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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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1. Top Tips for Imaging withthe D Floor Microscopes
Peter Grabowski
3. The D Floor microscopes
4. Microscope capabilities DMI4000B AF6000LXFluorescence + +
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 -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 on40x 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 wont 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