1
Q
Instrumentation
A
BASIS: EPI FLUORESCENCE MICROSCOPE
- wide field microscopy
- TIRF microscopy
- confocal microscopy
- light sources
- filters
- detection
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)
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
4
Q
Prism TIRF
A
-> NOT Epi fluorescence
+ Flexible choice for angle of incidence (air)
- Emission into “wrong” direction: low signal
5
Q
Objective TIRF
A
+Detection from the „right side“: High signal
- angle of incidence limited (e.g. N.A.=1.4, 67°)
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)
7
Q
Light sources (Laser)
A
- Mode of operation
-> Cw (continuous wave)
-> Pulsed (pulse durations ≈ 100 ps) for lifetime, pulsed interleaved excitation
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
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
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
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
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
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
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)
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
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
Wide field epileptic fluorescence microscopy
- 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
TIRF microscopy
- 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
Prism TIRF
- 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
Objective TIRF
- 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
Confocal microscopy
- 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
Light sources
- 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
Filters
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
