Instruments for checking physical characteristics

Description

The Francis Crick Institute (‘the Crick’) is a biomedical research institute dedicated to understanding the scientific mechanisms of living things. Its work is to understand why disease develops and to find new ways to treat, diagnose and prevent illnesses such as cancer, heart disease, stroke, infections and neurodegenerative diseases. The observation of living cells and their dynamics is of fundamental importance across all areas of Crick research. The most simple forms of light microscopy involve observation of cell behaviour using transmitted light (e.g. wound healing, cell migration, lineage tracing), although information about the location of specific proteins and the identity of cells within a population can also be obtained using fluorescence microscopy. There are many suppliers of conventional transmitted light technologies such as phase contrast and DIC. These approaches are generally useful for many applications, and can to some extent serve as the basis for quantitative analysis. The Crick has an in-house a microscopy facility that provides services in multi-dimensional imaging with fixed and live specimens using laser scanning confocal microscopy and low-light-level imaging and supports users in image processing, motion analysis, statistical analysis and sample preparation. The facility is heavily used for various applications throughout the Crick including studies on deconvolution, co-localisation, cell segmentation and intracellular structures (automatic or interactive), morphometry and tracking. The experiments explore the complexities of cells in health (including stem cell and developmental biology) and specific disease states including malaria, HIV and Toxoplasmosis. Current available systems have been configured for contrast enhancement, high-resolution 3D imaging, dynamic imaging of biological specimens, multiple fluorescence channels and optical sectioning using motorised focus and multiple fields. The Microscopy Facility works at the heart of the Crick in pursuing the development of light microscopy techniques and their applications in collaborative projects with the research laboratories of the Francis Crick Institute and it's many partners and collaborators. To this end, the Crick requires several (max 10 subject to budgetary approval, though current approval is limited to 3) a high-end wide field time lapse microscope systems over the coming 5 years which it intends to build in-house using several components. The equipment will be built and serviced in house using the extensive expertise of the facility in this area. This approach will enable the Crick to maintain its cutting-edge international reputation and success at the highest international level and supports product standardisation for these systems to customised highly specified Crick requirements, allowing the Microscopy staff as technical experts of this self-build to closely monitor usage and performance of sophisticated and fragile equipment and perform maintenance operations where necessary. The purchase of these microscope components will allow Crick researchers to make best use of the collaborative facilities at the Crick towards improving human health with a particular focus on development, immunity and infection. The components are essential on the basis of their exceptional technical performance which we believe cannot be reproduced or integrated with each other to the specification we require. The system would be comprised of the following essential components: 1. The heart of this system is the Nikon inverted Ti2 microscope, incorporating Nikon's patented Perfect Focus System (PFS) auto-focusing mechanism. This is a hardware autofocus mechanism which continuously maintains the focal position of the sample, even when it is not being imaged. The Nikon system benefits from use of two patented technologies. The first is that the PFS is able to detect and lock on to a suitable axial reference plane, while adjusting focus with an offset lens to maintain focus on the sample (i.e. the focus mechanism ‘sees’ one plane while the camera sees another). The second feature is the rapid feedback between the CCD line-detector and the microscope Z-drive. These features combine to produce an accurate and responsive mechanism which is highly robust over time and when traversing large XY distances. 2. The continuous feedback aspect of this feature which is patent protected is absolutely critical for long-term focal maintenance. The technology used by Nikon is proprietary and it exceeds the performance of conventional transmitted light microscopy. We therefore do not believe there is another supplier capable of fulfilling this requirement. 3. The Lumencor SpectraX by Nikon is superior to the other LED light sources in this price/performance category because of the greater number of illumination wavelengths which can be used at one time without the need for an excitation filter wheel. The SpectraX is capable of instantly switching between 6 different illumination wavelengths, which are fine-tuned through the use of individual built-in excitation filters (note that a 7th wavelength can be accessed by automatic switching of an internal filter). The design of the SpectraX allows coupling of individual LEDs with narrower excitation filters, to enable more specific excitation of fluorophores in multi-colour experiments. In contrast, other LEDs such as the pe4000 use broad-band excitation filters in conjunction with motorized banks containing multiple LEDs per filter. This approach results in poor excitation specificity when using LEDs on the same bank to illuminate different fluorophores, in addition to potentially significant delays in acquisition time. The SpectraX does not require use of an additional excitation filter wheel, as suggested by some manufacturers for their products, which reduces cost and complexity of the overall imaging system. 4. The Photometrics Prime camera has the lowest read noise of any scientific grade CMOS camera. The camera also incorporates patent protected de-noising algorithms to reduce the impact of photon-shot noise, which can lead to 3-5 times improvement in signal to noise ratio. These features in combination are critical for improving the quality of low light fluorescence images, which is essential for certain types of live cell imaging experiments conducted at the Crick. Reduced photo-damage of live samples is essential and state of the art rapid scanning of samples on 96-well plates is a feature of the setup requirement. The Prime camera achieves this though an improved S:R, which enable data of the same quality to be acquired using shorter exposure times. 5. The Photometrics ASI stage has a strong integration with the open source software Micro-Manager to which the Crick is committed as this can be provided for multiple users free of charge and is already the main training platform for many end users. ASI write Micro-Manager drivers for all the hardware they sell, including complex multi-component systems such as the diSPIM, which is required by the Crick. Again, we are currently unaware of any provider that can fulfil this level of integration with open source software as well as the components listed above. Type of procedure The Francis Crick Institute intends to procure microscopy components we believe cannot be reproduced or integrated with each other to the specification we require. The key requirements of the components are: — Hardware autofocus mechanism, which robustly maintains the focal position of the sample over long ranges of time and space. — Use of multiple (≥6), specific (individual filters), rapidly accessible (≤100 µs) illumination wavelengths. — Scientific grade CMOS camera with lowest read noise on the market encompassing complex de-noising algorithms to reduce the impact of photon-shot noise — Stage which encompasses complete integration with the open source software micromanager as well as the hardware components listed above.