Con-focal and Multi-photon Microscope Experiment Fundamental. Qian Hu, Lab of Laser Scanning Confocal & Two-Photon Microscopy, ION, CAS

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1 Con-focal and Multi-photon Microscope Experiment Fundamental Qian Hu, Lab of Laser Scanning Confocal & Two-Photon Microscopy, ION, CAS

2 1. Light is Electromagnetic Wave ν = c / λ

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5 2. Image of a Point Source and Point Spread Function (PSF) FWHM later FWHM axi

6 3. Objectives

7 Decrease in size of Airy disks accompanying an increase of numerical aperture

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9 4. Optical Aberrations of Objective Spherical aberration Chromatic aberrations Curvature of field

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11 5. Köhler illumination and Conjugate Planes

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13 6. Image Formation

14 7. Contrast Enhancing Techniques Percent Contrast (C) = ((I(s) - I(b)) x 100)/I(b)

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21 8. Fluorescence Ground states, Excited states Three important events Excitation (femtoseconds), Vibrational relaxation (picoseconds), and Emission (nanoseconds) Planck's Law: E = hν = hc/λ Absorption Spectrum, Excitation Spectrum, Stokes Shift, Mirror Image Rule Quench reversible, non-rediative relaxation, collisional quench (oxygen, halogens, amines), static or complex quench (form non-fluorescent complex), dipolar resonance energy transfer mechanism Photobleach - irreversible, destruction of fluorescence, irreversible covalent modification, photodynamic(light and oxygen, free radical oxygen), fluorescence recovery after photobleaching(frap)

22 Extinction Coefficient, Quantum Yield, Fluorescence Lifetime Environmental factors - interactions between the fluorophore and surrounding solvent molecules (dictated by solvent polarity), other dissolved inorganic and organic compounds, temperature, ph, and the localized concentration of the fluorescent species, solvent relaxation, red shift Resonance energy transfer (RET), acceptor, donor, long range dipole-dipole interactions, 10 nanometers

23 9. Fluorescence Microscope Bandpass filters transmit a band of wavelengths and block all light above and below the specified transmission range. Longpass filters transmit long wavelengths and block short wavelengths, while shortpass filters transmit short wavelengths and blocking others. Dichroic filters is special filters which reflect certain wavelength away but permit other wavelength to pass.

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25 10. Confocal Microscope A point light source for illumination, a point light focus within the specimen, a pinhole at the image detecting plane - These three points are optically conjugated together and aligned accurately to each other in the light path of image formation, this is confocal. Void of interference from lateral stray light: higher contrast. Void of supperimpose of out-of-focal-plane signal: less blur, sharper image. Images derived from optically sectioned slices (depth discrimination) Improved resolution (theoretically) due to better wave-optical performance.

26 11 Multiphoton Laser Confocal Microscope Mean features: Using infra-red laser for excitation High energy demanding -only pulsed laser can meet the requirement. Excitation of fluorophore occurs only at the focal point - in single photon system, the excitation is on the whole illuminated length, although only emission from focal point is detected. Detecting pinhole is not need in this configuration Advantages: Low photo-cytotoxicity - long wavelength infra red laser has less toxicity than short wavelength Less bleaching than LSCM - only the focal point is affected Suitable for living cell imaging, point bleach experiment, and other physiologic studies Working with even thicker specimen than single photon confocal - long wave length pulse laser has less energy loss when penetrating specimen Disadvantages: High price for building-up and maintaining the system Image quality is a little bit deteriorated since using long wavelength infra red light

27 Wt GFP DAPI DsRed EGFP Fluo-3 FITC Fura(free) Fura-Ca

28 12. Fluorochromes and Stains DAPI, Hoechst, SYTO 16: semipermeant and permeant DNA stain, apoptosis GFP: used in living systems as a gene/protein reporter Lucifer yellow, Fluorogold: neuronal tracer MitoFluor green, MitoTracker green: mitichondrial marker

29 DiI: lipophilic membrane marker Rhodamine 6G, MitoTracker red: mitochondrial and ER marker Propidium iodide, Ethidium Bromide, SYTO 25: impermeant and permeant nucleic acid stain

30 13. Physiology Probes Indo-1, Fura-2: dual emission or dual excitation calcium probe Calcium green, Fluo-3: single wavelength calcium probe BCECF: single or dual excitation wavelength ph probe JC-1: potential sensitive mitochondrial dual emission Calcein: volume changes, gap junctions, liposomes, permeability Lysosensor Blue DND-192, LysoSensor Green DND-153: lysosomal marker NBD-C6 ceramide: Golgi marker Rhodamine-123: mitochondrial marker

31 SNARF: ph indicator emission ratio FM1-43, RH-414: plasma membrane Fura red: calcium indicator, decreased fluorescence on binding DIC18(3), DIC16(3): ER marker LysoTracker Yellow DND-68: lysosomal marker MitoTracker Orange, Rhodamine B Hexyl ester, Tetramethylrosamine: mitochondrial marker

32 14. DIC, Phase Constrast, and Fluorescence Combination

33 15. Multi-fluorescence Imaging

34 (1) Fluorescence Crosstalk Fluorescence Crosstalk or Bleed-through refers to the overlap of emission spectra between two or more fluorescence markers. Bleed-through can occor when samples are labelled with more than one fluor and/or when there is significant autofluorescence. Whilst sequential imaging and high cut-on/off band pass emission filters can be used to minimise bleedthrough, it is not always a complete solution (1) Multi-Tracking for Crosstalk Reduction Multi-tracking is the technique to minimize the cross talk of the multi colored samples between channels, by exciting each dye at a time. (2) META-Tracking - Emission Fingerprinting A lambda stack of the emission is acquired by the META detector simultianeously. Then, by means of the individual reference spectra of the fluorochromes, these are separated into dedicated channels by the Linear Unmixing function. As a result, even greatly overlapping emission spectra will be separated without crosstalk.

