Concepts for Photomicrography

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ConceptsforPhotomicrography
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Introduction

• Microscopy is a visual art. It encompasses the
  recording, storage, and transmittance of
  information in the form of images.
• Microscopists are as concerned about context as
  they are about content.
• Visual communication is about sharing key
  information, not necessarily all information.

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Introduction

• The ultimate function of the microscope and its
  illumination is to produce the best possible
  image of the specimen.
• The ultimate function of the camera is to capture
  the best possible image of the specimen.

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Photography

• 1. The science which relates to the action of
  light on sensitive bodies in the production of
  pictures, the fixation of images, and the like.
• 2. The art or process of producing pictures by
  this action of light.

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Imaging

• A basic image capture system using film contains
  a lens and a detector. The detector
  is the film.

• Digital photography also uses a lens, the
  detector is a solid state image sensor called a
  charge coupled device or CCD.

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Film Photography
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Photography

• We owe the name "Photography" to Sir John
  Herschel , who first used the term in 1839, the
  year the photographic process became public.

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Introduction

• Photography
• From the Greek
• PHOS Light
• GRAPHE Drawing

Literally, photography is Drawing with Light.
Traditionally, photography is considered to mean
the recording of images on light-sensitive
media, using a camera.
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Photography

• The first successful picture was produced in
  June/July 1827 by Niépce, using material that
  hardened on exposure to light. This picture
  required an exposure of eight hours.

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Film Types
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Film Types

• Black and White
• Colour
• There are two different types of colour film.
• 1.) Print film
• 2.) Slide film

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Film Types
In colour film, the emulsion is made up of layers
sensitive to blue, green, and red light.
Black and white film is made up of a chemical
mixture (called an emulsion") protected by a thin
plastic layer.
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Film Types

• Black and White Film
• Black and white film selection tends to be more
  complicated than color.
• Different brands produce vast differences in the
  finished picture.

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Film Types

• 1.) Print film
• Also called negative film, it is the most popular
  type.
• Print film ranges typically from ISO 100 to ISO
  400, but can also be found in higher and lower
  speeds.
• Print film is recommended most for the amateur
  photographer because the colour balance is
  generally better and the risk of exposure damage
  is lower than with slide film. 
• Slower color print film tends to be better for
  color, with more vibrant colours and contrasts,
• Faster color print film is noticeably more
  grainy, and has less colour in the picture.

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Film Types

• 2.) Slide film
• Also called reversal film, has a much wider range
  of use than Print Film.
• Available anywhere from less than ISO 25 to ISO
  6400 and up.
• For good results in your pictures using slide
  film, you should be really experienced in
  photography.
• Exposure mistakes are much more dramatic with
  slide film than with print film, because slide
  film is much less forgiving.
• Slide film is much easier to make prints from in
  a darkroom. The colours that result from using
  slide film are much more intense and vibrant.

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Film Types

• The principle factor that determines selection of
  film is the subject to be photographed.
• a film that is ideal for one subject may be
  completely unsuitable for another.
• Other factors include
• the type of camera to be used,
• how a photographer wishes to photograph the
  subject to achieve the kind of images he or she
  wishes to capture,
• the subjects illumination,
• whether the final image will be a black and white
  or colour print, or a slide, and so on.

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Film Types

• BASIC DIFFERENCES IN FILMS
• The fundamental ways in which films differ from
  one another are
• 1. Format (principally roll and sheet film),
• 2. Size,
• 3. Sensitivity to colour,
• 4. Film speed,
• 5. Graininess, and
• 6. Gradation (contrast).

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Film Types
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Film Types

• Example Film Spectral Curves
• Kodak
• HS IR
• Konica
• 750
• TMAX
• 400

32 Light Source
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Film Types
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Film Types
Filter Comparison
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Film Types
Effective speed of all films, grouped by filter
Light Source 32
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Film Types
Understanding Colour InfraredTo understand why
colours appear completely different from what you
would expect we need to examine how colour
infrared film works.Colour IR film comes with
three colour sensitive layersInfrared and
blueGreen and blueRed and blue
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Film Speed
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Film Speed

• Film Speed is a measure of a films sensitivity
  to light.
• The more sensitive the film, the higher its
  speed-rating.

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Film Speed

• The speed is based on the size of the silver used
  to cover the film.
• If large (relatively speaking) chunks of silver
  are used, there are less chunks that need to be
  exposed to light. Thus, the image is produced
  very quickly, and the film is called a fast film.
  
