Content LSM 510 META in Chang ung University LSM 510 META 路 理 The features and applications of LSM 510 META 01-09 Introduction of the hardware 10-12 Fluorescence observation in conventional microscope 13-22 in laser scanning microscope 23-33 行 Transmitted observation in conventional microscope 34-41 in laser scanning microscope 42-44 Accessories with the system bjectives 45-53 1 2 LSM 510 META Features of LSM 510 META Solutions for signal separation Filter-based emission signal separation Spectra-based emission signal separation Application of LSM 510 META ene expression studies and protein localization Multiple fluorescence in situ hybridization (M-FISH) Autofluorescences Elimination Long term observation of fluorescent images Integrated Bleach with Time Lapse Experiment Colocalization Analysis FRET Analysis Ion Imaging FRAP FLIP Analysis PA-FP and KAEDE 3 4
Solutions for signal separation Multifluorescence Imaging Filter-based emission signal separation part Dichroic mirrors and conventional detectors Spectra-based emission signal separation part rating and META detector The Problem Spectral properties of the available dyes limit the experimental freedom ften it is even difficult to clearly separate two fluorescence markers With more markers, the problem grows increasingly complex (spectra published by Clonetech) Cross-talk between the FP variants at the excitation and emisson level 5 6 Crosstalk Solution Emission Fingerprinting - The basic principle The META idea Every dye has its characteristic emission spectrum that can be used as an identification tag. Because different dye has its characterized emission spectrum, we called these emission spectrum as emission fingerprinting. A general solution to the problem is to acquire the complete emission spectra of all dyes and separate them in a second step. META detector PMT array with 32 elements Reflective grating for even dispersion No moving parts Capture full emission spectra Simultaneous illumination with 8 channels Highly parallel detection FP 450 500 550 600 relative weighs (e.g.) + 450 500 550 600 450 500 550 600 0,68 0,32 YFP Reference spectra Combined emission spectrum (experimental data) Linear unmixing determines relative contribution of each component Emission signal Linear unmixing is performed for each pixel individually 7 8
Features of LSM 510 META System Drawing Solutions for signal separation Filter-based emission signal separation Spectra-based emission signal separation Application of LSM 510 META ene expression studies and protein localization Multiple fluorescence in situ hybridization (M-FISH) Autofluorescence Elimination Long term observation of fluorescent images Integrated Bleach with Time Lapse Experiment Tile scanning or stitch Colocalization Analysis Ion Imaging FRET Analysis FRAP FLIP Analysis PA-FP and KAEDE 3D Image measurement 1 Scan detection module 2 Upright or Inverted Microscope 3 LSM VIS Laser module 4 Electronic Control Unit (ECU) 5 LSM Software 6 Computer hardware Blue diode 405 DPSS 561 HeNe 633 Argon 458/477/488/514 AM ATF 3 1 Scan Module 2 4 Electronic Control Unit 5 diff.time:0.030 µs 6 9 10 Whole system The first step Turn on the system Inverted microscope 4 2. Power for internal PC and components 4. Power for user PC Scan module 1 1. Power for the system rack 2 3 Laser 3. Power for FL observation with naked eyes Electronic control unit User PC 11 12
