1. 6. Fluorescence Spectroscopy1

2. 6. Basic concepts in fluorescence spectroscopy
6.1 Stokes-Shift
= Stokes-Shift due to vibrational energy relaxation within electronic excited state
Energy differences between vibrational states which determine vibronic band intensities are very often the same for ground and electronic excited state
Emission spectrum = mirror image of absorption spectrum
Emission bands are shifted bathochromically i.e. to higher wavelengths 2

3. 6. Basic concepts in fluorescence spectroscopy
6.2 Fluorescence life-time
The following transitions will be considered: S1 T1 S0
: rate constant for radiative S1S0 decay via fluorescence;
: rate constant for internal conversion (S1S0);
: rate constant for intersystem crossing;
: rate constant for radiative decay via phosphorescence (T1S0);
: rate constant for non-radiative decay (T1S0).
Non radiative transitions originating from S1 are combined in: 3

4. 6. Basic concepts in fluorescence spectroscopy
6.2 Fluorescence life-time
Dilute solution of fluorescent species A.
Short d-laser pulse excites certain fraction of molecules A at t = 0.
Decay rate of excited molecules A*:
Integration: together with: number of molecules A promoted in the excited state at t = 0 and life-time of excited state S1:
Fluorescence intensity is number of photons emitted per time and volume:
Fluorescence intensity IF at time t after excitation by a short light pulse:
Part of molecules can end up in triplet state.
Life-time of triplet state is defined as:
1A*
1A + Photon 4

5. Integration over complete decay
6. Basic concepts in fluorescence spectroscopy
6.3 Fluorescence quantum yield
Fluorescence quantum yield: Emitted Photons per Excitation events
It follows:
The quantum yields for ISC and phosphorescence can be expressed in analogy:
Integration over complete decay
bzw. 5

6. 6. Basic concepts in fluorescence spectroscopy
6.3 Fluorescence quantum yield
Life-times & quantum yields
Attention:
Quantum yield is proportional to life-time
but
other non-radiative decay processes change lifetime
radiative rate depends on refractive index of medium 6

7. 6. Basic concepts in fluorescence spectroscopy
6.4 Steady-State fluorescence emission
It is advantageous to define the steady-state fluorescence per absorbed photon as photon flux in dependence of wavelength (Photon spectrum) :
Emission photon spectrum expresses the probability distribution of the different transitions from the vibrational ground state of S1 down to the various vibrational states of S0.
The normalized steady-state fluorescence IF(lF), recorded for the wavelength lF is proportional to as well as to the number of absorbed photons at the excitation wavelength lE.
Number of absorbed photons is given by:
Intensity
irradiated
transmitted 7

8. 6. Basic concepts in fluorescence spectroscopy
6.4 Steady-State fluorescence emission
Fluorescence intensity can be expressed as follows:
Considering the intensity of the transmitted light by Lambert-Beer‘s law yields:
Recording the intensity IF as function of the wavelength lF for a fixed excitation wavelength lE yields fluorescence spectrum.
For low concentrations it follows:
Higher terms can be neglected for diluted solutions.
Thus it follows: A(lE) = absorbance at lE
Proportionality between fluorescence intensity and concentration for diluted solutions only
with k = proportionality constant dependent on numerous experimental values like e.g. collection angle, band width of monochromator, slid width, etc. 8

9. 6. Basic concepts in fluorescence spectroscopy
6.4 Fluorescence excitation spectroscopy
Recording fluorescence intensity as function of excitation wavelength lE for a fixed observation wavelength lF yields fluorescence excitation spectrum.
According to: the fluorescence intensity recorded as a function of the excitation wavelength reflects the product
In case the wavelength dependency of the incoming light can be compensated the fluorescence excitation spectrum depends only on what corresponds to the absorption spectrum.
As long as only one ground state species exists the corrected excitation spectrum is identical to the absorption spectrum. Otherwise a comparison between fluorescence excitation and absorption spectrum yields valuable information about the sample species present. 9

10. 6. Basic concepts in fluorescence spectroscopy
6.4 Fluorescence excitation spectroscopy
Cinoxacin in H2O 10

11. 7. Fluorescence microscopy
7.1 Fluorochromes
Fluorescence microscopy differentiates between two kinds of fluorochromes:
Primary fluorescence (autofluorescence)
Secondary fluorescence (fluorochromation)
Fluorescence dyes
Immunofluorescence (using Antibodies)
Molecular tags (SNAP Tag, ...)
Fluorescent Proteins
Applications of fluorochromes
Identification of otherwise visible structures
Localization and identification of otherwise invisible structures
Monitoring of physiological processes
Specific detection of a protein
Using Photophysical properties of dyes (e.g. switching) for superresolution 11

12. Porphyrin ring – central unit in Chlorophyll
7. Fluorescence microscopy
7.1 Primary fluorescence (autofluorescence)
Most samples fluoresce when excited with short-wave light
Fluorescence very often occurs for systems containing many conjugated double bonds:
e.g. chlorophyll exhibits dark red fluorescence when excited by blue or red light
Porphyrin ring – central unit in Chlorophyll
Moss reeds – green excitation 12

13. 7. Fluorescence microscopy 13

14. 7. Fluorescence microscopy
7.1 Primary fluorescence (autofluorescence)
Further examples:
Riboflavine (550nm)
NAD(P)H (460nm, 400ps)
Elastin und Collagen ( nm)
Retinol (500nm)
Cuticula (blue)
Lignin (> 590nm)
DNA 390nm)
Aminoacids:
Tryptophane (348nm, 2.6ns)
Tyrosin (303nm, 3.6ns, weak)
Phenylalanine (282nm weak)
Resins, Oils
Eucalyptus leaf section – UV excitation
Nematode living sample – UV excitation 14

