both image and build biological tissues with greater ease, further expanding the user base for these cutting-edge techniques.
Benefits to Students and the University
The systems at the Garvey Imaging Facility are among the most user-heavy microscopes on campus and are utilized by undergraduate students, graduate students, staff scientists, post-doctoral students, and faculty alike. Training conducted with Mr. Ron Seifert (Garvey Imaging Facility Manager) allows any ISCRM associated staff or faculty the ability to image their samples using the facility equipment. Multiphoton microscopy is a unique capability available to UW students and the microscope at the Garvey Imaging Lab is the only microscope of its kind on campus. The capabilities of the multiphoton microscope allow researchers to probe questions about large tissues that cannot be explored with other forms of light microscopy.
While the confocal and fluorescence microscopes are under heavy use and are often booked weeks in advance, the multiphoton microscope (the newest and most expensive microscope in the facility) gets somewhat less use. The device was purchased with the intent and capacity to penetrate deeper into samples, able to reconstruct 3D images of tissues and cells, and able to generate images of higher resolution than capable on either the confocal or fluorescence microscopes. While still capable of such feats, the multiphoton has become less popular due to its untapped potential and limited versatility. Although capable of greater functionality, it is currently equipped with a single non-adjustable 25x water immersion objective lens warranting only a small field of view. The proposed purchase of a 4x dry objective lens would greatly enhance the area that can be explored using the microscope and offer users greater flexibility in imaging capacity. It is our hope that by expanding functionality of the microscope, we can increase usage of the existing multiphoton microscope while simultaneously freeing up availability of the confocal systems.
From a broader standpoint, confocal and multiphoton microscopy are ubiquitous tools in cellular biology and critical to any high-impact cellular biology, tissue engineering, or material science scientific article. As such, training and utilization of such technologies are crucial to the learning, teaching, and implementation of biomedical research and are imperative skills in research and development careers. Although the 4x objective lens is limited to use on the multiphoton, implementing such a fitting would have significant effects on the operations of the entire Garvey Imaging Facility. By encouraging researchers to use the multiphoton instead of the confocal systems, we anticipate a significant reduction in the confocal systems’ workload and demand. With greater availability and shared usage, more users will be capable of training on and utilizing all the systems at the Garvey Imaging Facility without the purchase of another $500k+ microscope.
I am writing in support of the proposal to augment the capabilities of the Olympus FV1000 MPE BX61 Multi-photon Microscope. I manage this instrument which is owned and supported by the Institute for Stem Cell and Regenerative Medicine and has provided imaging services for multiple faculty, post-doctoral fellows and graduate students within this collaborative and productive group of investigators. The addition of low power objectives would markedly enhance the ability of many groups to survey larger fields of cells and tissues both ex vivo and in situ to determine the fate and efficacy of stem cells in reparative and regenerative processes.
ISCRM - Garvey Cell Imaging Lab
Room S530, Box 358056
850 Republican St
Seattle, WA 98109
As faculty members of the Institute for Stem Cell and Regenerative Medicine (ISCRM), we (L. Crisa and V. Cirulli) herein endorse with the highest level of enthusiasm this proposal for the acquisition of a 4x dry objective lens, and compatible nosepiece, to be installed in in the multi-photon confocal microscope housed in our Garvey Imaging Facility at the ISCRM. Addition of this lens will dramatically increase the ability of this microscope to serve a much larger user-base by providing an expanded functionality for larger view areas, analysis and optical-based biochemical modification that would be otherwise impossible to achieve with the current repertoire of 25x and 40x objectives.
Thanks to the establishment of our recent collaboration with Dr. Cole DeForest in the Department of Chemical Engineering, both the Cirulli and Crisa laboratories will make heavy use of this new lens to conduct studies of pancreatic islet encapsulation, as part of our Diabetes Stem Cell Program efforts to cure diabetes.
Presently, up to seven team members (between faculty, graduate students, and staff research assistant) from the Crisa and Cirulli labs will benefit from the acquisition of the 4x dry objective lens, and compatible nosepiece.
