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1 MICROSYSTEMS 光學顯微鏡簡介 Our mission is to be the world's first-choice provider of innovative solutions to our customers' needs for vision, measurement, lithography and analysis of microstructures. 美嘉儀器股份有限公司

2 Introduction of Microscopes LEICA DM6000B Automatic Upright Microscope LEICA DMI3000 Semi- Automatic Inverted Microscope LEICA M205 Stereomicroscope

3 Eyesepiece Fluorescence cube DIC prism Objectives Condenser 光源 Stages and specimen holders PH Rings and DIC prism Focus drive Variable function buttons SmartTouch

4 顯微鏡的成像與柯氏照明

5 顯微鏡的成像 - Image formation - 二次放大 ( 物鏡, 目鏡 ) 與中間影像 ( Intermediate Image ) 聚焦鏡幻燈片投射影像樣本中間影像物鏡目鏡最終影像

$Direct light ) 即為背景光亮 Deviated light ( Diffracted light ) 因通過樣品而產生$ $+ Diffracted light YA = direct light YC = diffracted light YB = Image YB$

6 影像的形成 - Image formation - 光線在透過物體時, 因物體的阻擋而產生二種情況〆 Undeviated light ( Direct light ) 即為背景光亮 Deviated light ( Diffracted light ) 因通過樣品而產生二種光線產生破壞性的干擾 ( destructive interference ) 而觀察到物體的影像 Image = Direct light + Diffracted light YA = direct light YC = diffracted light YB = Image YB = YA + YC

7 - Koehler s Illumination - Key points: 每一個光源共軛面必在影像共軛面之前光源上的每一點均需照射到所有影像的範圍三處調整部分 - Aperture diaphragm - Field diaphragm - Condenser height

8 - Field diaphragm - 視野光圈 (Field diaphragm, FD) 位置〆一般位於孔徑光圈與燈源之間作用〆提供正確的柯氏照明提供正確的柯氏照明保護觀測物體避免受不必要的光熱阻隔不必要的光線照射, 加強對比效果視野光圈的調節〆使用高倍率物鏡時, 因其視野較小, 故視野光圈需調小々反之, 低倍率物鏡視野較大, 其視野光圈需放大每更換不同倍率物鏡時, 孔徑光圈與視野光圈均應做調整〆高 NA 物鏡〆 AD 々 FD 々低 NA 物鏡〆 AD 々 FD 大視野光圈小視野光圈視野光圈的調整應以恰可涵蓋觀察視野的範圍為準!

9 - Aperture diaphragm - 孔徑光圈 (Aperture diaphragm, AD) 位置〆光源路徑第一次投影處 ( S ), 一般在聚光鏡 (Condenser) 下方作用〆提供正確的柯氏照明影響〆孔徑光圈的調整會影響下列幾項結果〆側向解析度 (lateral resolution)- 孔徑光圈一旦小於物徑的孔徑值 (NA), 解析度便開始下降, 應避免將孔徑光圈縮小至物徑孔徑值的 0.6x 以下視野景深 (depth of field)- 孔徑光圈, 視野景深影像之對比 (contrast)- 孔徑光圈, 影像之對比, 解析度孔徑光圈的調節〆使用高孔徑值 (NA) 的物鏡, 其孔徑光圈需調大々而使用低孔徑值的物鏡時, 其孔徑光圈需縮小大孔徑光圈小孔徑光圈絕對絕對不要用孔徑光圈來調整影像的明亮度!

10 - NA, Numerical Aperture - Aperture Diaphragm vs. Resolution and Contrast AD fully open AD 2/3 open AD closed more than half

Close the field and aperture diaphragm to minimum 4.

11 - Koehler s Illumination - Koehler illumination is absolutely fundamental and necessary to get a good brightfield image. 1. Focus the specimen as clearly as possible ( use low magnification objective, eg. 10x ) 2. Turn the condenser turret to brightfield (H) 3. Close the field and aperture diaphragm to minimum 4. Raise or lower the condenser with the condenser focus knob until the edge of the field diaphragm is sharpest 5. Centre it. 6. Open the field diaphragm until light fills the field of view, and check the center again. 7. Open the condenser diaphragm until the resolution and contrast are as desired

12 穿透光顯微鏡影像的種類

13 Two kind of beam path Transmission Light 明視野 Bright Field 暗視野 Dark Field 相位差 Phase Contrast 偏光 Polarization 干涉相位差 Differential Interference Contrast Incident Light 螢光 Fluorescent

