Spotlight 理 The Basic Concept of Light Microscope 行 Basic Introduction of Light Microscopy What is the difference between the light microscope and the magnifier? Two kinds of beam path: reflected light and transmission light Transmitted Techniques in Light Microscope Bright Field Dark Field Phase Contrast Polarization Difference Interference Contrast Reflected Techniques in Light Microscope What is crosstalk? How to resolve the crosstalk problem? 2005-9-14/ The basic concept of light microscope 1 2005-9-14/ The basic concept of light microscope 2 The incident angle determines the size that we see All the magnifier are designed for extending the incident angle Microscopy means seeing a large image of something small. A, microscope beam path B, see the object directly from a distance of approx. 25 cm. 1, object 2, objective (image projected at infinity) 3, tube lens (produce a magnified intermediate image) 4, intermediate image 5, eyepiece 6, eye ICS principle (Infinity Color-corrected System) The objective projects an image at an infinite distance, the tube lens with its fixed focal length then forms the intermediate image from these parallel beams. Let us assume that nothing decisive for the image formation happens in the space between objective and tube lens. The light rays coming from the focused specimen plane are parallel in this space anyway. 2005-9-14/ The basic concept of light microscope 3 2005-9-14/ The basic concept of light microscope 4 The incident angle is magnified by lens. Microscopy means seeing a large image of something small. A, microscope beam path B, see the object directly from a distance of approx. 25 cm. 1, object 2, objective (image projected at infinity) 3, tube lens (produce a magnified intermediate image) 4, intermediate image 5, eyepiece 6, eye ICS principle (Infinity Color-corrected System) The objective projects an image at an infinite distance, the tube lens with its fixed focal length then forms the intermediate image from these parallel beams. Let us assume that nothing decisive for the image formation happens in the space between objective and tube lens. The light rays coming from the focused specimen plane are parallel in this space anyway. The incident angle is magnified by lens. Microscopy means seeing a large image of something small. A, microscope beam path B, see the object directly from a distance of approx. 25 cm. 1, object 2, objective (image projected at infinity) 3, tube lens (produce a magnified intermediate image) 4, intermediate image 5, eyepiece 6, eye ICS principle (Infinity Color-corrected System) The objective projects an image at an infinite distance, the tube lens with its fixed focal length then forms the intermediate image from these parallel beams. The light rays coming from the focused specimen plane are parallel in this space anyway. It makes it possible to project intermediate images in the same Z position from different objectives. 2005-9-14/ The basic concept of light microscope 5 2005-9-14/ The basic concept of light microscope 6 1
Besides the multi-lens, there are still other parts important for the resolution. What does resolution actually mean? The light incident from the objects is deflected from the original direction. To obtain sharp images of small structures, the objective in the microscope must collect as much of this diffracted light as possible. This works particularly well if the objective covers a large solid angle. The term aperture (opening) describing this property. Numerical aperture: a measure of the solid angle covered by an objective. A bright disk with shapely defined edges, but as a slightly blurred spot surrounded by diffraction rings, called Airy disks are formed from a spot light through the lens. The resolving power, the limit up to which two small objects are still seen separately. 2005-9-14/ The basic concept of light microscope 7 2005-9-14/ The basic concept of light microscope 8 Plan-Apochromat Plan-Neofluar N.A.=1.518* sin 67.3 =1.4 N.A.=1.518* sin 59 =1.3 Plan-Apochromat -Top of the objectives -Perfect image flatness for fields up to 25mm -Oil immersion objectives of highest numerical apertures -With the chromatic correction from 420 to 670 nm 2005-9-14/ The basic concept of light microscope 9 Plan-Neofluor -an universal objective -perfectly designed for general fluorescence microscope -chromatic correction from 435 nm to 670nm -transmission starts at 365 nm -brilliant view of field up to 25mm -made by low auto- fluorescence glass type 2005-9-14/ The basic concept of light microscope 10 C-Apochromat N.A.=1.33* sin 64.5 =1.2 The way of increasing the optical resolving power Choose a large angle of the ray cone on the illumination side. To use immersion liquids between the front lens of the objective and the cover slip. The aperture of the objective and the resolving power would be reduced by the reflection. C-Apochromat -special water immersion objectives -using water immersion is the closest match to the refractive index of biological tissue and popular embedding media -with chromatic range from 360~700nm, therefore they work especially well for extended Z-scans in biological tissue and for spectral imaging with the MEAT detector. -compensation of different temperature (21 ~37 ) Condenser 2005-9-14/ The basic concept of light microscope 11 2005-9-14/ The basic concept of light microscope 12 2