Laura Crisa, MD, PhD, Associate Professor (Department of Medicine, UW)
Kristin Mussar, Graduate Student
Stephany Pardike, Research Staff
Vincenzo Cirulli, MD, PhD, Associate Professor (Department of Medicine, UW)
Jeff Vercollone, MD, Fellow Resident in Training
Wendy Yang, Graduate Student
Pat Kensel-Hammes, Research Staff
I am writing in support of obtaining funding for the purchase of a 4x objective for the multi-photon microscope system within the imaging core of ISCRM. This system is unique in that it offers users to probe deep within samples with high resolution. Our current research is focused on developing 3D hydrogel systems with topography as the next-generation culture systems that mimic features of the native tissue for neural stem cells, with the goal of developing regenerative strategies for the central nervous system disorders. As such, we utilize a number of 3D culture systems in combination with human as well as rodent stem cells. The use of this two-photon system has been essential in assessing phenotypical and morphological analyses of our cultured cells. However, the use of 25x objective alone limits the area users can examine. Therefore, the availability of a 4x objective will greatly enhance this microscope by allowing users to develop and examine engineered 3D hydrogels on a larger scale.
Zin Z. Khaing, PhD
Department of Neurological Surgery
Institute for Stem Cell & Regenerative Medicine
850 Republican Street, Rm S440
Seattle WA 98109-4725
Phone: (206) 685-4109
The Murry lab has a longstanding interest in studying the physiological properties of engineered cardiac tissues. In cardiovascular physiology, the cardiomyocyte contractile cycle is a fundamental measure of the state of the cell. Just as the electrocardiogram and pressure-volume loops describe the contractile physiology of the whole heart, measurement of cell length and contractile shortening describe the physiology of the cardiomyocyte. Measurement of contractile function of single cardiomyocytes as well as whole constructs is a key end point in our studies. A 4x objective would greatly support achieving this goal.
Hans Reinecke, Ph.D.
University of Washington Medicine at South Lake Union
Center for Cardiovascular Biology
Institute for Stem Cell & Regenerative Medicine
850 Republican Street, Brotman Bldg., Room 454
Seattle, WA 98109
The pieces can be installed the same day as they arrive on site.
Resources Provided by Department
The new objective will be made available to all users of the Garvey Imaging Center, the shared microscopy suite located on the South Lake Union campus that is available to all University of Washington researchers. Ron Seifert will continue to oversee the facility, providing training and best practice expertise in using the proposed 4x objective.
As stated in the abstracted, Prof. Cole DeForest in Chemical Engineering will contribute $1,000 of the total purchase cost of the objective.
Access Restrictions (if any)
All researchers capable and trained by Dr. Seifert to use the Multiphoton microscope will be able to utilize both the new 4x or existing 25x objective lenses. As mentioned, the Garvey Imaging center is available to any ISCRM affiliated researcher and can be made available to any UW researcher as determined on a case-by-case basis.
I strongly support this grant application for the purchase of a 4x objective lens for the OlympusFv1000MPE BX61 Multiphoton Microscope. I am a joint M.D./Ph.D. student from the UW School of Medicine and the Department of Bioengineering and a trained user of both the confocal and multiphoton instruments at the Garvey Imaging core. I use the multiphoton on a very regular basis and utilize its unique capabilities as a way to 3D print vascular networks in 3 dimensions. The microscope is perhaps one of the most unique and powerful tools at the UW and it enables the exploration of truly innovative and ground breaking ways to simultaneous image and design new materials.
While the microscope equipped with a 25x objective is very powerful for small delicate designs and samples, its same advantages can be limitations. A 25x objective allows one to probe very precisely in a small field of view, however to image larger areas takes lots of time and can be quite cumbersome. The purchase and installation of a 4x objective would provide the microscope the option to image much larger areas much faster and more efficiently. While lacking the resolution which might be provided by the 25x, the choice to use either objective lens would very significantly increase the multiphoton’s versatility, allowing researchers to image both large scale and small scale.
Moreover, as a user of both the confocal and multiphoton, I am certain that by increasing the functionality of the multiphoton one could greatly increase its popularity among users and free up user time on the confocal systems. It is not uncommon for researchers, like myself, to stay until midnight or later, simply to reserve time on the confocal systems to acquire images. Should capabilities of large field of view (a capability of the confocal) be available on the multiphoton in conjunction with its many unique functionalities, I and others would be able to conduct research much more efficiently.
