Lecture 4: Fluorescence Microscopy

Transcription

1 Lecture 4: Fluorescence Microscopy Basic concepts Fluorescence: a phenomenon that some molecules absorb energy from light and emit another light of longer wavelength. FL-Microscopy: use fluorescence light from specimen to form image Fluorophore or Fluorochrome: A functional group which absorb energy and re-emit energy as light wave. It is now often refers the molecule which has the functional group attached to it. Fluorophore excitation and emission Excitation: absorb energy from light wave, raise to higher energy state. Emission: release the absorbed energy in photon, return to ground state 1

2 Excitation and emission Properties of fluorophore Jablonski Diagram 2

3 Stokes shift and emission Single excitation and emission Multiple excitation and single emission Emission additive effect 3

4 Fluorescence Microscopy Types of Stokes shift Usual Stokes shift is nm Too short shift: difficult to separate Em from Ex Too long shift: waste limited spectra range, but good for separation without cross-talking 4

5 Other physical parameters of fluorophores Not all light from Excitation source are absorbed molar extinction coefficient EC: EC= C.L A (c: concentration, L: light path length of measuring, A: absorbance OD) usually: (liter/mol.cm) are expected Not all absorbed light yield emission Quantum yielding QY (quantum efficient Qe): 10% - 80% Not all emission can be utilized Quenching FRET Bleaching: photobleaching quantum efficient (Qb) 5

6 Types of fluorophores Small molecule: like FITC, TRITC, Texas Red, AlexaFluor. (conjugated to protein, antibody, amino acid, sugar: like lectin ) Fluorescent Protein: GFP and derivatives: BFP,YFP; DsRed and others newly engineered fluorescent proteins Nanocrystal Qdot (Quantum Dot) Natural auto-fluorescent material: Riboflavin, NADH, lipofuscin, 6

7 Methods for fluorescent labeling of specimen Immunofluorescence staining: Direct immunofluorescence labeling: conjugate fluorophore to 1 st Ab against antigen (rarely in use now) Indirect immunofluorescence labeling: conjugate fluorophore to 2 nd Ab Non-immuno-binding (affinity): fluorophore labeled lectin to bind cell membrane, contour of cells. Fluorescent proteins: GFP and derivatives, RFP and derivatives: expression of fusion protein inside living cells, live cell imaging is possible Cell organelle markers: Mitochondria, lysosomes, ER, Golgi, Nuclei., Other markers within live cells: indicator for ion: Ca, K, Na, for PH for membrane potential, for cell viability (Molecular Probes have a extensive list of them) 7

8 Detecting Fluorescent signal by microscope Early development of FL-Microscope Transmitted light fluorescence microscope Transmitted light bright field FL-Mic Transmitted light darkfield FL-Mic 8

9 Reflected light fluorescence microscopy (diagram of an Epi-Fluorescence Microscope) 9

10 structure of epi-fluorescence Microscope Epi-fluorescence: Reflected light illumination Inverted microscope Upright microscope 10

11 objective lens NA and Fluorescent signal collection Oil lens increase emission collection efficiency to 2-3 times 11

12 Excitation light sources Mercury/Xenon arc lamp : a mixture of different wavelength Xenon 75 Laser: discrete single peak of wavelength Gas laser: Ar. 351, Ar. 488, Kr. 568, HeNe. 633; Diode laser and Solid state laser DPSS LED: Light-emitting Diode: Single band of wavelength Purchase of separate lines of light source is needed 12

13 Filter cube and its components Beam Splitter: the Pivotal of Filter-set Filter cube 13

14 Single-band BSP in a FITC cube An ideal filter-set: 1. Excitation filter of band-pass type with narrow band 2. BSP with a cut-off threshold just behind λexc, long-pass for λem w/o obstruction 3. Emission filter of long-pass type allow all emission pass-through 14

15 Multiple-channel, simultaneous detecting fluorescent signal when time delay for multi-channel sequential excitation and imaging is not allowed, simultaneous multi-channel detecting must be used. Microscope must provide RGB color image: multi-band BSP Camera must take RGB at once: Color camera must be used. DAPI single excitation: no emission loss DAPI/FITC/TRITC simultaneous excitation: Emission leak at shaded area 15

