20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 Module 3: Fluorescence Optical Microscope Contents 1 Objectives and Learning Goals 2 2 Roadmap and Milestones 2 3 Microscope Construction 3 3.1 Major microscope parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.1.1 Optical “Legos” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.1.2 Simple lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.1.3 Objective lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1.4 Sample holder/stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1.5 Fluorescence illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1.6 Dichroic mirror and barrier filter . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.1.7 CCD camera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2 Construction giudelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2.1 White light microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2.2 Fluorescence microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4 Experiment 1: Microscope Characterization and Fourier-Plane Imaging 8 4.1 White light calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4.2 Fluorescence characterization and imaging . . . . . . . . . . . . . . . . . . . . . . . . 8 4.3 Fourier optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4.3.1 The “light-scattering microscope” . . . . . . . . . . . . . . . . . . . . . . . . 8 4.3.2 Bio-photonic crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 5 Experiment 2: Microrheology Measurements by Particle Tracking 11 5.1 Introduction and background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5.2 Experimental details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 5.2.1 Stability and setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 5.2.2 System verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 5.2.3 Live cell measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 6 Experiment 3: Fluorescence Imaging of the Actin Cytoskeleton 15 6.1 Cell fixation and labeling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 6.2 Actin imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 7 Report Requirements 17 7.1 Microscope construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 7.2 Experiment 1: microscope characterization and Fourier-plane imaging . . . . . . . . 17 7.3 Experiment 2: microrheology measurements by particle tracking . . . . . . . . . . . 17 7.4 Experiment 3: fluorescence imaging of the actin cytoskeleton . . . . . . . . . . . . . 17 120.309: Biological Instrumentation and Measurement Laboratory Fall 2006 1 Objectives and Learning Goals Become familiar with the optical ”Lego” block set. • Construct a fluorescence and white light microscope. • Understand Fourier optics and its implications in microscopy. • Become familiar with basic cell labeling procedures using fluorescent probes. • Understand microrheology measurements in cells. • This laboratory sequence is three weeks long. At the end, you will have constructed and characterized a working fluorescence microscop e. You will understand basic optical principles such as ray tracing, fluorescence detection, and resolution. You will have also applied this microscope to characterize a bio-photonic crystal, and to measure the mechanical properties of living cells. 2 Roadmap and Milestones The AFM will be the basis of three weeks’ worth of experiments, and the roadmap below is subdi-vided into weeks to help you gauge your progress. Week 1: 1. Design and Build an inverted white light microscope based on ray tracing. 2. Measure the magnification and field of view using an air force target and a Ronchi ruling with bright-field white light illumination. 3. Add a laser illumination beam path. 4. Measure the diffraction of a Ronchi ruling …