Transcript Microscopy Techniques for Biomaterials: A Primer

Basic Microscopy and Imaging
Methods
in Biomedical Engineering
Prabhas V. Moghe, Lecturer
Rebecca Hughey, Eric Wallenstein, Mingjung Song
November 2, 2004
125:315 Biomedical Measurements and Analysis Lab
WWW Bibliography -- Supplementary Review Materials*
General Resources
Primer in Microscopy by Olympus: http://www.olympusmicro.com/primer/opticalmicroscopy.html
Molecular Expressions
A comprehensive resource maintained by Florida State University. Includes a Microscopy Primer
with tutorials on optical microscopy, digital imaging: http://micro.magnet.fsu.edu/primer/
microscopy.info
An information source for microscopy and microanalysis including a hyperlinked glossary and links
to professional societies and educational resources. http://www.mwrn.com
World Wide Web Virtual Library: Microscopy: http://www.ou.edu/research/electron/www-vl/long.shtml
Vast set of links covering all aspects of light, electron, and other types of microscopy, including
extensive lists of imaging and microscopy labs, image galleries, and educational sites.
Live Cell Imaging Applications in Confocal Microscopy: A lecture by Dr. J. Paul Robinson at Purdue Univ.
http://www.cyto.purdue.edu/flowcyt/educate/confocal/524Lec11/sld001.htm
NIH Office of Extramural Research: Links to Bioimaging Sites
http://grants2.nih.gov/grants/bioimaging/links.htm
Standard Microscopy Terminology: http://resolution.umn.edu/glossary/FrameGloss.html
A comprehensive glossary of microscopy terms made available by the University of Minnesota
Characterization Facility.
Microscopy/Imaging Resources on WWW: http://swehsc.pharmacy.arizona.edu/exppath/micro/index.html
A very well-organized, extensive collection of educational and informational resources, including links
to numerous online image galleries, microscopy sites, and an introduction to digital imaging.
* Science Magazine, April 2003, Special Issue on Biological Imaging
Outline
• Physics of Optical Microscopy
Bright Field Microscopy
Phase Contrast Microscopy
Fluorescence Microscopy
Confocal Microscopy
• Biomedical Image Processing
Image Acquisition
Image Processing Steps
Image Analysis
• Cellular Properties Derived via Biomedical Imaging
Electromagnetic Spectrum
The
wavelength
required to
“see” an
object must
be the same
size of
smaller than
the object
Choice of Microscopy Depends on the Scale of Imaging
Principle of Compound Light
Microscopy
Physics of Optical Microscopy
• The ability of a microscope
objective to "grasp" the various
rays coming from each
illuminated part of the specimen
is related to the angular
aperture of the objective.
N.A. = n . sin (u);
n= refractive index; u=1/2
subtended angle
- Max theoretical N.A. of a dry
objective is 1
- Max theoretical N.A. of oil
immersion objectives is 1.5
Optical Microscopy Issues:
Resolution
• Resolution is defined as the ability of an objective to separate
clearly two points or details lying close together in the specimen
0.61 
R
N. A.
where R=resolution distance; , the wavelength of
light used; N.A. = the numerical aperture.
- As N.A. increases, resolution gets better (R smaller).
- Longer wave lengths yield poorer resolution.
Bright and Dark Field Contrast
Bright Field vs. Dark Field
Microscopy
Principle of Phase Contrast
Microscopy
• Zernicke: Greatest advance in Microscopy (1953)
• Phase microscopy requires phase objectives and a phase condensor.
Contrast Microscopy
Phase Contrast; DIC
Fluorescence Microscopy:
Principle of Fluorescence
Fluorescence Microscopy
Barrier
Filter
Dichroic Mirror
Exciter
Filter
Objective/
Condensor
Specimen
Mercury
Light Source
Microscopy to Image Cell Structure
And Cell Architecture on Biomaterial Substrates
Use of fluorescence microscopy and transmitted/reflected imagin
A
B
C
D
Cytoskeletal Organization
Rounded
Phenotype
Mildly
Polarized
Phenotype
Selected Basic Definitions in Biomedical
Imaging & Image Analysis
1.
2.
3.
4.
5.
6.
7.
Pixels: 2-D Picture Elements
Mean Gray Level: Gray-scale Intensity (0-256)
MGL Histogram
Segmentation and Thresholding (MGL
Contrast)
Filtering
Densitometry
Morphometry (Area, Shape factor,
Polarity/Aspect Ratio)
Spatial and Gray Level Digital
Resolution
• Spatial resolution:
– # of samples per unit
length or area
– DPI: dots per inch
specifies the size of
an individual pixel
– If pixel size is kept
constant, the size of
an image will affect
spatial resolution
• Gray level
resolution:
– Number of bits per
pixel
– Usually 8 bits
– Color image has 3
image planes to yield
8 x 3 = 24 bits/pixel
– Too few levels may
cause false contour
Same Size, Different Pixel Sizes
Example of Biomedical Image Processing
• Biomedical polymer sponges were saturated with a fluorophore
(fluorescein isothiocyanate - FITC) & imaged on a microscope using
fluorescence microscopy.
• Images of each porous field were digitized and stored on the computer.
• Digitized images were analyzed using biomedical image processing to
identify the number, shape, and location of pores. (Similar approaches can
be used to identify fluorescently labeled cells and cell number, cell viability,
cell morphology)
1. Shading
Correction
3. Open
4. Scrap
& Fill
2. Segmentation
5. Measurements
Tjia and Moghe, J. Biomed. Mater. Res (Appl Biomat) 43: 291, 1998
Outline of the Lab Exercises
Microscopy and Cellular
Imaging
• Learn how to :
use a computer-interfaced optical microscope
archive images
process images and quantify results
identify cells via fluorescence labeling of the cytosk
show cells are viable via fluorescence labeling of D
visualize cell borders
quantify cell morphology (phenotyping)