14 - Bright-Field & Dark-Field Microscopy - Bright Field 適用於已染色強對比的樣本 Dark Field 小未染色對比差的樣本

15 - Phase Contrast Microscopy - 為什麼要有 Phase Contrast? 觀察的限制受觀測物體可區分為〆 Amplitude objects 已染色或吸光率高的高對比樣品 Phase objects 未染色之活細胞人的眼睛只能觀察到〆 1. 顏色的變化波長 wavelength. 2. 光強度的改變振幅 wave amplitude. 未染色之活細胞通常不能吸收光線, 他們只是改變入射光線約 1/4 的位相差, 而這樣變化人眼是幾乎無法察覺的.. 我們只能觀察 Amplitude objects 而無法看到 Phase objects 解決的方法將原本只有 phase 變化的物體, 藉由顯微技術的設計, 將其轉換為人眼可觀察的 amplitude 變化, 此即為 Phase Contrast Microscopy 年代,Fritz Zernike 成功的將 phase objects 的影像以 amplitude 方式展示出來而可被人眼容易的觀察, 奠定了 Phase contrast microscopy 的基礎,Zernike 也因此於 1953 年獲得諾貝爾物理獎

16 Phase shifter Conjugate Phase plate and light ring alignment Phase Contrast Microscopy 應用上的限制解析度限制. Phase 影像周圍的光暈現象不適合用於過厚的樣品觀察

Nomarski 所提出類三維影像的展現 (pseudo 3-dimensional image) 適用於 DIC

17 - Differential Interference Contrast - Differential Interference Contrast 的應用 DIC 顯微技術的確立是在 1950 年代中期由 Georges Nomarski 所提出類三維影像的展現 (pseudo 3-dimensional image) 適用於 DIC 觀察的樣本〆已染色的組織切片未染色的活細胞不適用於 DIC 觀察的樣本〆雙折射率物質, 塑膠製品位相差由 1/10 ~ 的樣本均可用 DIC 觀察

18 - Differential Interference Contrast - DIC 與 Phase Contrast 的比較 DIC 並沒有 phase light ring 的光環限制, 故物鏡的最大 NA 可被完整的運用解析度提高 DIC 影像沒有 phase contrast 的光暈現象物體邊界及細節較明顯 DIC 可用於較厚樣本之觀察 DIC 應用上的限制與缺點雙折射率物質或置於塑膠製的培養皿的樣本不能以 DIC 觀察對於非常薄的樣本,Phase contrast 是較好的選擇 DIC 觀察所需的配備十分昂貴

19 - Polarization Microscopy - Polarization 有偏光反應的樣本 Crystal, starch,, polymer, mineral Urate crystals

20 物鏡的種類與讀取

21 - Optics_Aberration - Monochromatic aberration Spherical aberration Coma Astigmatism Image curvature

22 - Optics_Aberration - Chromatic aberration Image lenses are never free of aberrations, but most of these can be eliminated by a combination of lenses. Axial chromatic aberration colored fog Lateral chromatic aberration color frignes

23 Objectives - Optics_Objective - HI PLAN: Achromatic objectives with a field performance up to 20 mm. Magnification: from 4x to 100x oil N PLAN: Planachromatic objectives with a field performance up to 22 mm. Magnification: from 2.5x to 100x oil PL FLUOTAR: Semi-apochromats with a field performance of at least 25 mm. Magnification: from 1.6x to 100x oil. PL APO: Plan apochromats with a field performance of up to 28 mm,. Magnification: from 10x to 100x oil, 150x and 250x are for semiconductor. Special Designed Objectives HCX PL APO CS: Some HCX PL APO objectives are re-calibrated for confocal system.

24 解析度 What is Resolution?

25 解析度 What is Resolution? R = 1.22* λ / (NAobj+NAcond) NAobj ~ NAcond R = 0.61* λ /NA 低 NA 物鏡高 NA 物鏡 => NA,R A general rule for a medium wavelength λ=0.55μfor visible light is: resolution =λ/ 2n.A = 0.55/2x μ

26 孔徑值 NA Numerical Aperture NA: Numerical Aperture n: Reflective index A: Sin of objective : The biggest angle that light can enter the objective

$light Refractive index$ n(air) = 1.

27 如何提高 NA? N,NA μ,na c speed of light Refractive index : n = v c in medium n(air) = n(glass) = 1.52 n(h2o) = 1.33 n(oil) = 1.515

- Optics - How to read your objectives? 1st line: e.g. /0.17/D : Infinite tube length objectives. (otherwise 160, 170.. ) 0.17: Coverglass thickness 0.

