SAPIENZA Università di Roma Laurea magistrale in Ingegneria delle Nanotecnologie A.A Biophotonics Laboratory Course

Transcription

1 SAPIENZA Università di Roma Laurea magistrale in Ingegneria delle Nanotecnologie A.A Biophotonics Laboratory Course Prof. Francesco Michelotti SAPIENZA Università di Roma Facoltà di Ingegneria Civile e Industriale francesco.michelotti@uniroma1.it

2 LECTURE 6 Super-resolution microscopies (STED, PALM, STORM) Test ELISA

3 Applications of optics and photonics Microscopic Techniques Conventional Wide-Field Fluorescence TIRF FLIM FRET, FRAP Confocal Two-Photon Second Harmonic Super-resolution (SNOM, STED, PALM, STORM) Non-Microscopic Label-free Surface plasmon Polaritons (SPP) Photonic crystals (PC) Raman, CARS Quantum dots Non-Microscopic Techniques Cytofluorimetry ELISA DNA-Chip Cycle-sequencing SOLID Other non Microscopic Techniques Southern Western Northern All of them make use of the emission of luminescent markers (labels)

4 STED STimulated Emission Depletion Microscopy In STED microscopy the specimen is excited and the fluorescence is collected as in conventional wide-field microscopy. A supplementary laser beam causes the relaxation of the chromophores inside an annular region around the central spot by stimulated emission.

5 STED STimulated Emission Depletion Microscopy S e t 1 S g t2>t1 t

6 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

7 STED STimulated Emission Depletion Microscopy

8 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

9 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

10 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

11 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

12 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

13 STED STimulated Emission Depletion Microscopy Diffraction would limit the resolution also in this case. The real mechanism that is responsible of the increase of resolution is the saturation of fluorescence reduction obtained by means of stimulated emission. I SAT The resolution is given by: x 2nsin 1 I I SAT

14 STED STimulated Emission Depletion Microscopy I SAT NOTE If there is no saturation of the fluorescence reduction (I SAT = ) the resolution is that given by the Abbe formula, i.e. it is limited by diffraction. x 2nsin 1 I SAT I 2nsin

15 STED STimulated Emission Depletion Microscopy Dye name (Manufacturer / Distributor) Exc. Exc. Wavelengt Pulse h Length STED Wavelengt h STED Pulse Length Repetition Rate Avg. STED Power Peak Irradiance Reported Spatial Pulse Resolution Energy (Direction) ATTO 532 (ATTO-TEC GmbH) 470 nm 80 ps 603 nm 280 ps 250 khz 0.5 mw 2 nj <25 nm (xy) Chromeo 488 (Actif Motif) 488 nm 140 ps 602 nm ~ 160 ps 250 khz 0.6 mw < 30 nm (xy) DY-485XL (Dyomics GmbH) 488 nm < 100 ps 647 nm ~ 200 ps 72 MHz (20 + 3) mw nm (xyz) GFP 490 nm 100 ps 575 nm 200 ps 80 MHz 7.2 mw ~ 70 nm (xy) ATTO 565 (ATTO-TEC GmbH) 532 nm ~ 90 ps nm ~ 90 ps 1 2 MHz nm (xy) MR 121 SE (Roche Diagnostics) 532 nm 10 ps 793 nm 107 ps 76 MHz 10.4 mw ~ 50 nm (z) NK51 (ATTO-TEC GmbH) 532 nm < 100 ps 647 nm ~ 200 ps 72 MHz Sulfonated & rigidized rhodamine derivatives (V. Boyarskiy, NanoBiophotonics, MPI Göttingen) Pyridine 2 / LDS 722 (Exciton, Radiant Dyes GmbH) 532 nm 100 ps 640 nm ~ 300 ps 80 MHz (20 + 3) mw 40 MW/cm nm (xyz) < 90 nm (xy) 554 nm 250 fs 760 nm 13 ps 76 MHz 33 nm (z) RH 414 (Invitrogen Corp.) 554 nm 250 fs 745 nm 13 ps 76 MHz 8.78 mw 30 nm (z) ATTO 590 (ATTO-TEC GmbH) 570 nm ~ 90 ps ATTO 633 (ATTO-TEC GmbH) 630 nm ~ 90 ps nm nm ~ 90 ps 1 2 MHz nm (xy) ~ 90 ps 1 2 MHz nm (xy) ATTO 647N (ATTO-TEC GmbH) 635 nm cw 750 nm cw cw 423 mw ~ 50 nm (xy) JA 26 (K.H. Drexhage, Siegen University) 635 nm 68 ps 775 nm 300 ps 76 MHz 800 MW/cm 2 16 nm (x)

16 STED STimulated Emission Depletion Microscopy By playing with the STED beam focusing one can obtain a 3D reduction of the fluorescence and increase the axial resolution.