Besides the multi-lens, there are still other parts important for the resolution. The light incident from the objects is deflected from the original direction. To obtain sharp images of small structures, the objective in the microscope must collect as much of this diffracted light as possible. This works particularly well if the objective covers a large solid angle. The term aperture (opening) describing this property. Numerical aperture: a measure of the solid angle covered by an objective. The way of increasing the optical resolving power Choose a large angle of the ray cone on the illumination side. To use immersion liquids between the front lens of the objective and the cover slip. The aperture of the objective and the resolving power would be reduced by the reflection. Condenser 2005-9-14/ The basic concept of light microscope 13 2005-9-14/ The basic concept of light microscope 14 Upright Light Microscope Inverted Light Microscope Illumination equipment for fluorescence Eyepiece Binocular tube Lamp for fluorescence Illuminator Filter turret Specimen stage Transmitted Condenser with aperture diaphragm Luminous-Field Diaphragm Luminous-field and aperture diaphragm for fluorescence Nosepiece Objective Light Control Illuminator Stand Eyepiece Binocular tube Nosepiece Light control of transmitted illuminator Transmitted Condenser with aperture diaphragm Specimen stage Objective Lamp for fluorescence Stand Filter turret 2005-9-14/ The basic concept of light microscope 15 2005-9-14/ The basic concept of light microscope 16 The difference between the upright and inverted microscope The difference between the upright and inverted microscope 2005-9-14/ The basic concept of light microscope 17 2005-9-14/ The basic concept of light microscope 18 3
The difference between the upright and inverted microscope The Right Climate for Live Cell Imaging The design of Carl Zeiss leaves all the control icons out of the incubation system. Brings users the convenience about switch between electronic and PC control. The released space can be freely used to arrange other instruments. 2005-9-14/ The basic concept of light microscope 19 2005-9-14/ The basic concept of light microscope 20 Different Beam Path of Image Formation Transmitted Light Path of Upright Microscope Eyepiece Binocular tube Specimen stage Nosepiece Transmitted Condenser with aperture diaphragm Luminous-Field Diaphragm Objective Light Control Illuminator Stand Transmitted-light Reflected-light (Fluorescence) 2005-9-14/ The basic concept of light microscope 21 2005-9-14/ The basic concept of light microscope 22 Transmitted Techniques in Light Microscopy Bright Field Bright Field is the most universal technique used in light microscope. Usually used in samples with colorimetric staining or good contrast. 2005-9-14/ The basic concept of light microscope 23 2005-9-14/ The basic concept of light microscope 24 4
Operation of light microscope Dark Field Fine structures can often not be seen in front of a bright background. 2005-9-14/ The basic concept of light microscope 25 2005-9-14/ The basic concept of light microscope 26 Dark Field Adjust parts of dark field on upright microscope Objective Sample Condenser optics Annular stop It is necessary for the objective aperture to be a smaller than the inner aperture of the illuminating light cone. If there is no sample, the image seen in the eyepieces remains completely dark. If objectives are in the objective plane, light is laterally diffracted away from the straight path. The object becomes brightly visible in front of a dark background. Objectives with an integrated variable iris diaphragm are available to shutter out the indirect light even if it falls into the aperture cone of the objective. This permits the use of very high apertures for darkfield. Variable iris diaphragm DF 2005-9-14/ The basic concept of light microscope 27 2005-9-14/ The basic concept of light microscope 28 Phase contrast Phase Contrast Phase contrast is ideal for thin unstained objects, for example culture cells on glass, which are approx. 5 bis 10 um thick above the cell nucleus, but less than 1um thick at the periphery, and which barely exhibit any light absorption in the visible part of the spectrum. The eye can scarcely see them in bright field and dark field. However, very small differences exist between the refractive indices of the cells and the surrounding aqueous solutions and within the cells between the cytoplasm and the cell nucleus. The higher the refractive index of a medium, the smaller the speed or velocity of light in the medium. It translates the tiny differences into differences in intensity. New path Phase ring Phase stop 2005-9-14/ The basic concept of light microscope 29 2005-9-14/ The basic concept of light microscope 30 5
The characteristics of objectives Adjust parts of phase contrast on upright microscope PH1 PH1 2005-9-14/ The basic concept of light microscope 31 2005-9-14/ The basic concept of light microscope 32 Polarization Adjust parts of Polarization on upright microscope The polarizer privileges light source with specific direction of vibration. The analyzer arranged at an angle of 90 to the polarizer is located behind the objective. If no specimen is on the microscope stage, the image will remain completely dark. Analyzer When illuminated, some specimens, such as starch, minerals and polymers,turn the vibration direction of the polarized light out of the plane produced by the polarizer. Polarizer 2005-9-14/ The basic concept of light microscope 33 2005-9-14/ The basic concept of light microscope 34 Differential Interference Contrast (DIC) Adjust parts of DIC on upright microscope 7. Analyzer 6. DIC prism (slider behind the objective) 2. Condenser prism 1. Polarizer 7. Analyzer 6. DIC prism (slider behind the objective) DIC Decomposition and laterally shift the partial light beams 2. Condenser prism 1. Polarizer 2005-9-14/ The basic concept of light microscope 35 2005-9-14/ The basic concept of light microscope 36 6
