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1. Visible line pairs on CCD sensor FOV 1 414 1400 linepairs 800 linepairs Diagram 2 In the above example the lens is capable of better resolution than today s high resolution cameras can capture At higher magnification a lower megapixel camera is required Lets take a 50x 0 85 NA objective with a square FOV as an example 22m 2 50x V2 Achievable Optical Resolution 0 85 NA x 3000 2550 Ip mm Line pairs covering the camera s sensor 0 31mm x 2550 Ip mm 790 Ip Required monochrome camera resolution 3 pixels Ip 0 31mm gt 4 2 megapixels Required color camera resolution 4 pixels Ip gt 7 5 megapixels continued on page 3 A Powerful Vision continued from page 2 Do Need a High Resolution Digital Camera Now let s get back to our original question High resolution 8 plus megapixel cameras provide a resolution benefit at low magnification But as magnification increases they provide less of an advantage What is optimum depends on what kind of imaging you are doing More pixels will increase the size of the image file As the number of pixels increases each pixel is typically smaller which results in reduced dynamic range and light sensitivity Going beyond 8 megapixels makes sense for very low magnifications typically used in comparison forensic macroscopy if the entire available FOV is used But even with fewer pixels adjusting the magnification to2. A Powerful Vision January e 27 eica N 6 MICROSYSTEMS To See or Not to See By Rob Kimura Leica Product Manager As the evolution of digital photomicrography continues new genera tions of high resolution cameras have become available for forensic imaging Digital camera technology is mainly driven by the consumer market where the more pixels the better is the status quo In recent years the consumer market has seen color cameras jump from 1 3 megapixels to 12 megapixels and higher A common question people ask is How much higher will camera resolution get But the real question for forensic investigators should be What do gain by using a high resolution digital camera on my microscope Understanding Image Formation Due to the physics of the image formation process even a perfect mi croscope objective will blur two adjacent objects into a single object when placed close enough together One way to consider this limit ing resolution is to image a repeating pattern of adjacent black and white lines When the number of line pairs per millimeter Ip mm is increased beyond the optical resolution limit of the microscope the image will no longer form lines but instead will form a uniform gray background In addition to blurring an image an objective lens also magnifies an object At the camera this translates into an image spread across a larger area whenever magnification is greater than 1x
3. the camera with an intermediate optic accessory such as a 1x magnification c mount you can match the camera to the microscope s resolution across a limited field of view Comparison Illumination Achieving the Best Illumination for Your Comparison Microscope by Wayne Buttermore Leica Marketing Manager Forensic Microscopy One of the biggest challenges in comparison microscopy is balancing color and intensity of the light sources between the two microscopes Unlike reflected light applications the transmitted light comparison microscope has many more variables that affect both illumination intensity and color balance The setup of a compound microscope for diffraction limited examina tion of trace evidence Is best achieved by setting up Koehler illumina tion Experienced microscopists can reproducibly establish the settings for the field and aperture diaphragms on an individual microscope to achieve the best resolution and contrast for a sample However when a second microscope is added to the system match ing the setup becomes far more complex First let s look at the different illumination types that are used for comparison microscopy 1 Two separate lamphouses controlled by separate power supplies This was a common illumination setup found on older comparison microscopes from American Optical Leitz and Leeds Pre 1980 2 Two separate lamphouses controlled by separate power supplies with continuous variable filter sys
4. Also a microscope may be configured with intermediate optical components to change the net magnification to the camera for the purposes of this article we will assume that the microscope has a 1x magnification c mount attachment How Many Pixels One would expect that the ideal pixel correlation would place 2 pixels across each line pair so that one pixel can detect the white line and the other pixel the black line However this pixel ratio can produce a gray result because pixels can be placed between the white and black lines To resolve all line pairs in all cases there must be at least 3 pixels per line pair As you can see in the ideal case shown in 2 pixels across 2 pixels per line pair Ideal gt 3 pixels each line pair shifted 1 2 pixel per line pair Diagram 1 Diagram 1 with at least 3 pixels per line pair the camera can now detect the line pairs even if pixels shift to the left or right It is impor tant to note that further increasing the number of pixels can lead to over sampling where the additional pixels per line pair provide no gain in spatial information However the transition to over sampling depends on the wavelength of light used the objective s numerical aperture magnification to the camera and the camera s pixel size Determining Resolution as Defined by Line Pairs With simple assumptions we can estimate the limiting resolution for a microscope objective determine the number of line pairs a