28 - Optics - How to read your objectives? 1st line: e.g. /0.17/D : Infinite tube length objectives. (otherwise 160, ) 0.17: Coverglass thickness 0.17mm; 0: Coverglass thickness 0; -:With or without coverglass; 0-2:for 0-2mm coverglass. A,B1,B2,C,D: Pupil position in the objective. When using the DIC contrast devices make sure that the IC prism used above the objective has the same letter. 2nd line: the criteria of the objectives. Left: coverglass=0.14mm Centre: coverglass=0.17mm Right: coverglass=0.20mm

29 - Optics - How to read your objectives? 3rd line: e.g. 100x/1.40 OIL PH3 100x: Magnification of objectives. 1.40: Numerical aperture ( N.A. ) of the objective. It influences: Resolution, which also depends on the wavelength λof the light. A general rule for a medium wavelength λ=0.55μfor visible light is: Resolution =λ/ 2n.A = 0.55 / 2x μ Useful magnification: 500 x N.A x N.A., e.g. 100x/1.40 objective, its useful magnification: 700x x Total magnification: 375x Left: 10x/0.25 Right: 25x/0.50

30 - Optics_Eyepiece - How to read your Eyepiece? e.g. HC PLAN 10x/25 M HC PLAN: The new LEICA HC system eyepiece, it means the chromatic difference in magnification (CDM) made by objectives and tube lens can be fully corrected. 10x: Magnification of the eyepiece, 8x, 12.5x, 16x. Total magnification of the microscope = Mob x Tube factor x Meye /25: Field number (FOV) of eyepiece. The field number represents the diameter (in mm) of the intermediate image that can be viewed through the eyepiece. Object field diameter: e.g. 10x/25 eyepiece and 50x objective, an object field of 25/50=0.5mm can be viewed. FOV 20: Objectives C PLAN (20), or N PLAN (22) FOV 22: Objectives N PLAN (22), or PL FLUOTAR (25) FOV 25: Objectives PL FLUOTAR (25), PL APO (28) M: The eyepiece has a focusable eyelens, range = ±4 dioptres. : The eyepiece can be used both with or without spectacles.

31 螢光顯微鏡影像

32 Two kind of beam path Transmission Light 明視野 Bright Field 暗視野 Dark Field 相位差 Phase Contrast 偏光 Polarization 干涉相位差 Differential Interference Contrast Incident Light 螢光 Fluorescent

33 What is Fluorescence? Exc ite d Sta te G ro un d Sta te G ro un d Sta te Nu c le o u s In n e r O rb ita l In n e r O rb ita l In n e r O rb ita l

34 Fluorochrome DAPI Fluorescein Texas Red

35 Conventional Fluorescence Microscope Illumination System (Excitation) Light Source HBO XBO

36 Conventional Fluorescence Microscopy Wavelength Selection Light Source

37 Conventional Fluorescence Microscope

38 Conventional Microscope Confocal Microscope Drosophila leg, FITC

39 螢光轉輪及燈源 Sutter Sutter LS 175 watt Liquid Light auide Lambda 10B controller 10 position 25mm with smart shutter Light Guide or LAMBDA XL Liquid light guide Lambda 10B controller 10 position 25mm with smart shutter Light Guide

40 Sutter 螢光轉輪濾鏡組

41 螢光轉輪及燈源 Sutter Filter Wheel Lambda 10B controller 10 position 32mm with smart shutter Excitation adaptor for 32mm Light Source Leica 100W Hg Leica EL 6000 Chroma Photofluor Excitation unit Assy, Light Guide, Liquid, 5mm X 2M, Universal PhotoFluor, Microscope Adapter, Leica precisexcite Cool LED ( RGB Filter Cube )

43 顯微鏡上的電動模組

44 XY Stage LUDL BioPrecision2 Flat top rotary encode stage (Acc: 6 um) BioPrecision2 Flat top linear encode 50nm stage (Acc: 3um) MAC6000 XY stage controller incl. joystick Stage Insert slide /petri-dish max 65mm Marzhauser SCAN IM 127X83 Universal Stage Insert Tango 3 PCI-S incl. motor cable and 3-axes joystick

45 Motorized Stages for Upright Microscopes Motorized Stages for Inverted Microscopes

46 Z Focus LUDL Focus Drive include adaptor MAC6000 Z-step controller incl. digipot PIEZO Z 200/500um PIEZO Z controller Marzhauser MFD for DMI Piezo Z-Stage P-737 incl. Piezo Amplifier E-625.SR Tango 3 PCI-S incl. motor cable and 3-axes joystick