35 How do we determine bleed-through and autofluorescence? Check for bleed-through with a single-labelled sample: exciting sample with single laser and checking for bleed-through in different channels. If there is a bleed-through, perform sequential imaging or META tracking. Check for autofluorescence by exciting a completely unstained sample using the same instrument parameters. How to avoid bleed-through? Select (where possible) fluors whose emission spectra do not overlap eg. Alexa 488 and Cy5. Select fluors which can be separately excited with two different laser lines (eg. If you need to use FITC, do not use a second fluor which will also be excited by the 488 nm line). Use selected band-pass filters which optimise the combination of collection of specific fluorescent signal with emission separation Use laser lines which each excite only one fluorescent label. Use sequential laser excitation to minimise non-specific fluor excitation. Ensure that when you do multi-labelling, the fluorescence signals are of approximately the same brightness. Ensure that signal is brighter than autofluorescence in all channels.

36 (2) Multi-Tracking for Crosstalk Reduction Isolated sallvary gland of a cockroach. Cell nuclei are labeled with DAPI (blue), Na+/K+ ATPase with Cy2 (green), and F-actin with Alexa 568 phalloidin (red). The simultaneous recording of either DAPI and Cy2, or Cy2 and Alexa 568 shows strong bleedthrough of the DAPI signal into the Cy2 channel (middle), or of the Cy2 signal into the Alexa 568 channel (right). The same specimen recorded by Multitracking (with the same laser intensity). The channels are clearly separated without any crosstalk.

37 (3) META-Tracking Emission Fingerprinting

38 16. Localization and Co-localization

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40 17. Intracellular Ca2+ Measurement Indicator form: The salt and dextran forms are typically loaded by microinjection or infusion from a patchpipette. the cell-permeant acetoxymethyl (AM) esters can be passively loaded into cells, where they are cleaved to cellimpermeant products by intracellular esterases. Measurement mode: Ion indicators that exhibit spectral shifts upon ion binding can be used for ratiometric measurements of Ca2+ concentration, which are essentially independent of uneven dye loading, cell thickness, photobleaching effects and dye leakage. Dissociation constant (Kd): Indicators have a detectable response in the concentration range from approximately 0.1 Kd to 10 Kd. Fura-2 and indo-1 are UV light excitable, ratiometric Ca2+ indicators. Two-photon excitation imaging techniques used with fura-2 and indo-1 avoid the deleterious effects of conventional ultraviolet illumination on living specimens. Fluo-3 and fluo-4 are visible-light excitable non-ratiometric Ca2+ indicator. More recently, fluo-3 imaging has also been extended to include two-photon excitation techniques. Simultaneous loading of cells with fluo-3 and fura red indicator enable researchers to make ratiometric measurements of intracellular Ca2+ using confocal laser-scanning microscopy. Simultaneous loading of cell with fluo-3 and SNARF-1 permits the simultaneous imaging of Ca2+ transients and intracellular ph. Intracellular calibration of Ca2+ indicators may be achieved by manipulating Ca2+ levels inside cells using an ionophore (e.g. ionomycin, A and its nonfluorescent analog, 4-bromo A ).

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44 18. Z-Series and Three-Dimensional Imaging

45 19. Time Lapse Study of Receptor Transport

46 20. Fluorescence Loss in Photobleaching (FLIP)

47 21. Fluorescence Recovery after Photobleaching (FRAP)

48 22. Fluorescence Resonance Energy Transfer (FRET)

49 (1) FRET Detection by Direct Measurement of Intensity Changes Step1: Cells expressing YFP construct only - measure CFPex/YFPem, YFPex/YFPem In this case there is a real YFP signal but no CFP signal. Any signal in the FRET channel (CFPex/YFPem) is therefore due to crosstalk of the YFP signal into this channel. This crosstalk (measured as a ratio of the CFPex/YFPem signal to the YFPex/YFPem signal) will be subtracted from the FRET signal measured in experimental cells. Call this value a. Usually a= 20-30%. Step2: Cells expressing CFP construct only - measure CFPex/YFPem, CFPex/CFPem In this case there is a real CFP signal but no YFP signal. Any signal in the FRET channel (CFPex/YFPem) is therefore due to crosstalk of the CFP signal into this channel. This crosstalk (measure as a ratio of the CFPex/YFPem signal to the CFPex/CFPem signal) will be subtracted from the FRET signal measured in experimental cells. Call this value b. Usually b= 50-70%. Step3: Cells expressing both constructs - measure CFPex/YFPem, YFPex/YFPem, CFPex/CFPem Net FRET = FRET signal (a*yfp signal) (b*cfp signal) In this case there are both YFP and CFP signals in the cell, and any signal measured in the FRET channel must have the appropriate percentages of these two signals subtracted from it. Remember: It s important to let cells express the constructs in the appropriate relative levels. It works best if the YFP signal is equal to or slightly higher than the equivalent CFP signal.

50 Step1: EYFP-U1A exoressed alone. Approximately 20% of the signal ceosses over into the FRET channel. Step2: ECFP-NIPP1 expressed alone. Approximately 70% of the signal crosses over into the FRET channel. Step3: Example of a real FRET measurement. Cells are expressing both YFP-PP1g and CFP-NIPP1, and the NIPP1 has bound to and retargeted PP1 to nuclear speckles (it is normally found in the nucleolus). Signal remaining in the FRET channel after YFP and CFP crosstalk were subtracted. Image colorintensity scaled from pixels.

51 (2) FRET Detection by Acceptor Photobleaching EF = (Iafter Ibefore) / Iafter