5. a sensor is typically square or rectangular If the FOV for the microscope is 22mm then the FOV of the sensors reduced by the square root of 2 Example Using a 5x objective with a 15 NA and a 22mm FOV 44 v2 3 1mm Achievable Optical Resolution 0 15 x 3000 450 Ip mm Line pairs covering camera sensor 3 1mm x 450 Ip mm 1400 Ip Monochrome Camera The monochrome camera presents an ideal case as every pixel con tributes equally to the resolution The number of pixels required to capture every bit of spatial information coming from the objective is Pixels across line pair 1400 Ip x 3 pixels Ip 4200 pixels Camera resolution is specified in terms of the total number of pixels so assuming a rectangular 4 3 format the number of pixels needed for ideal digital image quality is 4200 pixels x 3150 pixels 13 megapixels Color Camera A pixel in a color Bayer Matrix camera performs two functions spatial sampling of the image and measuring the intensity for a specific position of the spectrum e g red green and blue Consequently it takes more pixels approximately 25 more per line pair to obtain the same resolution as a monochrome camera Required color camera resolution 4 pixels Ip gt 23 megapixels Compound Lens 5x 50x Size of visible spot Optical Resolution of Lens NA x 3000 Visible line pairs in FOV 22mm 4 4 x 450 1980 linepairs 0 44 x 2550 1122 linepairs
6. cross the field of view FOV and compare this to the number of C ontents pixels covering the same distance fora A Powerful Vision page 1 given camera There Comparison Illumination page 3 are many mathemat S Industry News page 5 ical definitions for GOSSA eeaeee sea eee page 5 optical resolution R but a simple continued on page 2 A Powerful Vision continued from page 1 approximation is A 2 NA where is the wavelength of the light in nanometers nm This relationship indicates that when using a high NA lens and white light illumination the smallest resolvable distance is about 300nm or 0 3um In terms of line pairs per mm at a mid wavelength of the visible light spectrum green 550nm Optical Resolution Ip mm NA x 3000 To calculate the number of line pairs required to cover the objectives FOV calculate the area visible through the lens by dividing the FOV of your microscope let s assume 22mm by the magnification factor A 5x objective allows you to observe an area of 4 4mm 22mm FOV 5 A 50x objective allows you to observe an area of 44mm only less than 0 5mm 22mm FOV 50 0 44mm So how many line pairs can be observed with a 5x objective Simply multiply the visible area by the optical resolution to calculate the FOV But please note that there is one caveat in calculating FOV While a microscope objective forms a circular image a camer
7. g the aperture diaphragm to Q T Figure 49 establish Koehler illumination match the objective in use Leica users can refer to the Leica DM R user manual Figure 49 Discussion of Koehler illumination is outside the scope of this article In microscopes with a bifurcated illumination system these steps should be all that is required to obtain visually balanced light intensity and color Two Lamphouse Systems Systems having two light sources and Variolux continuous illumina tion control may require further adjustment A Variolux contains three independent filter controls for continuous introduction of red green or blue filters with increasing gradients This introduces infinitely adjustable color to the illumination path on each microscope With practice it provides a perfect match of color and intensity during side by side examinations Procedure for Adjusting Variolux 1 When starting be sure to re move any colored filters from the optical path Essentially this means neutralizing the Variolux by turning the filters to the open position It is easiest to accomplish this by observing the field of view and turning each color filter control If col Color filter wheel or is added while turning the knob reverse direction until it reaches the stop position This needs to be completed on both microscopes If any colors were introduced by the Variolux during initial illumination setup as described above repea
8. in Colorimetry which serve to define the color white Depending on the application different definitions of white are needed to give acceptable results Color Temperature Visible light is commonly described by its color temperature A traditional incandescent light source s color tempera ture is determined by comparing its hue with a theoretical heated black body radiator The lamp s color temperature is the tempera ture measured in kelvins at which the heated black body radiator matches the hue of the lamp Color temperature is sometimes used loosely to mean white balance or white point Line Pairs Resolution Line pairs are often used to measure resolution instead of lines A line pair is a pair of adjacent dark and light lines while a line counts both dark lines and light lines A resolu tion of 5 line pairs per mm means 5 dark lines alternating with 5 light lines or 10 lines per mm Editorial Staff Editor in Chief Managing Editors Graphic Design Contributing Editor Molly Lundberg Pam Jandura Wayne Buttermore M N Kennedy Rob Kimura Note We are interested in your comments and thoughts about the newsletter Please feel free to email your comments to molly lundberg leica microsystems com
