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Top Tips for Imaging with the D Floor Microscopes Peter Grabowski

OutlineIntroduction to D floor microscopes and capabilitiesInitial microscope adjustmentsCommon brightfield image artefactsColour imaging issuesPhase contrast problemsConsiderations for fluorescence imagingImage processing and analysisAdvanced image processing - deconvolution. The D Floor microscopes.

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Top Tips for Imaging with the D Floor Microscopes Peter Grabowski

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    1. Top Tips for Imaging with the D Floor Microscopes Peter Grabowski

    3. The D Floor microscopes

    4. Microscope capabilities DMI4000B AF6000LX Fluorescence + + Phase contrast + + Brightfield + + Camera Colour B/W Software LAS AF, LAS LAS AF, LAS Live cell imaging - + Temperature control - + CO2 supply - + FRET - + * The colour camera on the DMI4000B works in B/W mode for fluorescence and is less sensitive than the camera on the AF6000LX

    5. Cell culture vessels TC plastics Chamber slides Coverslips in wells Glass bottom plates Ibidi “µ-Slide” Condensers not suitable for flasks! Glass >> Plastic for high quality images

    6. Initial microscope adjustments Adjust the microscope for best performance when starting an imaging session “The beginning is the most important part of the work” Plato “He was a wise man who invented beer” Plato “The microscope with its accessories is by far the least understood, the most inefficiently operated, and the most abused of all laboratory instruments” Charles P Shillaber Author of Photomicrography Theory and Practice

    7. Kohler illumination Lamp filament focused on condenser aperture diaphragm Field diaphragm plane focused on specimen Field and aperture diaphragms adjusted for even, glare-free illumination Light reaches specimen in parallel rays Lamp filament focused away from image plane Images are free of diffraction and refraction artefacts

    8. Important components

    9. Condenser focused and centred

    10. Brightfield artefacts

    11. Brightfield colour images What colour is white light? Lamp intensity determines tint of illumination Brain perceives tint and corrects to neutral Camera adjusts tint using “white balance” function

    12. Lamp intensity and image colour

    13. Shading correction

    14. Phase contrast – phase rings

    15. Phase contrast artefacts

    16. Phase contrast - meniscus

    17. Fluorochromes and filter cubes Filter cube constraints Excitation bandpass Emission bandpass Autofluorescence Crosstalk Stability Alexa Fluors Quantum Dots Use antifade agents

    18. Filter cubes

    19. Typical fluorochromes

    20. Immunofluorescence: sample preparation Use appropriate fixation and permeabilisation reagents for the antigen Use high quality blocking agents 3% BSA 10% fetal (avoid calf) bovine serum 10% dried milk 5% same species serum 1% purified immunoglobulins

    21. 1-step or 2-step labelling?

    22. Sources of background Primary and secondary non-specific binding Insufficient washing Autofluorescence Specimen Culture medium Fixative – e.g. glutaraldehyde Camera noise High gain, long exposures Incorrect objective focus depth adjustment

    23. Antibodies Select high quality validated antibodies Specificity, selectivity Check out a range of antibodies Control with pre-immune serum Store antibodies in aliquots Avoid multiple freeze-thaw cycles Centrifuge antibody solutions Primary and secondary reagents can produce unwanted backgrounds

    24. Live cells / transfected cells Avoid phenol red medium Work in darkened room Use low intensity illumination: High intensity light ? photodamage + phototoxicity Select long wavelength fluorochrome Use temperature control Use CO2 control or a CO2 buffered solution (HEPES)

    25. Fluorescence - exposure

    26. Focus adjustment collar on 40x and 63x objectives

    27. Multichannel live cell imaging

    28. Should you post-process images? “Adjustment of digital images with computer software is acceptable. However, the final image must remain representative of the original data…” “Adjustments applied to the whole image are generally acceptable if no specific feature of the original data is obscured as a consequence.” J Cell Science “Information for authors” “No specific feature within an image may be enhanced, obscured, moved, removed, or introduced.” “Adjustments of brightness, contrast, or color balance are acceptable if they are applied to the whole image and as long as they do not obscure, eliminate, or misrepresent any information present in the original, including backgrounds.” J Cell Biol “Information for authors” Disclosure of all image manipulations is required by some journals

    29. However… Capture images well and you won’t have to do much post-processing Limit to: background subtraction contrast enhancement widefield deconvolution methods

    30. Processing and analysis software Adobe Photoshop CorelDraw etc… LAS AF – very limited and idiosyncratic Qwin - complex but powerful for analysis ImageJ - EMBL FREE !! Bundle designed for microscopy imaging

    31. Widefield deconvolution

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