Plan-Neofluar Transmitted beam path on inverted light microscope 2005-9-14/ The basic concept of light microscope 37 2005-9-14/ The basic concept of light microscope 38 Adjust parts of dark field on inverted microscope Adjust parts of phase contrast on inverted microscope 2005-9-14/ The basic concept of light microscope 39 2005-9-14/ The basic concept of light microscope 40 Adjust parts of Polarization on inverted microscope Adjust parts of DIC on inverted microscope 7. Analyzer 6. DIC prism (slider in the objective) 2. Condenser prism 1. Polarizer 2005-9-14/ The basic concept of light microscope 41 2005-9-14/ The basic concept of light microscope 42 7
The reflected beam path on light microscope The Principle of Fluorescence Excited state Ground state 40X Stoke shift Absorption and fluorescence emission spectra of a protein conjugate labeled with fluorescein-5- isothiocyanate in ph 8.0 buffer. (www.molecularprobes.com) 2005-9-14/ The basic concept of light microscope 43 2005-9-14/ The basic concept of light microscope 44 The principle of fluorescence The excitation of conventional light microscope 1. Quartz Glass bulb 2. Cathode 3. Anode 4. Burning Chamber contains some Mercury 5. Light Arc Emission intensity depends on excitation efficiency The more efficient excitation induces the stronger signal of emitted light. How to get the best fluorescence image? Excite samples with the appropriate excitation wavelength. Detect the strong and pure signals. Eliminate the signals from the out-of-focus plane. 2005-9-14/ The basic concept of light microscope 45 2005-9-14/ The basic concept of light microscope 46 How to get the best fluorescence image? Excite samples with the appropriate excitation wavelength. Detect the strong and pure signals. Eliminate the signals from the out-of-focus plane. How to get the best fluorescence image? Excite samples with the appropriate excitation wavelength. Detect the strong and pure signals. Eliminate the signals from the out-of-focus plane. The spectra of FITC The emission spectra of FITC The emission spectra of FITC and other fluorophore 2005-9-14/ The basic concept of light microscope 47 2005-9-14/ The basic concept of light microscope 48 8
Conventional microscope use wavelength filters to segment the excitation light and emission light Conventional microscope use wavelength filters to segment the excitation light and emission light 2005-9-14/ The basic concept of light microscope 49 2005-9-14/ The basic concept of light microscope 50 Types of the filter The filters used in conventional light microscope Ex BP 450-490 BS FT 510 EM LP 515 1. Light from HBO Lamp 2. Monochromatic Light 3. Fluorescence Light returning from the Specimen A. Excitation Filter B. Dichroic Beam Splitter C. Emission Filter 2005-9-14/ The basic concept of light microscope 51 2005-9-14/ The basic concept of light microscope 52 Understanding the spectra of filters Band pass Filter of Excitation and Emission A. Excitation filter / B. Dichroic beam splitter / C. Emission filter The conventional fluorescence microscope identifies the true signal by these filters. 2005-9-14/ The basic concept of light microscope 53 2005-9-14/ The basic concept of light microscope 54 9
Adjust parts of fluorescence on upright microscope Then you will get fluorescence images 2005-9-14/ The basic concept of light microscope 55 2005-9-14/ The basic concept of light microscope 56 The crosstalk problem The crosstalk problem FITC / Rhod FITC / Rhod FITC FITC 2005-9-14/ The basic concept of light microscope 57 2005-9-14/ The basic concept of light microscope 58 The crosstalk problem The solution of crosstalk in conventional fluorescence microscope -Multichannel unmixing FITC / Rhod Raw CFP image Raw GFP image FITC Universal localized CFP that accumulated at nusleoli But In fact GFP coupled with a histone protein that localized in the entire nucleus and a lesser degree of nucleoli. Merged image composed by raw images Several signal in the GFP channel is bleeded from CFP dye. 2005-9-14/ The basic concept of light microscope 59 2005-9-14/ The basic concept of light microscope 60 10
The steps of multichannel unmixing Result of Multichannel unmixing 1. Prepare the CFP-stained sample and GFP-stained sample as reference and double stained sample as the processed image. 2. Get the image through the filters, respectively. CFP only sample GFP only sample Double stained sample CFP channel Raw CFP image Raw GFP image Merged image composed by raw images GFP channel 2005-9-14/ The basic concept of light microscope 61 Universal licalized CFP that GFP coupled with a histone accumulated at nusleoli protein that localized in the entire Merged image composed by unmixing nucleus and a lesser degree of images nucleoli 2005-9-14/ The basic concept of light microscope 62 11