9. ning conference The host committee comprises not only the San Francisco Police Department Firearms and Toolmark Unit but also firearms examiners from various agencies all over Northern California such as Contra Costa County Sheriff s Office Santa Clara County Fresno DOJ Sacramento DOJ BATFE Walnut Creek Sacramento County and Oakland PD More information www afte org The 2007 ASQDE Meeting will be held on August 11 16 2007 at the Boulder CO Millennium Harvest House More information will be provided as it becomes available www asqde org The 2007 Annual Meeting of the Southern Association of Forensic Scientists will be on September 9 14 2007 in Atlanta A full program is planned including Advanced Structure Elucidation DI LC Tandem MS use in Post mortem Cases and Statistics in DNA Analysis More information www southernforensic org Ge he age Glossary Koehler Illumination A characteristic feature of the light path provided by all high quality microscopes is the consistent imaging of the luminous spot on the light source and the illuminated field through all imaging stages of the microscope from the light source to the final image These conditions are met when the field and aperture diaphragm positions in the microscope light path are conjugated to the object plane and to the rear focal plane of the objective respectively White Point A white point is one of a number of reference illumi nants used
10. s to contend with In systems having a Leica Variolux adjustable filter device the process of balancing illumination and color is simplified How do I ensure balanced intensity and color Initially it is critical to set up the two light sources as uniformly as possible 1 Prepare two identical slides with slides and coverglass from the same box Place a slide on each microscope stage 2 Place the comparison bridge in the side by side image mode 3 Select the 10x objective for each microscope a When using a two lamphouse system adjust lamp intensity so the voltage is the same for both sides Adjust the collector lens for each lamphouse so that intensity and homogeneity of the field of view is as uniform as possible b In microscopes with bifurcated fiber optic systems adjust lamp intensity to a comfortable level for the objective in use If intensity or homogeneity is not consistent rotate the fiber bundle at its interface with the microscope Then secure it in place Intensity can be influenced by moving the fiber optic guide forward or backward in the mount 4 Be sure to remove any colored glass filters polarizers or polarization compensators from the light path 5 Remove the first two slides from step 1 and place two new slides each having a sample and coverglass on the stage of each of the microscopes 6 Establish Koehler illumination by setting the condenser height gt a centering the field diaphragm and adjustin
11. t the illumination setup 2 Carefully examine the color of the light on each microscope side Keep in mind the complimentary color If a yellow tint is viewed on one side the addition of an equal amount of blue would be required to offset the effect If the color match gets worse as the process continues start over 3 When any component in the optical path is changed such as the sample objective or aperture setting you may need to readjust the Variolux Balancing illumination intensity and color takes practice It is much easier to set up a system for visual examination A camera system whether 35mm or digital is far more sensitive to color and density differences than the human eye so fine tuning the camera may still be necessary Next issue Achieving the best illumination for your digital camera Industry News The AAFS American Academy of Forensic Sciences will hold its 59th Annual Scientific Meeting on February 19 24 2007 at the Henry B Gonzalez Convention Center in San Antonio Texas The Academy s annual scientific meeting presents over 500 scientific papers breakfast seminars workshops and other special events The AAFS represents a wide range of forensic specialties More information www aafs org The 38th annual AFTE Training Seminar will take place at the Hyatt Regency San Francisco California on May 27 June 1 2007 AFTE welcomes everyone to this beautiful city and to what promises to be a fantastic trai
12. tems in the illumination path Leica Variolux Leitz and Leica used this configuration for many years on comparison systems using the Laborlux Dialux Dialux 20 DM R and DM4000 microscope platforms 1980 s to present 3 Randomized bifurcated fiber optic light guides from a single cold light source 150W or 250W Adapted to the illumination path in place of standard halogen light bulb sources Mid 1980 s to present A microscope is designed with many optical elements apart from the illumination source that affect color As light travels through the microscope it passes through a collecting lens diffuser field diaphragm glass cover for the light exit port filter holder for colored glass or interference filters aperture diaphragm condenser element slide mounting media with sample coverglass and finally to the objective eyetube lens and eyepieces Each of these components can be responsible for changing the light path intensity and color of the light passing through the optical system In the design and manufacture of modern comparison microscopes care is taken to select matching optical components to reduce these continued on page 4 Comparison Illumination continued from page 3 influences by decreasing manufacturing tolerances and ensuring that components originate from the same production lot Ideally this leaves only lamp intensity condenser height position and field and aperture diaphragm settings as variable
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