1. Chapter 4 - microscopy BME 310 Lecture 20

2. Types of microscopy 1.Optical (Huygens, Galileo, Hooke, Leeuwenhoek - early 17th cent.) 2.Fluorescence ( Stokes - 1852) 3.Confocal (Minsky - 1955) 4.Electron (Ruska - 1931, 1986 Nobel) 5.Scanning probe (Binnig & Rohrer - 1981, 1986 Nobel)

3. 1.1 Optical www.leica-microsystems.com Eyepiece Objective lenses Stage Fine and coarse adjustment knobs - move stage up and down

4. 1.2 Optical Close-up of objectives - bulk of magnification Condenser - series of lenses to illuminate sample with parallel beams of light to reduce glare and ensure even lighting www.leica-microsystems.com

5. 1.3 Optical Sample must transmit light –Or reflect light in stereo optical microscope Staining to add contrast as most cells are transparent (e.g., bacteria and Gram stain) Resolution determined by light (0.3  m) –Eye resolution <100  m Maximum magnification = 1000X

6. 2.1 Fluorescence Adjunct to optical microscopy Sample must be thin Sample is stained with fluorescent dye or marker (fluorophor) Marker selectively attaches to specific cell structures When light shines on marker, it emits light Triple fluorescence staining of endothelium cells from a pulmonary artery. www.nobelprize.org

7. 2.2 Fluorescence Dye or marker initially in ground state, E 0 Photon hits marker, sends it to excited state, E 1’ Marker relaxes to E 1 Marker returns to E 0 emitting photon E 1 excited Energy 0 1’ 1

8. 2.3 Fluorescence Emitted light is low intensity Excitation light is at lower wavelength and can be filtered Multiple dyes may be used on same sample Triple fluorescence staining of division of a Chinese hamster ovary cell. www.nobelprize.org www.microscopyu.com

9. 3.1 Confocal Laser shines through pinhole Light focused by objectives to a point in sample Fluorescence from sample passes filter Only light from focal point passes pinhole aperture to PMT Laser scanned in XYZ, process repeats to build image Laser Sample PMT microscope objectives pinhole apertures filter mirror

10. 3.2 Confocal Image thick samples (laser may be focused at depth) Combine with fluoroscopy Eliminate fluorescent glare from out-of- focus sample Better resolution then optical Sloooooow (scanning, not widefield) Computer reconstruction of image

11. 3.3 Confocal www.leica-microsystems.com

12. 3.4 Confocal Human osteosarcoma (bone cancer) - nuclei are red, 60X zoom, illuminated with laser light at 488 nm, 543nm, 633 nm www.olympusconfocal.com

13. 3.5 Confocal Drosophila (fruit fly) brain - 3D rendering from multiple optical sections, mushroom bodies (cerebral cortex of insects) stained green www.olympusconfocal.com

14. 4.1 Electron Transmission electron microscopy –Similar to optical microscope –Electrons have much shorter than light –Resolution 100000X –Sample under vacuum (electrons hit air) –Source is tungsten wire cathode –“Lenses” are magnetic coils –Image created on phosphor screen

15. 4.2 Electron Transmission electron microscopy –No live samples –Samples are fixed, washed, dehydrated, infiltrated with resin, cured, and sectioned –Thicker or denser parts of sample block more electrons -> dark image –Thinner or less dense parts of sample pass more electrons -> light image

16. 4.3 Electron bacteriophage lambda www.biochem.wisc.edu

17. 4.4 Electron Scanning electron microscopy –Similar to confocal microscopy (1 point at a time) –Electrons focused to point (2 nm) –Samples made conductive w/ metal coating –Incident electrons displace secondary electrons –Secondary electrons detected by scintillator and PMT –More secondary electrons when beam more parallel to surface –More secondary electrons -> brighter –Thus images have 3D appearance

18. 4.4 Electron Isolated mitochondria from cultured bovine lymphocytes. These mitochondria are either in the late stage of dividing, or an early stage of fusing. White dots are 18 nm gold particles conjugated to an antibody. www.ansci.wisc.edu

19. 5.1 Scanning probe Many types in 2 main categories –Scanning tunneling microscope –Atomic force microscope Probe scans lines across sample surface Probe is displaced up/down as it is scanned Surface topography reconstructed from many scanlines by computer

20. 5.2 Scanning probe mrsec.wisc.edu Sample moved nanometers at a time by piezoelectric positioners Resolution ~1 pm possible scanlines x z y1 y2 y3 y4 y5 y6 y7 height (z) -> brightness x y

21. 5.3 Scanning probe Scanning tunneling microscope –Sample must conduct –Voltage between tip and sample creates tunneling current (quantum) –Current magnitude related to distance between tip and surface –Two methods - Keep current constant by moving tip up/down Keep tip steady, measure changing current –Mainly used in materials research –Tissue adhesion to biomaterials in part dependent on nanoscale topography Pure silicon cut at 4 degree angle showing individual silicon atoms in layers mrgcvd.engr.wisc.edu

22. 5.4 Scanning probe Atomic force microscopy –Probe tip displaced up/down physically –Deflection measured by movement of light reflected off cantilever – At initial position, equal photons on photodetector quadrants, no differential current –When light deflects, more photons on one quadrant, measurable differential current piezoelectric positioners mrsec.wisc.edu

23. 5.5 Scanning probe Atomic force microscopy in biology –Can image living cells in solution –Drag tip across cell to measure friction/elasticity –Move cell to measure adhesion to surface –Try to pull ligand from receptor on cell surface –Measure volume change of cell

24. Combined imaging Phase contrast opticalFluorescence Atomic force heightAtomic force deflection (derivative of height) Living fibroblast cell - image size is 80 x 60  m www.jpk.com

25. Sources Bioinstrumentation, John Webster, Ed. www.microscopyu.com (Nikon edu. site, excellent resource) www.olympusconfocal.com www.leica-microsystems.com www.nobelprize.org www.wisc.edu www.jpk.com