16 Fluorescent signal detecting Color CCD or B/W CCD: why? CCD is inherently color insensitive (it is intrinsically monochrome, B/W) Charge Coupled Device: convert received photon into electric charge, that s all, no color information. Color is added artificially by using filter on top of CCD cells and digital image processing unit on the CCD-chip. Mosaic filter (Bayer filter) Red: 1/4 Green: 1/2 Blue: 1/4 only part of CCD area is used for each color 16

17 Color reproduction in matrix filter Missing information in Mosaic filter Interpolated virtual pixel value for missing information 17

18 Disadvantages of Mosaic filter 1. Reduced sensitivity: actual photon sensing area decrease. sensitivity is very important for detecting FL-signal as FL-field has very few photons FL (<100 photons/field) compare to BF (1000 to photons/field) 2. Reduced spatial resolution: four photon sensing cells combined into bigger unit. 3. Reduced edge sharpness: abrupt intensity change at edge has been averaged or neutralized. 4. Make quantification measurement difficult: guessed, interpolated virtual value replaced real intensity value. 5. multiple-band BSP plus mosaic filter color camera (Color camera needs true color info from microscope: multiband BSP needed) The worst combination for signal detecting sensitivity 18

19 Any alternatives for color? Yes, very simple for FL-MIC by Pseudo-color When single-band BSP is used, it delivers one color to CCD per exposure all signal can be assigned to one color by Lookup table (LUT) in software Pseudo color: LUT 19

20 Conclusion: monochrome, pseudo-color is best for FL-mic Whenever possible: Use single band filter-set. Use monochrome CCD camera (black and white) Sequential detecting signal one channel per time. Images captured by monochrome CCD camera. two channel gray-scale images saved, or with LUT embedded pseudo color images rendered by software, two channel merged Advantage: high sensitivity, high resolution, high accuracy of color location Disadvantage: time delay of a few seconds for taking two images 20

21 More about LUT operation LUT can handle more complex colors by assigning color value to pixels at certain index value Pixels are indexed by their gray intensity value, coloring operation affects all pixels with the same intensity value The color value can be edited based on index LUT operation handle 8-32 bits grayscale image, or 8 bit color, but not RGB image 21

22 Other methods for color imaging 3-ccd configuration with prism: No loss of spatial resolution, all pixels are used. No interpolation artifacts. Good color fidelity. Simultaneous three color, no time delay. But more expensive Less sensitive, even less sensitive than mosaic filter because of loss on prism Image distortion due to prism 22

23 Three shot color setup Use three filters in tern instead of prism. One color per shot: all pixels are used. No interpolation, color fidelity good. One camera can be used for both monochrome and colored one. Disadvantages: Less sensitive due to filter absorption Low temporal resolution: time delay Mechanical filter: seconds delay Turnable RGB-LCD filter: ms delay Bad color fidelity if white balance is not set properly 23

24 When color camera is needed? 1. Bright-field microscope of color stained slides Impossible to render millions of color tones Illumination is more than enough, no sensitivity problem Three-shot, tunable filter is an option when both BF/FL on the same Microscope are needed 2. Dynamic event, no time delay allowed multi-color calcium study: ms or µs time rate. Mosaic or Three-CCD color needed. 3. Multi-labeling living cell imaging depending on event monitored 1) monochrome camera with motorized FL-filterset 2) 3-shot-tunable filter 3) 3-CCD color 24

25 The way of increase CCD sensitivity CCD pixel binning Increase CCD sensitivity: n x binning, surface area increase n 2 speed up ccd reading process: n x binning, pixel number decrease n 2 4 x binning, 4 mega reduce to 0,25 mega, 16 x faster reading. Back-thinned CCD: high sensitivity for very low light apps EMCCD: electron multiplying CCD: single photon counting 25

26 Total Internal Reflection Fluorescence (TIRF) Microscopy Total internal reflection When light passed from a high RI media to a low RI media at an angle larger than a critical angle on the interface, no light can pass through. The light is reflected back. The critical angle is defined by From glass to air: θ c = arcsin n 2 n 1 θ c = arcsin = 41.8 θ c is valid only when n 2 n 1 < 1 when a critical angle 41.8 reached, the light is turned back: In ordinary microscopy, TIRF is a problem which should be avoided. TIRF brings a good side effect: evanescent wave (Vanishing wave) It is exponentially attenuated in z-direction: nm then dissappear This short penetrating is utilized for selective illumination in TIRF microscopy. 26