• If the silver is in very fine particles, more
  need to be exposed before the image is produced,
  and the film is much slower.

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Film Speed
ISO - International Standards Organization - a
group of standards setting bodies from many
countries.
ASA - American Standards Association - the
standards setting body in the United States -
became the American National Standards Institute
(ANSI) in 1970 - ANSI is a member of ISO
DIN - Deutsches Institute Für Normung - the
standards setting body in Germany - DIN is a
member of ISO
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Film Speed

• Film Speeds are given as ISO Numbers
• ISO 400 / 27
• The first number is the old ASA speed, an
  arithmetic measure of sensitivity.
• The second number is the European DIN speed, a
  logarithmic measure.

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Film Speed

• With ISO numbers, each doubling of the number
  indicates a doubling of the film sensitivity
• ISO 100 ? ISO 200 ? ISO 400
• ISO rating is how sensitive (fast) the film is.
• ISO 200 is twice as sensitive as ISO 100, and
  only half as sensitive as ISO 400.
• ISO 200 also has less image quality than 100, but
  more than 400 (image quality is not proportional,
  nor is it constant across brands of film.)

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Film Speed

• With DIN numbers, every three steps up the number
  scale indicates a doubling of the film
  sensitivity
• DIN 21 ? DIN 24 ? DIN 27
• DIN rating is how sensitive (fast) the film is.
• DIN 24 is twice as sensitive as DIN 21, and only
  half as sensitive as DIN 27.
• DIN 24 also has less image quality than 21, but
  more than 27 (image quality is not proportional,
  nor is it constant across brands of film.)

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Film Speed

• Film Speed is important for several reasons
• Proper exposure of the film
• Affects shutter speeds and lens apertures
• Determines the degree of sharpness
• Determines the depth of field

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Film Speed

• Films with an ISO rating of 400 or more are
  considered fast.
• Fast films are generally
• Grainier
• Less sharp
• Less contrasty
• Lower in colour saturation
• Fast films require less light and are therefore
  better for moving subjects.

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Light Source
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Light Source

• Light Source Dominant Color Example Use
• Natural Sunlight (5400K) WhiteUVIR NA
• Florescent (7500K) Cyan Room lighting
• Florescent (5000K) White Color
  viewing
• Florescent Black-Light UV Dylux
  (bluelines)
• Incandescent (2500K) Yellow NA
• Mercury Vapour Blue-green Plates most proofs
• Metal Halides Blue Plates, proofs, contacts,
  duplication
• Quartz-Iodine Yellow B/W proc. cam.,
  contacts, duplication
• Pulsed-Xenon White Color process camera
• Carbon Arc Blue No longer used
• Lasers Vary Scanners, imagesetters

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Light Source

• For ordinary purposes the visible range lies
  between 400 and 700nm
• In microscopy the possible photographic range is
  between 250 to 1100nm
• UV 250 ? 400nm
• Vis 400 ? 700nm
• IR 700 ? 1100nm

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Light Source
The spectral energy plots for tungsten and
sunlight illumination are different. Tungsten
illumination is much "warmer" and contains much
more energy in the red portion of the spectrum.
Sunlight has more energy in the "cooler" or blue
portion of the spectrum.
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Light Source

• Spectral energy plots for sunlight and two types
  of tungsten illumination.

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Focus and Exposure
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Focus and Exposure

• Camera exposure is dependent on four variables
• the aperture
• controls the intensity of light entering the
  camera,
• the camera shutter
• controls the duration of light entering the
  camera ,
• the sensitivity of the film to light
• Different films require more or less exposure to
  light to produce usable images,
• the intensity of the light source.

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Focus and Exposure

• Lens Focal Length
• It is the distance from the lens to the film,
  when focused on a subject at infinity

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Focus and Exposure

• F-Stop
• F-stop is the focal length divided by the
  diameter of the lens. ie. a 200mm f/4 lens will
  be 50mm wide.
• 200mm/50mm f/4
• F/4, meaning "focal-length over 4" or
  "focal-length divided by four".

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Focus and Exposure

• Shutter Speeds
• exposure controls run through a sequence of
  settings which involve doubling and halving the
  amount of light reaching the film.
• Shutter speeds are measured in seconds and
  fractions of a second and so the doubling and
  halving is self-evident. One quarter second is
  half as long as one-half second but is twice as
  long as one-eighth

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Focus and Exposure

• On the Nikon FE, for instance, the shutter speed
  sequence is 
• 8 sec    4 sec    2 sec    1 sec    1/2 sec
  1/4    1/8    1/15    1/30    1/60    1/125    1/2
  50    
• 1/500    1/1000
• Each of these settings is clearly half/double the
  length of time of its immediate neighbours.
• This doubling/halving is thus pretty simple to
  comprehend for this exposure setting.

