Instruction

Here is a collection of instructional videos and documents for working with microscopes and with images that come from microscope samples. Also included are guidelines for how images are to be post-processed according to commonly accepted rules among scientific publishers (Image Integrity, Image Ethics). An additional section can be found in the image integrity overview regarding how images are best acquired in order to preserve accurate visual information.

The post-processing methods in the instructional videos show steps to eliminate over-manipulation of images to the point at which visual data is obscured or removed. It is HIGHLY recommended that all steps be followed.

The following contains links to scientific imaging categories. The Image Integrity Overview should be read by EVERY researcher in the life sciences!

IMAGE INTEGRITY OVERVIEW

SETTING UP MICROSCOPE FOR COLOR BRIGHTFIELD (HISTOLOGY) SAMPLES

POST-PROCESSING IMAGES (Photoshop, ImageJ, GIMP)
see below

MAKING FIGURES/PLATES (\Inkscape, Illustrator)
see below

POST-PROCESSING IMAGES : Order of Steps

  • History Log (audit trail) and Set Up
  • Opening Files (Scaling Bit Depth, Movies, Extended Focus, Montages or Panoramas, Z-project or Merging)
  • Converting Color to Grayscale
  • Rotate, Flip, Straighten, Crop
  • Uneven Illumination Correction (Phase, DIC, EM, Flatfield Correction)
  • Noise Reduction and Removal (Shot Noise, Color Fringing, Hot Pixel, Deinterlacing)
  • Tonal Adjustment and Linear Histogram Matching (Gamma, Equalize, Match Color)
  • Visualization (Pseudocolor, Colorize, Colocalization, Co-Existence, Merge)
  • For Publication (Scale Bars, Resolution, Figures and Plates)
  • Sharpening

POST-PROCESSING IMAGES: METHODS

KEY FOR METHODS:
B for Brightfield (histology, pathology slides)
F for Fluorescence (confocal, fluorescent dyes)
EM for Electron Microscopy
Q for quantitation (image analysis, quantization, densitometry, density)
A for all and for Figures, Plates

ESSENTIAL METHOD
A History Log (a means to record all your post-processing steps)

OVERVIEW OF PHOTOSHOP
A Set Up Photoshop Environment, Color Settings and Overview for Newbies

OPENING AND MERGING IMAGES
F Problem Files (Files that open too dark or light as a result of bit depth issues)
A Opening Movies
F Opening a series of images, Z-project (merge confocal images)

F Merging Channels, All-in-One Steps (Image J)

B Extended Focus (pictures at different focus to get single in-focus image)
A Panorama or Montage of several images (correct for uneven illumination)

IMAGE CONVERSION COLOR TO GRAYSCALE
B Brightfield, Conventional Images (Plants, Ecology, etc)
F Fluorescent including Confocal Images

GELS, BLOTS, FOR FIGURES
A Rotate, Flip, and Straighten
A Cropping more than one image to the same dimensions

UNEVEN ILLUMINATION
A Q Testing for Uneven Illumination (Darker in some areas, lighter in others)
A Spotlit Center (Typical with inexpensive camera tubes and cheap objectives)
A Flatfield Correct (When a flatfield image has been saved)
A Q Using Image Itself to Correct (Typical for Fluorescence, for quantitation)
A Correction for Phase, DIC, EM Microscope Images

NOISE
F Remove Outliers (Shot Noise, Hot Pixels, Dim Fluorescent Signal images)
Q Median Filtering (Without significant effect on feature borders)
F Surface Blur Method (Salt and Pepper Noise)
F Image Averaging (When several images have been saved)
A Image Size Method (Recommended when destined for Quantization)
F Deinterlacing (Laser Scanning Confocal images, video images)
B Color Noise (Color Fringing)

TONAL AND COLOR ADJUSTMENT
B Manual Method for Matching Images in Figures, Plates
B Auto Method

B All-in-One Method for Brightfield (GIMP)
EM All-in-One Method for EM (Image J)

A Histogram Matching (for densitometry: images taken over time)
EM Gamma or Midtone Adjustment (EM, Brightfield)
B Brightfield Color Adjustments
B Match Color or Grayscale Tones (Brightfield, EM)
A Equalize (Useful for Second Harmonic Imaging, etc.)

VISUALIZATION
A Psuedocolor with Color Bar
A Reduce Number of Colors, Tones (Posterize)
F Colorize Fluorescence
F Colocalization, Co-Existence (Confocal and Fluorescent Channels)
F Merge Fluorescent Images

FOR PUBLICATION
A Scale Bars (Photoshop) Scale Bars (Image J)
A Set Resolution and Dimensions

SHARPENING
A Sharpening Method

MAKING FIGURES/PLATES

INKSCAPE (free program)

Cropping Multiple Images to make figure or plate
Make Figure or Plate
Make Arrows and Boxes

ILLUSTRATOR

Setting Up the Illustrator Environment
Creating a New Page in Illustrator
Making Figure from Images at Same Aspect Ratio
Cropping at Common Crop for Pictures at Different Sizes
Add Lettering to Figures and Plates
Making Inserts and Rectangles
45 Degree Lettering for Arrays, Blots, Gels
Saving Figures and Plates as EPS