27 Application of TIRF Use TIRF: Z-resolution at 100 nm, while confocal has nm z-resolution Application: study event near the cell membrane. Vesicle trafficking, cortical cytoskeleton dynamics. TIRF can be combined with wide-field FL, phase contrast, DIC, and recently with super resolution microscopy 27

28 Fluorescent dye fading Fading: fluorescent emission become weaker and weaker during observation Reasons of dye fading: Photoblinking: intensity fluctuation in mini-seconds scale. Photoblinking: Reversible losses of fluorescence leading to a dark state. Photoresurrection: photon induced transition from dark to bright -state. Quenching Energy transfer between two very close (1-10 nm) dye molecules: FRET Collisional quenching: due to dye overlap. Static quenching: ground state complex, dye dimer self quenching. Photo-bleaching (photolysis): chemical modification of the dye and irreversible loss of ability to fluoresce. 28

29 Mechanism of bleaching Not fully understood: Inter-system crossing Increased ratio of excited triplet Fluorophore high Qb Molecule oxygen O2 Reacts with excited dye triplet state, Yielding singlet oxygen 1 O2 oxidize and irreversibly damage dye ROS: O 1 2, H2O2 Dual action of molecule oxygen O2 As triple state quencher (TSQ) Oxidize dyes: bleaching. 29

30 The ways of combating fading (1) Choose right fluorophores by their properties EC (Molar extinct coefficient): Photons absorbed at a wavelength by unit molar of dye: how easy a fluorophore can be excited. High EC means less excitation light for more emission, but not always. Qy must be considered together. Qy (Quantum yielding): Number of photon emitted per absorbed excitation photon. Qb (photobleaching quantum efficiency): Number of molecules bleached Number of photon absorbed 30

31 The ways of combating fading (2) anti-fading mounting media Traditional time-honored anti fading mounting PVA (polyvinyl alcohol; Mowiol): become solid after drying: permanent mounting media Prolong fluorescent dye storage time in dark, 4 C for months, years. Lower down background fluorescence. Limited anti-fading ability: must be used with PPD or npg or DABCO Birefringent: cannot be used for DIC-Fluorescence combined application. PPD (paraphenylenediamine): good anti-fading but cytotoxic. PPD added to PVA oxidize within a few days, must be used in a few days PPD added to glycerol can be kept longer in dark, -20 C up to three months. npg (n-propyl gallate): Anti-fading next to PPD but less cytotoxic DABCO(diazobicyclo-octane): less effective anti-fading but good storage property. 31

32 The ways of combating fading (3) (only for high photon influx application) For Two photon excitation, FRET, TIRF, STED, Not for ordinary application Triplet state quenching (TSQ)by bio-reducing reagent: BME β-mercaptoethanol, MEA mercaptoethylamine, DTT (Dithiothreitol), COT cyclooctatetraene, Trolox (water-soluble Vit.E), NBA (nitrobenzyl alcohol) Anti-oxidant: Vit.C and E; plus npg, PPD Deoxygenating by N2 bubbling improve survival time of dyes and cells. Remove molecular oxygen by oxygen scavenger PCA/PCD protocatechuate acid/protocatechuate-dioxygenase. GODCAT Glucose oxidase and catalas 32

33 Commercial Anti-fading reagent comparison Gelvatol-nPG, Slowfade, Prolong look good from the curve (Gelvatol is a PVA equivalent) 33

34 Final comment on FL-microscopy Developed rapidly. All super-resolution microscopy use FL Special marker detecting variety of cellular structures. Reveal morphology and detecting chemical composition High sensitivity, specificity, detecting single molecule now Study protein interaction: co-localization or FRET, FLIM. Limitation: Many labeling methods need fix cells, permeablize cells. Detected cellular structure does not reflect its true morphology but distribution pattern of positive signal. Other contrast modes should be combined to get a true morphology and localization information. 34

35 FL-positive area is not consistent with cell shape Fluorescence only Fluorescence + DIC 35

36 Comparison of fluorescence microscopy and other color contrasting methods Immuno-FL IHC Special Staining Sensitivity high Low, mid or high low Specificity High (molecule) High (molecule) Low (material category affinity) Stability and durability low, fade with use and storage time Good, not fade with use. Long storage time Good, not fade with use. Long storage time Easy of use More demanding Yes, standardized Yes, standardized Quantification accuracy Good, but must be controller properly Semi-(graded) quantification: -++ Semi-(graded) quantification: -++ Device demanding FL-MIC Ordinary MIC Ordinary MIC 36