17 STED STimulated Emission Depletion Microscopy Visualization of single vesicles in a synapsis. More precisely it is visualized the Synaptotagmin protein, which is embedded in the membrane of the vesicles. The vesicles have an average dimension of 40 nm and are filled with neurotransmitters.

18 STED STimulated Emission Depletion Microscopy Confocal (below) and STED (above) images obtained by means of a Leica microscope

19 STED STimulated Emission Depletion Microscopy

20 PALM Photo-Activated Localisation Microscopy STORM - STochastic Optical Reconstruction Microscopy The basic mechanisms underlying both techniques are: Label the specimen with a chromophore that is characterized by a long lifetime dark state; Bring all chromophores in such a dark state by means of a first light pulse. When in the dark state the chromophores are activated and brought again to a fluorescent state by means of a second light pulse; Due to the stochastic nature of photoactivation, only few and well separated chromophores will be lighted up. The probability to have two close chromophores is very small; The chromophore emission is fitted by Gaussian curves, retrieving the position of the emitter; At the end of the emission the few active molecules are brought back to the dark state and another activation pulse lights up randomly a different set of emitters; The process is repeated many times, reconstructing the imag molecule by molecule.

21 PALM Photo-Activated Localisation Microscopy STORM - STochastic Optical Reconstruction Microscopy These are two basically identical techniques that make use of a massive statistical analysis of the fluorescence data. CCD n The position of a fluorophore is determined with high precision, by fitting the diffraction pattern that is generated after many cycles on a CCD detector.

22 PALM Photo-Activated Localisation Microscopy STORM - STochastic Optical Reconstruction Microscopy The specimen is illuiminated with light pulses that activate stochastically few chromophores at a time. The probability that two close chromophores are activated simultaneously is minimized. The postions are fitted at every cycle. CCD In PALM and STORM we are never under the conditions in which the diffraction curves are superimposed more than the Rayleigh condition. The process is repeated up to image reconstruction.

23 PALM Photo-Activated Localisation Microscopy STORM - STochastic Optical Reconstruction Microscopy Comparison between TIRF and PALM, obtained by means of a Zeiss microscope. Labelling by means of tubulin antibodies in cultured cells.

24 STED STimulated Emission Depletion Microscopy PALM Photo-Activated Localisation Microscopy STORM - STochastic Optical Reconstruction Microscopy Summary of the Techniques

25 Applications of optics and photonics Microscopic Techniques Conventional Wide-Field Fluorescence TIRF FLIM FRET, FRAP Confocal Two-Photon Second Harmonic Super-resolution (SNOM, STED, PALM, STORM) Non-Microscopic Label-free Surface plasmon Polaritons (SPP) Photonic crystals (PC) Raman, CARS Quantum dots Non-Microscopic Techniques Cytofluorimetry ELISA DNA-Chip Cycle-sequencing SOLID Other non Microscopic Techniques Southern Western Northern All of them make use of the emission of luminescent markers (labels)

26 Non microscopic techniques ELISA - Enzyme-Linked ImmunoSorbent Assay ELISA is an immunology test used in biochemistry to reveal the presence of a given antigene, typically pertaining to a pathogenic micro-organism, and makes use of a specific antibody. There exist two variants of the ELISA test: non competitive ELISA and competitive ELISA. The non competitive ELISA test can be executed according to two different procedures: direct method and indirect method. The tests are colorimetric and the read out is performed by means of a spectrophotometer.