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Focus and Exposure
The f/stop is a ratio. The ratio is between the
diameter of the aperture in the lens and the
focal length of the lens. The progression of
f-stops, 1 1.4 - 2 - 2.8 - 4 - 5.6 - 8 - 11
16 - 22, are powers of the square root of 2.
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Vignetting
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Vignetting

• Vignetting
• A photograph or drawing whose edges gradually
  fade into the surrounding paper is called a
  vignette.
• Optical vignetting causes a gradual darkening of
  the image towards the corners.

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Vignetting
Optical vignetting is also known as artificial or
physical vignetting. Its origin relates to the
simple fact that a lens has a length.
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Vignetting

• Entrance pupil, the aperture stop seen through
  all lens elements in front and from a position on
  the optical axis.
• Half the full angle of view of the lens. Here,
  the white openings correspond to the clear
  aperture for light that is heading for the image
  corner

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Vignetting

• When mechanical extensions to a lens protrude
  into its field of view, the image corners receive
  less light than they would in the absence of the
  extension and vignetting occurs.

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Digital vs. Film
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Digital vs. Film
57 Digital Camera Technology
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Dynamic Range

• We can distinguish about 100 levels of luminance
  but logarithmically spaced.
• Colour slide films can record as many as 10,000
  discrete levels (213) and do a good job of
  representing what our eyes see.
• Digital cameras must sample at least 213 levels
  and present at least 256 levels to the eye per
  colour to rival film.

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Pixel Equivalents
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Camera Comparisons
Cooled, c-mount, and base software in CDN
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Microscopes Resolved
Sensor 8.5mm x 6.4 mm (2/3 inch) at 500 nm
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Higher Adapter Magnifications

• Benefits
• Less pixels required to fully resolve image
• Smaller stored image
• Very high object magnification

• Costs
• Longer exposures required
• Less image area recorded (loss of context)
• Very high object magnification

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DigitalPhotoMicrography
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Digital Camera Terminology
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Camera Terminology

• Pixel Size
• Size of each CCD element
• Sensor Size
• Size of whole imaging area of the camera
• Spectral Range
• Wavelength dependant sensitivity of sensor

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Camera Terminology

• Full Well
• Number of electrons of charge each element can
  hold related to number of luminance levels which
  can be recorded.
• Dynamic Range
• Bit depth at which signal is digitized.
• Readout Rate
• The rate at which the signal is digitized.

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Camera Terminology

• Dark Current
• The signal created in the absence of any light
  (thermal effect).
• Readout Noise
• Amplifier or F.E.T noise that is rate dependant
  and created once at time of readout.
• Binning
• Clustering of groups of pixels to increase
  sensitivity at the cost of resolution.

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CCDCharge Coupled Device
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CCD

• Global Phenomenons
• Dark Current
• Temperature dependent increasing by factor of
  two when temperature is increased about 7 degree
  celsius
• Readout Noise
• Dependant on readout speed
• Photo Response Non Uniformity
• Cosmetic Defects
• Column Defects
• Bright columns
• Dark columns
• Pixel Defects (Blemishes)
• Bright pixels (hot spots)
• Dark pixels (black spots)

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CCD
Column Defect
Dark Pixels
Hot Spots
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Cooling and Noise
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Cooling and Noise

• Cooled Cameras
• integration time is limited by dark current
• thermally generated electrons not distinguishable
  from optically generated electrons
• while long integration times, pixels will
  saturate due to dark current
• rule of thumb
• reducing sensor temperature by 7 Celsius reduces
  the dark current by the factor of 1/2
• methods of cooling
• single stage peltier element with radiator
• double stage peltier element with fan
• double stage peltier element with liquid cooler

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Cooling and Noise

• Advantages of cooling
• one method of increasing the low light capability
  
• Disadvantages of cooling
• heavy cooling causes condensation effects
• sophisticated design of the sensor housing
  required
• expensive technology

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Digital Cameras

• Advantages
• cheap
• portable
• can be operated without PC due to Memory Cards
• Disadvantages
• image compression JPEG or FLASHPIX
• loss of resolution, image artifacts
• often low spatial resolution
• all single chip cameras
• integrated lenses difficult adaptation
• often noisy images

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Digital Imaging
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Digital Imaging

• Digital imaging is any pictorial or graphical
  information stored or presented as binary
  information in an electronic device.
• The original information could be digitized
  from many devices.
• Analogue CCD cameras
• Digital CCD cameras
• Scanners
• Laser / Flatbed / Slide

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Digital Imaging

• On a CCD, a matrix of hundreds of thousands of
  microscopic photocells creates pixels by sensing
  the light intensity of small portions of the film
  image.

