光学显微成像（PPT课件讲稿）光学显微术 Optical Microscopy

Lecture-3 Optical Microscopy Introduction Lens formula, image formation and Magnification Resolution and lens defects Basic components and their functions Common modes of analysis Specialized microscopy Techniques Typical examples of applications http://www.youtube.com/watch?v=p2tee17zt4i&list=plkstg-8vpwkzoe4tkVa7f6qmig2hh8meXatn0:46-1:33

Lecture-3 Optical Microscopy • Introduction • Lens formula, Image formation and Magnification • Resolution and lens defects • Basic components and their functions • Common modes of analysis • Specialized Microscopy Techniques • Typical examples of applications http://www.youtube.com/watch?v=P2teE17zT4I&list=PLKstG-8VPWKzOe4TkvA7F6qMlG2HH8meX at~0:46-1:33

Review Problems on Optical Microscopy 1. Compare the focal lengths of two glass converging lenses, one with a larger curvature angle and the other with a smaller curvature angle. 2. List the parameters that affect the resolution of optical microscopes 3. a student finds that some details on the specimen cannot be resolved even after the resolution of the microscope was improved by using the oil immersion objective. The student thinks that the details can be resolved by enlarging a photograph taken with the microscope at maximum magnification Do you agree? Justify your answer. http://www.doitpoms.ac.uk/tlplib/optical-microscopy/questions.php

Review Problems on Optical Microscopy 1. Compare the focal lengths of two glass converging lenses, one with a larger curvature angle and the other with a smaller curvature angle. 2. List the parameters that affect the resolution of optical microscopes. 3. A student finds that some details on the specimen cannot be resolved even after the resolution of the microscope was improved by using the oil immersion objective. The student thinks that the details can be resolved by enlarging a photograph taken with the microscope at maximum magnification. Do you agree? Justify your answer. http://www.doitpoms.ac.uk/tlplib/optical-microscopy/questions.php

http://micro.magnetfsuedu/primer/java/microscopy/immersion/index.html Resolution of a Microscope (lateral) The smallest distance between two specimen points that can still be distinguished as two separate entities dmin =0.61\/NA NA=nsin(a) 入-ⅲ urination wavelength(ight) NA -numerical aperture a-one half of the objective angular aperture n-imaging medium refractive index dmin n 0. 3um for a midspectrum 2 of 0. 55um https://www.youtube.com/watch?v=nzasdncmymoatn5:35-6:00

The smallest distance between two specimen points that can still be distinguished as two separate entities dmin = 0.61l/NA NA=nsin() l – illumination wavelength (light) NA – numerical aperture -one half of the objective angular aperture n-imaging medium refractive index dmin ~ 0.3m for a midspectrum l of 0.55m Resolution of a Microscope (lateral) http://micro.magnet.fsu.edu/primer/java/microscopy/immersion/index.html https://www.youtube.com/watch?v=n2asdncMYMo at~5:35-6:00

Numerical Aperture(NA) NA=1-theoretical Aperture NA=n(sin a) maximum numerical aperture of a lens n: refractive index of the operating with air as imaging medium between the imaging medium the front lens of objective and specimen cover glass Objective lens A Angular aperture Light (≤72 degrees Cone a One half of A-A Specimen cover glass— na of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance https://en.wikipedia.org/wiki/angular_aperture http://micro.magnetfsuedu/primer/java/microscopy/immersion/index.html

NA = n(sin ) n: refractive index of the imaging medium between the front lens of objective and specimen cover glass Numerical Aperture (NA)  Angular aperture One half of A-A NA=1 - theoretical maximum numerical aperture of a lens operating with air as the imaging medium (72 degrees) https://en.wikipedia.org/wiki/Angular_aperture http://micro.magnet.fsu.edu/primer/java/microscopy/immersion/index.html NA of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. Objective lens Specimen cover glass

Numerical Aperture NA=n(sin a) Oil Immersion and Numerical aperture Imaging Medium Objective Numerical Aperture Comparison Air n=1.0 NA.=0.25 5 NA.=0.75 345 Immersion oil n=1.515 http://ww.youtube.com/watch?v=rskb0j1srnu oil immersion objective use in microscope at0: 33