27 ELISA non Competitive

28 ELISA non Competitive Preparation of the wells of a polystyrene multi-well plate by injecting of a solution of the primary antibody, which is specific for the antigen that we intend to detect. The antibodies adhere at the base of the well by means of a layer of fish gelatin. The excess is washed out by rinsing. Addiction of the human samples in which we wish to detect the presence of the antigen that is characteristic of the pathogenic microorganism. Wash with a buffer solution. The antigen, if present, binds specifically the antibody. The excess is washed out by rinsing. Addiction of the secondary antibodies. Such antibody are conjugated to an enzyme, typically either peroxidase or alkaline phosphatase.

29 ELISA non Competitive The secondary antibodies bind selectively to the antigen, if present. The excess is washed out. Alternatively, if the specific antigen is absent, the secondary antibody (and the conjugated enzyme) will be removed during the washing step. Addiction of a p-nitrophenyl phosphate substrate, triggers a reaction with the enzyme conjugated to the secondary antibody, that produces yellow-colored para-nitrophenol. Addiction of sodium hydroxide blocks the reaction between the enzyme and the p-nitrophenyl phosphate. Read out of the result by means of a spectrophotometer.

30 ELISA non Competitive

31 ELISA Competitive Unlabelled antibodies coming from a sample are incubated in the presence of their antigens; Such antibody/antigen complexes are injected in the wells of a plate, which were previously functionalized with the same antigens. The plate is washed so as to remove all unbound antibodies. Secondary antibodies that are specific for the first antibody are injected. Such secondary antibody are conjugated to tan enzyme. A substrate ia added and the bound enzymes cause a chromatic change. In the competitive ELISA test, the larger it is the antigen concentration in the sample the weaker it is the signal. The advantage is that we can use impure samples and that we still have the possibility to bind selectively any present antigen.

32 ELISA Competitive Quntity k-casein B The well is functionalized with k-casein B Sample pipetting Pipetting of a know quantity of the specific antibody for k- casein B The antibody binds to all k-casein B of the sample and the excess binds to the k-casein on the bottom of the well The excess antibody bound to the bottom of the well is colored k-casein B k-casein A Color Intensity The larger it is the color intensity the smaller it is the k-casein B content in the sample Antibody k-casein B Seconday antibody

33 Non microscopic techniques ELFA - Enzyme-Linked Fluorescence Assay ELFA is an evolution of the ELISA test. The enzyme that is conjugated to the secondary antibody converts the substrate to a luminescent compound The read out is carried out by means of a spectrofluorometer Figure 1: Fluorescence image of a 96-well platereader plate filled with an ELISA. The red framed wells contain known concentrations and the blue framed wells the unknowns. The wells marked with other colours contain control solutions.

34 Non microscopic techniques ELFA - Enzyme-Linked Fluorescence Assay

35 Non microscopic techniques ELFA - Enzyme-Linked Fluorescence Assay

36 Non microscopic techniques DNA sequencing Introduction to the Sanger methd The Sanger method permits to sequence DNA, making use of the biochemical techniques associated to the use of radio-active markers. It exploits the reaction of polymerization of aminoacids sequences (Nobel Chemistry 1980) and the gel electrophoresis technique (Nobel Chemistry 1958) sangerseq.exe

37 Non microscopic techniques Cycle sequencing Evolution of the Sanger method The Sanger method permits to sequence DNA, making use of the biochemical techniques associated to the use of radio-active markers. It exploits the reaction of polymerization of aminoacids sequences (Nobel Chemistry 1980) and the gel electrophoresis technique (Nobel Chemistry 1958) cycseq.exe

38 Non microscopic techniques Cycle sequencing Example

39 Non microscopic techniques Cycle sequencing Evolution of the Sanger method

40 Non microscopic techniques Cycle sequencing SOLID Further evolution (already old!) SOLID.wmv

41 Applications of optics and photonics Microscopic Techniques Conventional Wide-Field Fluorescence TIRF FLIM FRET, FRAP Confocal Two-Photon Second Harmonic Super-resolution (SNOM, STED, PALM, STORM) Non-Microscopic Label-free Surface plasmon Polaritons (SPP) Photonic crystals (PC) Raman, CARS Quantum dots Non-Microscopic Techniques Cytofluorimetry ELISA DNA-Chip Cycle-sequencing SOLID Other non Microscopic Techniques Southern Western Northern All of them make use of the emission of luminescent markers (labels)