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Strukturanalyse > Instrumentation > Flashcards

Instrumentation Flashcards

1
Q

Instrumentation

A

BASIS: EPI FLUORESCENCE MICROSCOPE
- wide field microscopy
- TIRF microscopy
- confocal microscopy

  • light sources
  • filters
  • detection
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2
Q

Wide field microscopy

A
  • Illumination of the sample with parallel light (no Köhler illumination, because fluorescence is isotropic)
  • Fluorescence detection with camera
  • Laser is focussed in back focal plane of microscope objective
    + parallel (simultaneous) detection of many molecules
    + ms tracing possible
  • out of focus molecules are excited: high background
  • suited for thin film samples, e.g. membranes or molecules immobilized on a cover slip
  • time resolution limited by camera (ms)
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3
Q

TIR Microscopy

A
  • Total internal reflection: critical angle glass/water ≈ 61°)
  • Evanescent field at interface, exponentially decaying
  • Fluorescence detection with camera
    -> wave fronts that are getting reflected
    + Illumination of thin layer: molecules above are not excited
    + Parallel detection
  • no imaging depth
  • all other camera limitations

Dipole emission at interface -> most of the fluorescence is emitted into the glass because it has a higher emission

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4
Q

Prism TIRF

A

-> NOT Epi fluorescence
+ Flexible choice for angle of incidence (air)
- Emission into “wrong” direction: low signal

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5
Q

Objective TIRF

A

+Detection from the „right side“: High signal
- angle of incidence limited (e.g. N.A.=1.4, 67°)

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6
Q

Confocal microscopy

A
  • Excitation and detection in one point
  • Imaging by scanning
  • Realizations: Laser scanning confocal microscope and Sample scanning confocal microscope
    +Out-of-focus fluorescence suppressed
    +Measurement depth not limited
    +High time resolution (down to nanoseconds)
    – No parallel detection (time consuming)
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7
Q

Light sources (Laser)

A
  • Mode of operation
    -> Cw (continuous wave)
    -> Pulsed (pulse durations ≈ 100 ps) for lifetime, pulsed interleaved excitation
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8
Q

Diode Lasers

A
  • Wavelength 400 – 14000 nm
  • single wavelength (slightly tunable)
  • Cw and pulsed (100 ps, flexible pulse rates)
  • elliptical beam profile
  • good efficiency (230 Volt, air cooling)
  • compact
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9
Q

Gas (ion) laser

A
  • Wavelength 325 – 676 nm
  • several lines from one laser
  • Cw and mode locked
  • excellent beam profile (efficient fiber coupling)
  • low efficiency (kW power consumption, water cooling)
  • bulky
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10
Q

Solid state lasers

A
  • lambda = 266 - 1444 nm
  • one line
  • Cw and mode locked (down to fs pulse duration)
  • excellent beam profile (efficient fiber coupling)
  • diode laser pumped: high efficiency, compact
  • fiber laser: very compact, rugged
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11
Q

Supercontinuum light source (laser)

A
  • Quasi continuum 400 – 2000 nm
  • Choice of wavelength with filter or grating
  • only pulsed (ps)
  • reasonable beam profile
  • high efficiency
  • rather compact
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12
Q

Filter

A
  • Purpose:Transmit one (two…) wavelength region(s), reflect (suppress) other(s)
  • Based on interference of waves reflected at interfaces between dielectric layers
  • reflection depends on polarization and angle, interference depends on angle
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13
Q

Production

A
  • Vapor deposition (soft coating)
  • less defined layers
  • less stable (humidity degrades filters!)
  • cheaper
  • Sputtering (hard coating)
  • well defined layers robust
  • more expensive
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14
Q

Most important properties

A
  • Transmission (as high as possible, close to 1)
  • Edge steepness (as steep as possible,T=0.1 to T=0.9 within few nm)
  • Optical density in stop band (>5, i.e. 105 suppression)
  • Polarization dependence (as small as possible)
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15
Q

Detectors

A

General principle: Generation of one charge by one photon
Requirements:
- high quantum efficiency (charge per photon) over broad wavelength range
- high linearity (no saturation)
- high time resolution
- low dark count rate

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16
Q

Detection

A
  • Point detectors: avalanche photo diode (APD) and photomultiplier tube (PMT)
  • Integral intensity on detector surface
  • Cameras: Charge coupled device (CCD) and electron multiplying CCD (EMCCD)
  • Imaging
17
Q

APD vs PMT

A

APD:
+ single photon detection
+ good linearity
+ good quantum efficiency (> 60% in the red)
+ reasonable time resolution (400 ps)
- afterpulsing
- high dark count rate

PMT:
+ single photon detection
+ high time resolution
+ large detector area
- bad linearity
- small spectral range
- low quantum efficiency (< 25%)

18
Q

Cameras: CCD

A
  • Charge coupled device (Nobel Prize Physics 2009)
  • Charges are accumulated in pixel (maximum number [full well capacity] determines dynamic range)
  • Charges are „counted“, typ. noise 4-5 charges (!)
  • quantum efficiency reaches 100 %
19
Q

Cameras: EMCCD

A
  • electron multiplying charge coupled device
  • Charges are accumulated like in CCD
  • Special area on chip (gain register) serves amplification by generation pf secondary charge by high voltage
  • but: this gain is introducing multiplication noise
20
Q

Properties EMCCD

A
  • Single photon detection capable
  • Quantum efficiency close to 100 %
  • dark noise – reduced by cooling (-90 °C) to 0.001/pixel/s
  • readout noise – not relevant due to amplification (0.02 charges effectively)
21
Q

Cameras: CMOS

A
  • Charge-to-digital conversion within pixel
  • Improved speed, lower readout noise
  • Higher pixel-to-pixel noise
22
Q