47 Z section control Galvo z stage -- accuracy:10nm Microscope focus -- traveling range : mm~cm

48 Variable Microscopes LEICA DM6000B Automatic Upright Microscope LEICA DMI3000 Semi- Automatic Inverted Microscope LEICA M205 Stereomicroscope

49 Upright Microscopes & Samples

50 Inverted Microscopes & Samples

51 Stereomicroscope 由一個共用的物鏡, 對物體成像後的兩光束被兩組中間物鏡聚光鏡分開, 並成一體視角再經各自的目鏡成像, 它的倍率變化是由改變中間鏡組之間的距離而獲得的, 因此又稱為 " 連續變倍顯微鏡 "(Zoom stereo microscope). 隨著應用的要求, 目前體視鏡可選配豐富的選購附件, 如螢光, 照相, 攝像, 冷光源等等.

52 Stereomicroscope

53 Stereomicroscope 特性 1 雙目鏡筒中的左右兩光束不是平行, 而是具有一定的夾角體視角 ( 一般為 12 度度 ), 因此成像具有三維立體感 2 像是直立的, 便於操作和解剖, 這是由於在目鏡下方的棱鏡把像倒轉過來的緣故 3 雖然放大率不如常規顯微鏡, 但其工作距離很長 4 景深大, 便於觀察被檢物體的全層. 5 視野直徑大.

57 Advanced Fluorescence Microscopes

58 TIRFM Total Internal Reflection Fluorescence Microscopes Epi-Fluorescence TIRF

59 What is TIRF -- Phenomenon Snell s Law n 1 sinθ 1 = n 2 sinθ 2 sin c = n 2 /n 1 n2 θ 2 n2 θ 2 =90 n1 θ 1 n1 θ c Critical Angle n2 θ 2 n2 n1 n1 θ 1 Total Internal Reflection

60 What is TIRF Evanescent Field n2 Evanescent wave n1

61 What is TIRF Evanescent Filed n2 n1 E(z) = E(0)e (-z/d) E(z): Energy at z from surface d: penetration depth Evanescent Wave d = /4 (n 1 2sin n 22 ) -1/2 1. Energy exponentially decay in the z position -- excitation only at the interface ( 70~300 nm) 2. 1, d, decay

62 Applications and Solution Application examples Membrane research Vesicle transport Single molecule detection

Advantages and Application Excitation within

of minute structure on the surface Membrane

63 Advantages and Application Excitation within 70~300 nm in Z Low Energy Evanescent filed Low photobleaching High S/N ratio High Contrast No out of focus information Imaging of minute structure on the surface Membrane Research Vesicle Transport Single Molecular Detection

64 Objective TIRF 1.NA = n sin max 2. max > 1 3. n sin max > n sin 1 = n 2 sin 2 4. n sin max > n 2 sin 2 5. When TIRF Occur : 2 =90,sin 2 = 1 6. n sin max > n 2 7. NA > n 2 n 2 : n cytosol = 1.38 NA > 1.4 n2 θ 2 n1 θ 1

65 Leica TIRFM Working Station Applications and

66 Main components TIRF module Multiple laser TIRF Objective/Optics TIRF Filter cubes Camera Features and Benefits

67 Leica TIRF Objective HCX Pl APO 100X, 1.46 Oil, CORR High color correction High numerical aperture 160 m Laserspot = 16 m 5,52 mm 5,84 mm Temperature corrected C Cover slip correction Features and Benefits

68 TIRF Filtercubes Special TIRF cubes (Chroma) with thicker dichroic Less fringes and cleaner TIRF image CFP GFP YFP Excitation Dichroic Emission Features and Benefits

69 Thank you!

Microsoft PowerPoint - Microscopy principle__台大生化.ppt [相容模式]

Microsoft PowerPoint - Microscopy principle__台大生化.ppt [相容模式] Leica 全自動正立螢光顯微鏡 DM6000 B 目鏡螢光濾鏡轉盤物鏡穿透光聚光鏡 LCD 觸碰式螢幕螢光燈箱螢光光路調節輪功能按鍵 C- Mount ( 轉接環 ) CCD 載物台穿透光燈箱功能按鍵光源亮度調整景深光圈調整視野光圈調整穿透光 / 螢光切換 - Image formation - 顯微鏡的成像二次放大 ( 物鏡, 目鏡 ) 與中間影像 ( Intermediate