Fundamentals of Digital Imaging

Dr Paul McMillan

Biological Optical Microscopy Platform

1

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

FIJI/Image J for Beginners

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 bit2 grey levels

21

2 bit4 grey levels

22

3 bit8 grey levels

23

4 bit16 grey levels

24

Bit depth – intensity coding

8bit

175 175

16bit

44679 44830

Bit depth – Example images

1-bit2-bit3-bit4-bit8-bit

Bit depth

0

50

100

150

200

250

1-bit

2 bit

3-bit

4-bit

8-bit

Bit depth - equipment

Bit depth Grey levels Use BOMP equipment

1 2 Binary masks for image analysis

FIJI, Imaris, Metamorph, Volocity

5.65 50 Late night reading NA

8 256 Scientific cameras, PMT, GaAsP

PMT, GaAsP (LSM800)

12 4096 Scientific cameras, PMT, HyD PMT, HyD (Leica SP8)

15 32,768 Scientific cameras sCMOS (OMX)

16 65,536 Scientific cameras, PMT, GaAsP

PMT, GaAsP (LSM800)

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

VECTOR RASTER

Resolution & Sampling

10x10

0.1

Frame size3x3

Pixel size 1/3 = 0.3

5x5

0.2

20x20

0.05

300x300

0.003

Resolution & Sampling

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 2.3x

Just enough

10x

oversampled

1.3x

Undersampled

Image courtesy of Cameron Nowell

• http://www.svi.nl/NyquistCalculator

• Pixel dimensions = Theoretical axial resolution/2.3

• Only use this if you need this resolution

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

Nyquist-Shannon sampling

Get the best representative image

Best representation of what you are seeing on the microscope

- No offset on your raw data- Set range with your (+)/(-) control sample

- Same setting between samples- Pilot analysis before/during

imaging

Any staining can be positive or negative depending on your imaging setting

Crap in Crap out

Basic Rules of Digital Imaging

Dr Paul McMillan

Biological Optical Microscopy Platform

Raw file

Basic rules of digital data

1. SAVE

Always save as the raw file extension

.lif or .lei (Leica)

.lsm or .czi (Zeiss)

.oif or .oib (Olympus)

.dv (Deltavision)

Metadata contains all image parameters

Image metadata

• Laser wavelength• Light path settings • Objective lens / NA• Pinhole• Frame Size

• Voxel dimension• Gain/offset• Bit Depth• Scan speed• Average

Raw file

Basic rules of digital data

2. ExportImage metadata

TIF

Keeps original informationfor data analysis

JPG, GIF, PNG

Compressed, lossyfor PowerPoint only

Basic rules of digital data

3. Modification

Never process/modify on the raw files. Make a copy to process.

Raw file

Image metadata

Exported

Image

Exported

modified

Image

DataGraphs

Modification

details

Raw file

Image metadata

Exported

Image

Exported

modified

Image

DataGraphs

Modificationdetails

4. Backup

Always double back up your data (NHMRC guidelines)

Never use the Microscope computer to store your data

Image = scientific data

“oops, I was not aware of this”

“sorry, but I thought it was okay to make the images clearer”

Image beautification = data manipulation

Modification on a specific feature

Image from Mike Rossner, and Kenneth M. Yamada J Cell Biol 2004;166:11-15

Original image Manipulated image

-The gold particles have been enhanced-Background dot has been removed

Modification on a specific feature

Image from Mike Rossner, and Kenneth M. Yamada J Cell Biol 2004;166:11-15

Manipulated image ?Manipulation revealed

By contrast adjustment

Cells from other image has been added

Modification on a specific feature

➢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

Original image Manipulated image

Modification on the background

Background Shading in one cornercleaned up

Acceptable only if- No change occurred in the actual tissue- A statement is made in the figure legend

Original image Manipulated image

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

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)

Original image Manipulated image

Image from http://www.niehs.nih.gov/research/atniehs/labs/assets/docs/q_z/to_adjust_or_not_to_adjust_508.pdf

Generally acceptable

Modification to whole image

Original image

Misrepresented

Modification to whole image

exposure and contrast to the maximum

Images have not been processed identically

Completelyblack

colour balance differs

(-)Control (+)control sample

black

Image from Mike Blatt, and Cathie Martin Plant Physiol. 2013;163:3-4

GFP expression on the sample looks similar to (+)control

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)

Blue

Green

Red

0 255

Colour sensitivity

RGB RED GREEN BLUE

0

255

0

255 0

00

2550

0

0

255 255

255255

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