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Digital Imaging
A typical image sensor has square photocells
(pixels) arranged in rows and columns.
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Digital Imaging

• When an image is focused through the camera lens,
  it falls on the image sensor.

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Digital Imaging

• The quality of a digitized image is determined by
  
• pixel size spatial resolution pixel depth
  brightness.

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Capture

• When a camera samples the image, it divides the
  image into pixels. The size of pixel depends upon
  the number of photocells.
• Fewer photocells Lower resolution
• More photocells Higher resolution

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Capture

• In low resolution images the granular texture
  and blurred appearance tell us that pixelization
  has occurred.

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Capture

• A CCD with more photocells will sample at
  higher spatial resolution.In this kind of image
  individual pixels can no longer be seen.

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Capture

• Higher resolution produces images that are
  crisp and clear in which pixelization is not
  apparent.

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Digital Imaging
Colour?
Colour?
Colour?
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Digital Imaging

• To capture colour images red, green and blue
  filters may be placed over the photocells.

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Capture
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Capture
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Capture

• In a color image, each pixel is assigned three
  8-bit numbers for the red, green and blue
  brightness values.
• For example, this pixel is created by assigning a
  
• red brightness level of 227
• green brightness level of 166
• blue brightness level of 97.

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Visual Resolution
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Visual Resolution
Human Visual Resolution The maximum number of
black lines that can be crowded side-by-side with
equal width white spacing without appearing as a
single gray mass to the observer.
39 x 60 2340 lines horizontally 49.25 x 60
2955 lines vertically or 269 lines/inch 538
pixels/inch horizontally 275 lines/inch 550
pixels/inch vertically
Resolution eye 60
lines/degree (1 degree 290 microns 120 cones
on retina)
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Visual Detail

• Absolute resolution of black and white lines is
  only significant for printers.
• We view and recognize images through luminance
  and chrominance detail.
• The visual limit for luminance detail is about
  200 micron at 350mm.

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Capture

• Dynamic range indicates how well the camera
  can differentiate between light levels.

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Capture

• To accurately render highlights and shadows,
  camera exposure must be controlled precisely.

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Capture

• Noise is another factor. The information captured
  by a sensor contains both image data and noise.

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Capture

• Another factor in digital image capture are
  artifacts (distortions) such as the moiré pattern
  that occurs when an image is under sampled.

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• Processing

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Processing

• The most common analysis operation is the
  histogram which is a bar graph showing the number
  of pixels at each gray level.

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Processing

• An image with good contrast and good dynamic
  range generates a histogram with a pixel
  distribution across the brightness range from 0
  to 255.

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Processing
This brown moth on a grey card has most of its
values in the midrange. That's why there is a
number of high vertical lines grouped in the
middle of the horizontal axis.
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Processing
This high-key fog scene has most of its values
towards the highlight end of the scale.
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Processing

• A high contrast image generates a histogram
  with high pixel count at the white and black
  extremes of the range.

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Processing

• Image enhancement processes are based on
  fundamental ways in which image data can be
  changed mathematically.
• Three ways in which histogram information can be
  manipulated.
• Slide Mapping
• Stretch Mapping and
• Complement mapping

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Processing

• Slide Mapping changes brightness by adding or
  subtracting a constant value.
• For example, adding a constant of 50 to every
  pixel in this image, slides the histogram to the
  right by 50 gray levels.

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Processing

• Stretch Mapping improves poor contrast by
  multiplying or dividing each pixel by a constant.
  Multiplying "spreads" the pixel values out so
  that a greater range of gray is used.

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Processing

• Complement mapping changes the digital value of
  each pixel to reverse the image. Black pixels
  become white. White pixels become black. And gray
  pixels become their complement.

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Processing

• The mapping functions
• Pixel Point Processing.
• Pixel Group Processes, and
• Frame Processes.

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Processing

• In Pixel Point Processing a mathematical function
  "maps" the input value of each pixel to a new
  output value. This lightens or darkens the image,
  or changes contrast.

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Processing

• Example of Pixel Point Processing
• To make color corrections to 24-bit color images,
  mapping operations can be applied to the red,
  green and blue color planes.