M.D./Ph.D. Student- UW Medical Scientist Training Program
UW School of Medicine
UW Department of Bioengineering
University of Washington
I am writing in support of this application to purchase a second objective for the multi-photon microscope housed in the Garvey Imaging Core. I am a Bioengineering graduate student in Dr. Charles Murry’s lab, in the Institute for Stem Cell and Regenerative Medicine (ISCRM) and the Center for Cardiovascular Biology. I am currently a trained user of the both the confocal microscope and the live cell imaging microscope in the Garvey Imaging Core. Although I have only had limited experience with the multi-photon microscope to date, there are increasingly more opportunities to use this resource in my own research.
When I have used the Garvey multi-photon microscope in the past, the types of images I could acquire were limited by being restricted to a single objective. With the current setup, the use is restricted to the 25x objective with no option for low magnification imaging. In my own research designing engineered cardiac tissues, it is often beneficial to image the entire tissue at a low magnification before moving to a higher objective. I feel that the addition of a 4x objective is a relatively low-cost upgrade that would allow the user to optimize the imaging settings to best meet the needs of their experiment. The multi-photon microscope is available to many undergraduate and graduate students, and there is already a fantastic training and support system available to new and continuing users through the Garvey. Having the option to image a 4x and 25x objective would help expand the usage of the multi-photon microscope, and I look forward to including multi-photon imaging in my future experiments.
Ph.D. Student- Dept. Bioengineering
University of Washington
The addition of the 4x objective lens would vastly advance the usability of the two photon microscope in the Garvey Imaging Center. This new lens increases the frame size and operates without the limitation of water submersion. It is currently difficult to avoid contamination of samples while imagining. As innovative biological research transitions further into three dimensional applications, superior imagining technology is required for the success of many undergraduate, graduate, and post-doctorate students including myself. Being able to dive deeper into a sample and an increase in the efficiency of imagining 3D samples would benefit the many members of the UW community who use the Garvey Imagining Center.
Ph.D. Student - Chemical Engineering
University of Washington
The two-photon microscope allows researcher to image living tissues up to a very high depth and helps to understand the structure and function of tissues in real time. I am a first year Master's student in Chemical Engineering and my research is focused towards designing logic-gate based protein delivery systems in hydro-gels. Eventually, these hydro-gels will be tested on tissues. Two-photon microscopy will play a crucial part in study such systems in real time and in great detail. The present Two-photon microscope has only a 25x objective lens which severely limits the region of study. I believe that having a 4x objective lens will definitely make the Two-photon more versatile and increase the region within the tissues which can be studied. This would help many students and researcher to step up better experiments and study the tissues with enhanced versatility of Two-photon.
M.S. Student- Chemical Engineering
University of Washington
I humbly request that you consider the grant request for the procurement of a 4x Objective Lens and appropriate nosepiece for the Olympus Multiphoton Microscope currently housed at the Garvey Imaging Center at the South Lake Union Medical Research Campus. The Multiphoton Microscope is a powerful tool that is currently being under-utilized due to its current configuration. The current objective for this multiphoton microscope spatially limits current researchers’ ability to effectively and efficiently use this equipment with some experiments lasting several hours to perform. The smaller objective lens will significantly cut this time down while still maintaining excellent resolutions for ground breaking cellular and material science research. By providing the funds necessary to upgrade this piece of equipment, it will provide new opportunities for researchers to explore cellular biology and materials science with a higher throughput, thus leading to new innovations for the University of Washington. Thank you for your time in considering this grant proposal.
Michael P. Comerford, Lieutenant, USCG
M.S. Student- Department of Chemical Engineering
University of Washington
In my research, the precision afforded by the 2 photon microscope will be used to activate small molecules by cleaving photolabile groups in vitro. Having a wider field of view is important because it makes it much easier to sample individual cells and improves standardization when sampling multiple cells simultaneously. Having access to the 2 photon microscope is critical to my research, so improving the capabilities of this system facilitates my work tremendously.
PhD Chemical Engineering Student
University of Washington
As a future user of the two-photon microscope for 3D hydrogel photopatterning, I am interested in the expanded capabilities that a 4x objective lens will provide to the instrument. This objective will increase the quality of the photopatterning by reducing the number of viewing windows and reduce the time I need on the microscope by more than 80%.
Ph.D. Student- Chemical Engineering
University of Washington
4x objective and connecting nosepiece for existing Olympus FV1000 MPE BX61 Multi-photon Microscope.Justification
Primary item detailed in proposal.
Total requested: $8,000.00
Total funded: $0.00
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