Fluorescent labels

A
  • in general, the electrons involved in the fluorescence process in the dyes we use, belong to delocalized conjugated pi electron systems
  • there are intrinsically fluorescing amino acids; however, there low photo stability prevents their use in single-molecule applications
  • we distinguish between synthetic dyes and fluorescent proteins: While synthetic dyes are chemically attached to the mature protein, fluorescent protein like the green fluorescent protein GFP can be fused to the protein under investigation genetically
  • for in-cell applications, the genetic encoding has advantages, if biomolecules are to be studied in vitro, a labeling with synthetic dyes is preferred, because synthetic dyes are compared to fluorescent proteins
    1. less bulky
    2. more photo-stable
    3. less prone to blinking and spectral shifts
23
Q

Synthetic dyes

A
  • Rhodamines are highly photo-stable fluorophores with high fluorescence quantum yields and available in the range of absorption wavelength between 480 and 633 nm
  • Cyanines are also highly photo stable and have a high fluorescence quantum yield and are available in the range of absorption wavelength between 490 and 800 nm
  • they can reversibly photoisomerize leading to a dark state, show complicated photo physics
24
Q

Instrumentation

A
  • to detect the light of a single molecule, a microscope is paramount
  • there are two main types of microscope used: wide field (conventional) microscopes and scanning confocal (rarely: near-field) microscopes
  • with a microscope, the signal of distinct fluorescence molecules can be resolved
  • thus, the spatial separation between the molecules must be larger than the resolution limit of the microscope, which is roughly half the wavelength of the light
  • in solution, this requires a concentration in the sub-nano molar range
  • with microscopy techniques lifting the resolution limit of conventional microscopes like near-field microscopy or STED microscopy, higher concentrations up to micro-molar can be used
25
Q

Wide field epileptic fluorescence microscopy

A
  • wide field epileptic fluorescence microscopy is based on the excitation of the fluorescence through the microscope objective
  • The same objective is used to image the fluorescence signal onto a camera
  • Therefore, excitation and emission are parallel at the back pupil of the microscope objective
  • To split emission from excitation, a dichroic mirror is used, Dependent on the wavelength of the light, this dichroic either is reflective or transmissive
  • If (as usual) the mirror lets pass longer wavelength, it is called long-pass, otherwise short-pass
  • Using a camera enables the detection of the light of many molecules simultaneously in parallel, saving measurement time
  • In Wide field microscopy the whole sample volume is illuminated, including molecules that are not in the focal plane (out-of-focus molecules), leading to strong background
  • Wide field microscopy is therefore mostly suited for thin film samples like bio membranes
  • In membranes, the motion of molecules can be followed (single molecule tracing/tracking)
  • The temporal resolution is limited by the readout speed of the camera, down to 30 microseconds!
26
Q

TIRF microscopy

A
  • TIRF stands for TOTAL INTERNAL REFLECTION
  • If light is incident to an interface from a medium with higher refractive index to a medium with lower refractive index, for an angle of incidence above the critical angle it gets totally reflected
  • In TIRF microscopy, the total reflection occurs at the interface between glass (with n=1,518) and water (with n=1,33) with a critical angle of alpha=62°
  • Only fluorophors in the evanescent field on the water side get excited
  • The emission of these fluorophores is not isotropic: The larger part of the emission is directed towards the glass!
  • There are two realizations of TIRF microscopy: prism type and objective type TIRF
27
Q

Prism TIRF

A
  • Here, as the name says, the sample is illuminated via a prism (not epifluorescence)
  • Fluorescence is detected from the other side through the water (water immersion objective!)
  • Adavantages:
    + No dichroic necessary
    + Any angle of incidence can be realized: very flexible
    Disadvantages:
  • emission is detected from the “wrong” side, reducing the signal
28
Q

Objective TIRF

A
  • Emission laser is focussed in an epifluorescence scheme in the back plane of the microscope objective at the very rim of the aperture
  • collimated (parallel) light is leaving the objective under an angle limited by the numerical aperture of the oil immersion objective
29
Q

Confocal microscopy

A
  • In a confical microscope, there’s point illumination and point detector, and the illumination point and the detection point are focussed into one point in the sample, thus the term con focal
  • The main advantage is an enormous reduction in the background signal
  • The main disadvantage is that there is no parallel detection possible any more
  • For Imaging, the confocal point has to be scanned with respect to the sample
  • To this end, either the confocal point (laser scanning) or the sample (sample or stage scanning) has to be moved
  • The advantage of laser scanning is that the imaging area can be changed by choosing the appropriate microscope objective magnification
  • Also, bulky sample chambers can be used
  • Typically, also higher scanning speeds are achievable
  • The main disadvantage of laser scanning is that the microscope objective is an angle between the light pass and the optical axis, which gives rise to optical aberrations
  • In turn, in stage scanning the light pass is always parallel to the optical axis
  • Here, however, the field of view is limited by the scan range of the scanning stage, which is typically 100 µM in x and y
  • Also, bulky sample chambers like cryostats cannot be moved by stage scanners
30
Q

Light sources

A
  • Since single molecule fluorescence is very demanding with respect to background suppression, only spectrally very clean excitation light sources can be used
  • Therefore, lasers are the light sources of choice
  • This is particularly the case in confocal microscopes, since lasers can be focussed down to a diffraction limited spot and thus can be efficiently coupled into optical fibers
31
Q

Filters

A

In Single molecule fluorescence, the optical filters should have the following characteristics:
- High transmission in the pass region (>90%)
- An optical density in the blocking region of >OD 5 (i.e. 10^-5 or lower transmission)
- steep transitions between pass/blocking regions

Strukturanalyse (11 decks)

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