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Processing

• Reducing the red color plane by 50 levels moves
  the color balance towards cyan.

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Processing

• Reducing the green color plane by 50 levels moves
  the color balance towards magenta.

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Processing

• Reducing the blue color plane by 50 levels moves
  the color balance towards yellow.

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Processing

• In Pixel Group Processing, a mathematical process
  called a convolution changes a pixel's values
  based on the brightness of the pixel and its
  neighbors.

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Processing

• Some examples of Pixel Group Processing are
• Noise Filtering
• Sharpening
• Blurring

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Processing

• This example of noise filtering changes all black
  pixels surrounded by white pixels to white.
• This eliminates the noisy dots, but leaves the
  rest of the image unchanged.

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Processing

• Group Processes can be used to sharpen an image

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Processing

• Group Processes can be used to blur a portion of
  the image, such as a busy background.

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Processing

• In Frame Processing, the image is manipulated by
  changing the locations of pixels of the entire
  image or a portion of the image.

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Processing

• Some examples of Frame Processing are
• Image Rotation
• Scaling

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Processing

• Image Rotation rotates the image a specified
  value, such as 90 degrees or 180 degrees

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Processing

• Image Scaling reduces the size of the image
  through a process called decimation, or enlarges
  the size by replication or interpolation.

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Processing
Decimation removes pixels to reduce the size of
an image. To reduce it by half, every other line
and row of pixels is removed.
Replication enlarges images by duplicating pixels.

• Interpolation enlarges images by averaging the
  values of neighboring pixels to calculate values
  for the added pixels. This produces higher
  quality enlargement than replication.

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Processing

• Transforms are frame processes which place image
  data into another space, or domain, so that it
  can be more readily manipulated.

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Processing

• Transforms can also provide precise filtering by
  separating an image into its spatial frequency
  components, then manipulating specific
  frequencies.
• For example, edges can be enhanced by increasing
  the high spatial frequencies.

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Processing

• Image compression reduces image data by
  identifying patterns in the bit strings
  describing pixel values, then replacing them with
  a short code.
• For example, a scan line beginning with 9 black
  pixels followed by 5 white pixels could be
  encoded as "9b, 5W."

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Processing

• In much the same way, a color image can be
  compressed by grouping the data for similar
  pixels.
• For example, a group of 20 pixels can be encoded
  with one pixel address and color value.

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Processing

• There are two basic types of data compression
• lossless compression and
• lossy compression.

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Processing

• Lossless compression achieves only about a 21
  compression ratio, but the reconstructed image is
  mathematically and visually identical to the
  original.

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Processing

• Lossy compression provides much higher
  compression rates, but the reconstructed image
  shows some loss of data compared to the original
  image.

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Processing

• Visually lossless compression is based on
  knowledge about color images and human
  perception.

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Printing

• Printer resolution is stated as dots/inch (DPI)
• High resolution printers print 1200 DPI in BW
• Colors and gray levels require half toning
• Enhanced color printers can reach photo quality

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Printing

• DPI or dots-per-inch, refers to the size of the
  spots created by the output device.

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Printing

• Digital printers can be divided into two basic
  groups
• halftone printers and
• continuous tone printers.

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Halftones or Dithering

• A pattern of dots used together to produce a
  desired color or gray level.
• 64 gray levels require a grid of 8X8 dots to
  represent each image pixel.
• Dots may be different sizes and can be overlaid
  to produce more tones.
• Colors are produced by using 3-5 different
  colored dots that are dithered or overlaid.

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Halftones or Dithering

• Through a process called dithering, halftone
  printers group pixels to simulate continuous tone
  gray scale, or a continuous range of color.

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Halftones or Dithering

• For color printing, these printers increase color
  range by grouping dots of cyan magenta and yellow
  to simulate a third color. For example,
  alternating cyan and yellow dots look like green.

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Continuous Tone

• Since continuous-tone printers blend the colors
  as they are printed, they do not have to use
  dithering or half toning.That's why thermal
  prints with a resolution of 300 dpi can be
  superior to a 600 dpi halftone print.

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Information Management
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Information Management

• For collaboration on projects, digital images are
  being shared by teams working in the same
  building, via local area networks.

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Output

• Images are also being transferred between
  researchers around the world by linking computers
  via the internet.

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Information Management

• Digital images can be easily data based
• Software can automatically record
• Specimen location / Magnification
• Exposure / Filters used
• Date / operator
• Special Notes / Key Words
• Databases can be queried
• Network / Internet Access

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Thank You The End