Fundamentals of Digital Imaging
Dr Paul McMillan Biological Optical Microscopy Platform
1 FIJI/Image J for Beginners
Fundamentals of digital imaging • The Digital Image (pixels, bit depth) • Image Acquisition (dynamic range, resolution, sampling,) • Basic Rules of Digital Images (data handling & image ethics) • Image Presentation (guidelines, colour sensitivity/blindness & printing)
Applications (Demonstrations & hands on sessions) • Image Import • Image manipulation • Annotation • Visualisation & presentation of 2D data • Visualisation & presentation of 3D data • Visualisation & presentation of timelapse data The Digital Image
Dr Paul McMillan Biological Optical Microscopy Platform Pixels
Your image is recorded as a numerical array of pixels
Pixel has its defined - size (sampling resolution) - grey level (bit depth)
100nm
100nm Bit depth
1 bit 2 bit 3 bit 4 bit 2 grey levels 4 grey levels 8 grey levels 16 grey levels 21 22 23 24 Bit depth – intensity coding
175 175 44679 44830
8bit 16bit Bit depth – Example images
12348-bit Bit depth
250
200
1-bit 150 2 bit 3-bit 4-bit 100 8-bit
50
0 Bit depth - equipment
Bit depth Grey levels Use BOMP equipment 1 2 Binary masks for image FIJI, Imaris, Metamorph, analysis Volocity 5.65 50 Late night reading NA 8 256 Scientific cameras, PMT, PMT, GaAsP (LSM800) GaAsP 12 4096 Scientific cameras, PMT, HyD PMT, HyD (Leica SP8) 15 32,768 Scientific cameras sCMOS (OMX) 16 65,536 Scientific cameras, PMT, PMT, GaAsP (LSM800) GaAsP 32 4,294,967,296 FLIM & other high end techniques
Visualisation = 8 bit, quantification = 12 or 16 bit Image acquisition
Dr Paul McMillan Biological Optical Microscopy Platform Intensity graph
Intensity distribution of image x axis = intensity Y axis = # of pixels with that intensity Intensity graph
All image modification must be ETHICAL Can adjust Minimum & Maximum position DO NOT adjust gamma (non-linear) Be descriptive of modifications in methods Resolution & Sampling
VECTOR RASTER Resolution & Sampling
Frame size 3x3 5x5 10x10 20x20 300x300
Pixel size 1/3 = 0.3 0.2 0.1 0.05 0.003 Nyquist rate
In order to convert analogue signal to digital, signal needs to be sampled at least 2.3 times of half cycle (from top to bottom)
0.83x 1.3x 2.3x 10x
Undersampled Just enough oversampled
Image courtesy of Cameron Nowell Nyquist-Shannon sampling
• http://www.svi.nl/NyquistCalculator
• Pixel dimensions = Theoretical axial resolution/2.3
Magnification NA Type 405 nm 488 nm 561 nm 633 nm 5 x 0.1 Air 704 848 975 1100 10 x 0.3 Air 235 282 325 367 20 x 0.5 Air 141 169 195 220 40 x 1.25 Oil 57 68 78 88 63 x 1.4 Oil 50 60 70 79 100 x 1.4 Oil 50 60 70 79
• Only use this if you need this resolution Get the best representative image
Best representation of what you are seeing on the microscope
Crap in Crap out
Any staining can be positive or negative depending on your imaging setting - No offset on your raw data - Set range with your (+)/(-) control sample - Same setting between samples - Pilot analysis before/during imaging Basic Rules of Digital Imaging
Dr Paul McMillan Biological Optical Microscopy Platform Basic rules of digital data
1. SAVE Raw file Always save as the raw file extension Image metadata .lif or .lei (Leica) .lsm or .czi (Zeiss) .oif or .oib (Olympus) .dv (Deltavision)
Metadata contains all image parameters • Laser wavelength • Voxel dimension • Light path settings • Gain/offset • Objective lens / NA • Bit Depth • Pinhole • Scan speed • Frame Size • Average Basic rules of digital data
Raw file 2. Export Image metadata
TIF JPG, GIF, PNG
Keeps original information Compressed, lossy for data analysis for PowerPoint only Basic rules of digital data
Raw file Raw file 3. Modification Image metadata Image metadata Never process/modify on the raw files. Make a copy to process.
Exported Exported modified Exported modified 4. Backup Exported Image Image Image Image Always double back up your data (NHMRC guidelines) Never use the Microscope computer to store your data
Data Modification Data Modification Graphs details Graphs details Image = scientific data
Image beautification = data manipulation
“oops, I was not aware of this” “sorry, but I thought it was okay to make the images clearer” Modification on a specific feature
Original image Manipulated image
-The gold particles have been enhanced -Background dot has been removed
Image from Mike Rossner, and Kenneth M. Yamada J Cell Biol 2004;166:11-15 Modification on a specific feature
Manipulation revealed Manipulated image ? By contrast adjustment
Cells from other image has been added
Image from Mike Rossner, and Kenneth M. Yamada J Cell Biol 2004;166:11-15 Modification on a specific feature Original image Manipulated image
➢live with it since it isn’t too bad ➢crop it out ➢resection the block and take a new photomicrograph without the tissue fold
Image from http://www.niehs.nih.gov/research/atniehs/labs/assets/docs/q_z/to_adjust_or_not_to_adjust_508.pdf Modification on the background Original image Manipulated image
Background Shading in one corner cleaned up Acceptable only if - No change occurred in the actual tissue - A statement is made in the figure legend
Image from http://www.niehs.nih.gov/research/atniehs/labs/assets/docs/q_z/to_adjust_or_not_to_adjust_508.pdf Modification on the background
Original image Manipulated image
uneven illumination & brown fat has been erased NOT Acceptable because -large degree of background alteration (even if it is clarified and the interested area is not modified)
Image from http://www.niehs.nih.gov/research/atniehs/labs/assets/docs/q_z/to_adjust_or_not_to_adjust_508.pdf Modification to whole image
Original image
Generally acceptable
Misrepresented Modification to whole image
(-)Control (+)control sample black GFP expression on the sample looks similar to (+)control
exposure and contrast to the maximum
Images have not been processed identically
Completely colour balance differs black
Image from Mike Blatt, and Cathie Martin Plant Physiol. 2013;163:3-4 Image presentation
Dr Paul McMillan Biological Optical Microscopy Platform Data presentation guidelines
Cell – Instructions to authors • Any alterations should be applied to the entire image. • Clearly explain all alterations in the figure legend. • Only compare data that are appropriate to compare (e.g., data from the same experiment, acquired the same way). • Individual images should not be used in multiple figures (unless the figures describe different aspects of the same experiment) My 5 cents worth • Be aware of colour sensitivity & colour blindness • Be aware of printing issues Colour
• (Most) Fluorescence detectors are monochrome • Images are usually grayscale • Converted to colour using Look Up Tables (LUTs)
0 255 Blue
Green
Red Colour sensitivity RGB RED GREEN BLUE
255 0 0
0 255 0
0 0 255
255 255 0
255 0 255 RGB vs CMYK
http://wip.blackfox1985.com/wp-content/uploads/2016/03/rgb-vs-cmyk.jpg Colours – RGB vs CMYK
http://www.colorprintingforum.com/attachments/layout-graphics-prepress-software/406d1254902900-how-change-rgb-cmyk-cmyk-vs-rgb.jpg Use grayscale for panels