The Principle of Fluorescence The principle of fluorescence Excited state round state Stoke shift Absorption and fluorescence emission spectra of a protein conjugate labeled with fluorescein-5- isothiocyanate in ph 8.0 buffer. (www.molecularprobes.com) 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. 13 14 The fluorescence beam path in conventional microscope The fluorescence beam path in conventional microscope Light source Mercury lamp Conventional microscope 1. Quartz lass bulb 2. Cathode 3. Anode 4. Burning Chamber contains some Mercury 5. Light Arc 15 16
The fluorescence beam path in conventional microscope Filter should be used to define the excitation wavelength Excite samples with the appropriate excitation wavelength. Detect the strong and pure signals. Eliminate the signals from the out-of-focus plane. The fluorescence beam path in conventional microscope the wavelength of emission signal must be defined by filters 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 17 18 The fluorescence beam path in conventional microscope The position for the filter sets The fluorescence beam path in conventional microscope The composition of the filter set Ex BP 450-490 BS FT 510 EM LP 515 1. Light from HB Lamp 2. Monochromatic Light 3. Fluorescence Light returning from the Specimen A. Excitation Filter B. Dichroic Beam Splitter C. Emission Filter 19 20
Types of the filter The filter sets that can be used for conventional observation Filter name DAPI Excitation wavelength BP 365/12 Beamsplitter FT 395 Emission wavelength LP 397 FITC BP 450-490 FT 510 BP 515-565 Cy3 BP 546/12 FT 580 LP590 CFP BP 436/20 FT 455 BP 480/40 YFP BP 500/20 FT 515 BP 535/30 http://www.zeiss.de/micro 21 22 The fluorescence beam path in LSM The account of the blurred image The specimen is excited by lasers The specimen emits the emission signal Illumination from laser The detectors inside of the scan head scan the emission signal The emission signal is reflected back to the scan head 23 24
The account of the blurred image The account of the blurred image 25 26 The account of the blurred image The reason for better fluorescent images from confocal Pinhole A minute diaphragm, situated in a conjugated focal plane, prevents out of focus light to be detected. The pinhole diameter directly controls the thickness of the optical section 27 28
Pinhole eliminates the interference signal The reason for better fluorescent images from confocal Point scanning Pinhole Pinhole elimination in xz projection 29 30 The reason for better fluorescent images from confocal Point scanning Images from CLM or LSM Conventional microscope Laser scanning microscope Wide field illumination Point illumination Wide Field Confocal 31 32
What kind of fluorescent images could we scan now? Diode laser Multilin Ar laser DPSS laser HeNe laser Laser line 405 nm 458 nm 477 nm 488 nm 514 nm 561 nm 633 nm Examples of the fluorochromes DAPI, Hoechst, AMCA, BFP CFP, BCECF, Lucifer Yellow, Acridine range, Fura Red Fura Red, FP FP, BCECF, FITC, Cy2, Acridine range, YFP DsRed, Rhodamine, TRITC, Cy3, Propidium iodide, Texas Red Cy5, DiD, Alexa Fluor 633 33 34 Principle of phase contrast Principle of 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 35 36
Phase ring is inside of the specific objective Each phase ring has its corresponding phase stop The internal ring on the condenser and the objective must match to form the perfect phase image. 37 38 Select the specific objective and the corresponding phase stop Principle of DIC 7. Analyzer 6. DIC prism (slider behind the objective) Decomposition and laterally shift the partial light beams 2. Condenser prism 1. Polarizer 39 40
