Multi-Mode Imaging of Membranes, Gels and Plates
Please acknowledge the SIP core facility ( RRID: SCR_018986) in publications, on posters, or in talks if you use any instruments in the SIP core facility. Please include SIP's RRID (RRID: SCR_018986) and the grant numbers for instruments funded through instrumentation grants in your acknowledgements. This is a requirement from the funding agencies and is crucial for future funding. Find example text on theÌýÌýor the individual instrument pages.
Multi-Mode Imagingin Biochemistry, Biophysics and Structural Biology
The detection of biomolecules on gels, blots, and arrays is a foundational technique in biochemistry and biophysics, crucial for the separation, identification, and quantification of proteins, nucleic acids, and other macromolecules. These methods provide key insights into complex biomolecular processes including molecular interactions, cellular signalling pathways, and post-translational modifications, deepening our understanding of biological mechanisms in both health and disease.Ìý
The integration of multimode imaging, utilising multi-colour fluorescence, chemiluminescence, and colourimetric detection, significantly enhances analytical sensitivity, specificity, and dynamic range. Through multiplexing, researchers can combine these imaging techniques to analyse multiple molecular targets within a single experiment, adjusting detection methods and colour schemes to optimise sensitivity and dynamic range based on experimental requirements. This approach maximises data acquisition from a single sample, improving both the depth and accuracy of molecular analysis
SIP hosts two multimode imagers: the Amersham Typhoon 5 and the Cytiva IQ800. Additionally, we have multiple licences for the ImageQuantâ„¢ TL analysis software.
Amersham Typhoon 5
Example Text for Acknowledgements
We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the Amersham Typhoon 5 imager. The Typhoon 5 was funded by NIH S10OD034218.Ìý
The Typhoon 5 is a multi-mode, high-sensitivity imaging platform to capture high-resolution images from a multitude of samples. Ìý
Applications
The Typhoon has a wide range of applications including imaging of phospho storage screens (filmless autoradiography ), gel sandwiches, agarose and polyacrylamide gels, membranes, microplates, microarrays and even tissue slices.Ìý
Imaging Modes
The Typhoon 5 has 4 imaging modes that can be combined automatically. Fluorescence (5 channels), Phosphorimaging, Densitometry, and Chemiluminescence (dark scan)Ìý
Fluorescence Channels:
It has blue (488 nm), green (532 nm), red (635 nm), near IR (685nm) and far IR (785nm) excitation wavelengths and a wide choice of emission filters (IP,Ìý P 390BP, Cy2 525BP20, Cy3 570BP20, Cy5 670BP30, IRshort 720BP20, IRlong 825BP30, Cy2 LPB515, Cy3 LPG550, Cy5 LPR660) enable imaging of an extensive variety of fluorophores.Ìý
Automated multi-colour fluorescence scanning allows multiplexing of multiple targets in the same sample.
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Amersham IQ 800
The ImageQuant 800 imager from Cytiva/Fujifilm is equipped with a highly sensitive 8.3-megapixel, Peltier-cooled CCD detector and an F 0.74 lens, enabling high-resolution imaging of closely spaced bands. While it excels in the straightforward imaging of western blots and gels, it is versatile enough to support a wide range of additional applications.
Applications
The IQ 800 is capable of colour imaging a wide range of samples including imaging for various samples, including gels, membrane blots, multiwell plates, and petri dishes.
Imaging Modes
The IQ 800 has three imaging modes that can be combine automaticaly. Modes include chemiluminescence, fluorescence (5 channels), and colorimetric imaging.Ìý
Light Sources:
UV Epi 365 nm, Blue Epi 460 nm, Green Epi 535 nm, Red Epi 635 nm, IRshort Epi 660 nm, IRlong Epi 775 nm, White Epi 470 to 635 nm, White Trans 470 to 635 nm
Emission Filters:
Cy2 525 BP20 nm, Cy3(UV) 605 BP40 nm, Cy5 705 BP40 nm, IRshort 715 BP30 nm, IRlong 836 BP46 nm
Automated Signal-To-Noise Optimisation
The SNOW (Signal-to-Noise Optimisation Watch) imaging mode provides automated optimisation of parameters to detect faint bands in Western blot while ensuring image quality. SNOW increases sensitivity and extends dynamic range by preventing signal saturation. IQ 800 automatically stops once the optimal S/N is reached, removing the need for iterative manual adjustment of exposure and capture settings.
Analysis software
ImageQuant TL (IQTL)
We provide multiple licences for the ImageQuantâ„¢ TL (IQTL) analysis software, which is installed on both imagers as well as a dedicated workstation available for users to perform image analysis at any time.
In addition, we offer extra licences that can be allocated to individual laboratories upon request, allowing installation on shared workstations within their labs. The software supports various file formats, including TIF, JPG, IMG, and GEL, and is fully compatible with both the Amersham Typhoon 5 and the Cytiva IQ800 imagers.
ÌýAnalysis Modules in ImageQuant TL:
- Gel and Blot Analysis: Tools for lane and band detection, background subtraction, quantity calibration, normalisation, and lane comparison.
- Analysis Toolbox: Versatile drawing tools for general image analysis tasks.
- Array Analysis: Designed for analysing data from multiwell plates and arrays.
- Colony Counter: Enables detection and analysis of bacterial colony images.
ImageJ
ImageJ is a public-domain, Java-based image processing program, originally inspired by NIH Image for Macintosh. It is installed on both imager computers and the dedicated analysis workstation. ImageJ offers a wide range of applications beyond the quantification of gels, blots, arrays, and colony plates, making it a versatile tool for diverse imaging needs.
Support
SIP staff are readily available to provide support for both ImageJ and ImageQuant TL, helping users make the most of these powerful analysis software packages.