Numerical Aperture Immersion oil n=1.515 Air n=1.0 http://www.youtube.com/watch?v=RSKB0J1sRnU oil immersion objective use in microscope at~0:33 Imaging Medium NA = n(sin )

Axial resolution- Depth of Field Depth of focus (f mm) Depth of Field ranges(F um) Low Depth of focus Image plane High object specimen Plane NA fF 0.10.1315.5Deph 04385.8 of Field 9580.00.19 Object plane The distance above and below The axial range through which geometric image plane within an object can be focused without which the image is in focus any appreciable change in image MI NAIf F sharpness F is determined by Na http://www.matter.org.uk/tem/depth_of_field.htm http://www.youtube.com/watch?v=fvc2wluqeugatw3:40

Depth of focus (f mm) The distance above and below geometric image plane within which the image is in focus The axial range through which an object can be focused without any appreciable change in image sharpness (F m) M NA f F M NA f F Axial resolution – Depth of Field Depth of Field Ranges (F m) F is determined by NA. NA f F 0.1 0.13 15.5 0.4 3.8 5.8 .95 80.0 0.19 http://www.matter.org.uk/tem/depth_of_field.htm http://www.youtube.com/watch?v=FvC2WLUqEug at~3:40

Depth of Focus The distance above and below geometric image plane within which the image is in focus CCD camera Camera Tube Eye Observation Light Source Tube Lens Microscope Optical Objective Train Components Condenser Aperture aphragm Condenser Field Diaphragm Figure 2

Depth of focus (f mm) Depth of Focus CCD camera The distance above and below geometric image plane within which the image is in focus

Axial resolution Depth of Field The axial range through which an object can be focused without any appreciable change in image sharpness Depth of Field Ranges 寄 ject lane 25um Small Large F

Depth of focus (f mm) Axial resolution – Depth of Field NA f F 0.1 0.13 15.5 0.4 3.8 5.8 .95 80.0 0.19 Small F Large F The axial range through which an object can be focused without any appreciable change in image sharpness. 25m

Basic components and their functions http://www.youtube.com/watch?v=rka8_mif6-e Microscope Review(simple, clear) http://www.youtube.com/watch?v=b2pc]5s-iyk Microscope working in animation(How to use a microscope) http://www.youtube.com/watch?annotation_id=annotation_100990&featur e=iv&src_vid=L6d3zD2LtsI&v=ntPjuUMdXbg(I http://www.youtube.com/watch?v=vqtmhj3valg(ii) Parts and Function of a Microscope(details) http://www.youtube.com/watch?v=x-w98ka8uqu&feature=related How to use a microscope(specimen preparation at 1: 55-2: 30) http://www.youtube.com/watch?v=bgbgablev4g How to care for and operate a microscope

Basic components and their functions http://www.youtube.com/watch?v=RKA8_mif6-E Microscope Review (simple, clear) http://www.youtube.com/watch?v=b2PCJ5s-iyk Microscope working in animation (How to use a microscope) http://www.youtube.com/watch?annotation_id=annotation_100990&featur e=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbg (I) http://www.youtube.com/watch?v=VQtMHj3vaLg (II) Parts and Function of a Microscope (details) http://www.youtube.com/watch?v=X-w98KA8UqU&feature=related How to use a microscope (specimen preparation at~1:55-2:30) http://www.youtube.com/watch?v=bGBgABLEV4g How to care for and operate a microscope

Eyepiece Hooke Microscope circa 1670 sk Bearer Basic components and their functions Focusing Screw ctive Figure 1 Specime 1) Eyepiece(ocular lens) 2)Revolving nose piece(to hold multiple objective lenses) (3 Objective lenses (4)And(5)Focus knobs (4)Coarse adjustment Fine adjustment (6 Stage(to hold the specimen) 7) Light source (lamp 8) Condenser lens and diaphragm (9) Mechanical stage(move the specimen on two horizontal axes for positioning the specimen

Basic components and their functions (1) Eyepiece (ocular lens) (2) Revolving nose piece (to hold multiple objective lenses) (3) Objective lenses (4) And (5) Focus knobs (4) Coarse adjustment (5) Fine adjustment (6) Stage (to hold the specimen) (7) Light source (lamp) (8) Condenser lens and diaphragm (9) Mechanical stage (move the specimen on two horizontal axes for positioning the specimen)