Put 2 prisms and 2 polarizers in the beam path Beam path of phase contrast in LSM Detector for transmission images Polarizer II Corresponding phase stop must be in the beam path Prism II Laser line passes through the specimen Illumination from laser Prism I DIC Polarizer I 41 42 Beam path of DIC in LSM Transmitted beam path in LSM Put 2 prisms and 1 polarizers in the beam path Detector for transmission images Put the polarizer with 75 degree in the beam path Polarizer II Laser line passes through the specimen DIC prism must be in the beam path Prism II Illumination from laser Prism I Polarizer I Empty position 43 44
Accessories with the system bjectives What does resolution actually mean? Mag Description EC Plan-Neofluar 10x/ 0.3 Ph1 NA 0.3 Imm. Mat. WD (mm) /P 5.2 Ph DIC Special Notes 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. Fluar 10x/ EC Plan-Neofluar 20x/ Ph2 1.9 2.0 The resolving power, the limit up to which two small objects are still seen separately. Fluar 20x/ 0.75 0.75 LD Plan-Neofluar 20x/0.4 0.4 /P 7.9 LD Plan-Neofluar 40x/ EC Plan-Neofluar 40x/ 1.3 1.3 il /P 2.9 0.21 Plan-Apochromat 63x/ il 0.19 100X Plan-Apochromat 100x/ oil il 0.17 45 46 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. Accessories with the system bjectives Mag Description EC Plan-Neofluar 10x/ 0.3 Ph1 Fluar 10x/ EC Plan-Neofluar 20x/ Ph2 Fluar 20x/ 0.75 LD Plan-Neofluar 20x/0.4 LD Plan-Neofluar 40x/ EC Plan-Neofluar 40x/ 1.3 Plan-Apochromat 63x/ NA 0.3 0.75 0.4 1.3 Imm. il il Mat. WD Ph (mm) /P 5.2 /P /P 1.9 2.0 7.9 2.9 0.21 0.19 DIC Special Notes 100X Plan-Apochromat 100x/ oil il 0.17 47 48
Immersed objective Accessories with the system bjectives Mag Description NA Imm. Mat. WD (mm) Ph DIC Special Notes EC Plan-Neofluar 10x/ 0.3 Ph1 0.3 /P 5.2 Fluar 10x/ 1.9 EC Plan-Neofluar 20x/ Ph2 2.0 Fluar 20x/ 0.75 0.75 LD Plan-Neofluar 20x/0.4 0.4 /P 7.9 LD Plan-Neofluar 40x/ EC Plan-Neofluar 40x/ 1.3 1.3 il /P 2.9 0.21 Plan-Apochromat 63x/ il 0.19 100X Plan-Apochromat 100x/ oil il 0.17 49 50 Special objectives for different chamber including plastic ones Accessories with the system bjectives Mag Description NA Imm. Mat. WD (mm) Ph DIC Special Notes EC Plan-Neofluar 10x/ 0.3 Ph1 0.3 /P 5.2 Fluar 10x/ 1.9 0.17 mm for standard cover glasses Thickness value Correction ring for different chamber thickness When the plastic chamber or dishes are used, users can observe images with this kind of objective. It is usable as well when the container is glasses-made. 100X EC Plan-Neofluar 20x/ Ph2 Fluar 20x/ 0.75 LD Plan-Neofluar 20x/0.4 LD Plan-Neofluar 40x/ EC Plan-Neofluar 40x/ 1.3 Plan-Apochromat 63x/ Plan-Apochromat 100x/ oil 0.75 0.4 1.3 il il il /P /P 2.0 7.9 2.9 0.21 0.19 0.17 51 52
Modular culture chamber Accessories with the system bjectives Mag Description NA Imm. Mat. WD (mm) Ph DIC Special Notes EC Plan-Neofluar 10x/ 0.3 Ph1 0.3 /P 5.2 Fluar 10x/ 1.9 EC Plan-Neofluar 20x/ Ph2 2.0 Fluar 20x/ 0.75 0.75 LD Plan-Neofluar 20x/0.4 0.4 /P 7.9 LD Plan-Neofluar 40x/ EC Plan-Neofluar 40x/ 1.3 1.3 il /P 2.9 0.21 Plan-Apochromat 63x/ il 0.19 100X Plan-Apochromat 100x/ oil il 0.17 53 54 Accessories with the system bjectives Mag Description NA Imm. Mat. WD (mm) Ph DIC Special Notes EC Plan-Neofluar 10x/ 0.3 Ph1 0.3 /P 5.2 Fluar 10x/ 1.9 EC Plan-Neofluar 20x/ Ph2 2.0 Fluar 20x/ 0.75 0.75 LD Plan-Neofluar 20x/0.4 0.4 /P 7.9 LD Plan-Neofluar 40x/ EC Plan-Neofluar 40x/ 1.3 1.3 il /P 2.9 0.21 Plan-Apochromat 63x/ il 0.19 100X Plan-Apochromat 100x/ oil il 0.17 55