Biomedical & X-ray Physics 
                                 Kjell Carlsson 
                                                         
                                                    
            
                  Light Microscopy 
            
                                     
                     Compendium compiled for course SK2500, 
                                     
                    Physics of Biomedical Microscopy 
            
                                    by 
                                     
                               Kjell Carlsson 
            
                      ©Applied Physics Dept., KTH, Stockholm, 2007 
            
           No part of this document may be copied and distributed without the express written 
           consent of the author (kjell.carlsson@biox.kth.se). Copies and printouts for personal 
           use by students etc. are, of course, allowed. 
            
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                                                               Contents 
                            
                            
                           Introduction                                                       5 
                            
                           1. Wide-field microscopy                                           5 
                            
                              1.1 Basics of light microscopy                                  5 
                              1.2 Illumination systems                                        9 
                              1.3 Imaging properties (incoherent case)                      12 
                              1.4 Imaging properties (coherent case)                        17 
                              1.5 Contrast techniques                                       20 
                                  1.5.1 Fluorescence labeling                               21 
                                  1.5.2 Phase imaging techniques                            24 
                                  1.5.3. Dark-field imaging                                 29 
                              1.6 Microscope photometry                                     29 
                            
                           2. Confocal microscopy                                           31 
                            
                              2.1 What is confocal microscopy?                              31 
                              2.2 Imaging properties of confocal microscopy                 34 
                              2.3 Limitations and errors in confocal microscopy             42 
                              2.4 Illumination and filter choice in confocal microscopy     44 
                               
                           3. Recent microscopy techniques                                  48 
                            
                              3.1 Introduction                                              48 
                              3.2 Two-photon microscopy                                     49 
                              3.3 4π-microscopy                             51 
                              3.4 Stimulated emission depletion                             53 
                              3.5 Structured illumination                                   54 
                              3.6 Total internal reflection fluorescence microscopy         57 
                              3.7 Deconvolution                                             58 
                              3.8 Near-field microscopy                                     59 
                               
                               
                           Appendix I (Optical aberrations)                                 61 
                           Appendix II (Illumination systems for infinity systems)          65 
                           Appendix III (psf and MTF)                                       66 
                           Appendix IV (Photometry)                                         69 
                           Appendix V (Depth distortion)                                    71 
                           Historical note                                                  73 
                           References                                                       73 
                           Index                                                            74 
                            
                            
                            
                            
                            
                            
                                                                     
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