15. 7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation)
Staining (labeling) specific structures with fluorescent labels (dyes): fluorochromation
Small dye concentrations are sufficient due to high fluorescence contrast  fluorescence labels are superior than bright field dyes
Single molecule sensitivity
Fluorescence labels must selectively bind to structures or selectively accumulate in specific compartments
e.g. DAPI (= 4',6-diamidino-2-phenylindole) to label DNA (cell nuclei)
Fluorescence image of Endothelium cells. Microtubili are labeld in green, while actin filaments are labeled red. DNA within cell nuclei are stained with DAPI.
DAPI:
lexc = 358 nm lem = 461 nm 15

16. 7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer requires interaction between donor and acceptor
Emission spectrum of donor must overlap with absorption spectrum of acceptor.
Several vibronic transitions within donor have the same energy than in the acceptor
Resonant coupling of the transitions
RET = resonance energy transfer
Resonant transitions 16

17. 7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer
Assumption: 2 electrons one at the donor D and one at the acceptor A are involved in the transition:
Antisymmetric wavefunction (Fermions) for initially excited state i (D excited, but not A) and final state f (A excited, but not D): Overall Hamiltonian: Interaction energy: 
Coulomb term UC
Exchange term Uex 17

18. 7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer
Coulomb Interaction (CI)
Exchange Interaction 18

19. 7. Fluorescence microscopy
7.3 FRET microscopy
Radiationless excitation energy transfer
Different interaction mechanism lead to excitation energy transfer:
Dipolar (Förster)
„Long Range“
Coulomb interaction
Singlet
energy transfer
Multipolar
Electron exchange (Dexter)
„Short Range“
Inter molecular orbital overlap
Charge resonance interaction
Triplet
Energy transfer 19

20. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
D + hn1  D* Absorption
D* + A  A* + D Energy transfer
A*  A + hn2 Emission
The following conditions must hold:
D must be a fluorophore with sufficiently long life-time
Partial spectral overlap between emission spectrum of D and absorption spectrum of A
Transition dipole moments D and A must be oriented properly to each other;
Distance between D and A shouldn‘t be too large 20

21. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
Coulomb interaction can be developed in a multipole series in which the dipole term exhibits the term with the longest range
Energy transfer via dipole-dipole transfer has been first calculated by Förster and is therefore called Förster process
Energy transfer rate from molecule D to molecule A at a distance r:
kD = radiative decay rate of donor
tD0 = donor life-time in absence of energy transfer
r-6-dependency as a result of dipole-dipole interaction
R0 = critical distance or Förster-radius (distance at which intensity decrease caused by energy transfer and spontaneous decay are equal ( = kD)). 21

22. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
R0 can be determined via spectroscopic values:
For R0 in Å, l in nm, eA(l) in M-1 cm-1 (overlap integral in M-1 cm-1 nm4)
Typical values for Förster-radii R0, i.e. for distances, over which energy transfer is important lie in the range of Å
Overlap between fluorescence of donor and absorption of acceptor
k2 = orientational factor F0D = quantum yield of donor in absence of energy transfer n = average refractive index for wavelength area of spectral overlap ID(l) = normalized fluorescence spectrum of donor ( )
eA(l) = molar absorption coefficient of acceptor.  22

23. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
Transfer efficiency can be expressed by:
In combination with changed lifetime:
It follows:
distance dependency:
D und D0 are excited state life-times of donor in absence and presence of acceptor, respectively 23

24. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
Besides the distance between the two chromophores also the relative orientation of the transition dipole moments of the donor D and acceptor A plays a crucial role for the energy transfer efficiency
The orientation factor k2 is given by: mD mA
A: angle between D-A connecting line and acceptor transition dipole moment
D: angle between D-A connecting line and donor transition dipole moment
T: angle between donor and acceptor transition dipole moment 24

25. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
For systems where the orientation stays constant during the energy transfer (e.g. usage of highly viscose solvents or rigid coupling of chromophores to large and stiff molecules) k2 can reach values between 0 (transition dipole moments are orthogonal) and 4 (collinear arrangement); k2 = 1, for a parallel arrangement
If both acceptor and donor can rotate the orientational factor 2 must be replaced by an average value:
In case both chromophores undergo a fast isotropic rotation i.e. the rotation is considerably faster than the energy transfer rate the average orientation factor is given by k2 = 2/3
In case donor and acceptor are freely movable but the rotation is significantly slower than the energy transfer the orientation factor results in: 2 = 0.476 25

26. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
RET is utilized as „optical nano ruler“ (10 – 100 Å) in biochemistry and cell biology
Distance between donor and acceptor should be in the range of: because R0 is a benchmark for donor-acceptor distances which can be determined by FRET. 26

27. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
RET as „optical nano ruler“ in biochemistry and cell biology 27

28. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
RET as „optical nano ruler“ in biochemistry and cell biology 28

29. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
RET as „optical nano ruler“ in biochemistry and cell biology One requires appropriate method to label specific intracellular proteins with suitable fluorophores (fluorescent proteins genetics):
Green Fluorescent Protein (GFP) first isolated from the jellyfish Aequorea victoria
GFP can be combined with just about any other protein by attaching its gene to the gene of a target protein, thereby introducing it into a cell. Thus by recording the GFP fluorescence the spatial and temporal distribution of this target protein can be directly monitored in living cells, tissue and organism.
Several GFP mutants with altered fluorescence spectra exist. These mutants are named according to their color e.g. CFP (cyan) or YFP (yellow)
Excitation maxima at 395 und 475 nm Emission wavelength at 509 nm 29

30. Agar plate of fluorescent bacteria colonies
7. Fluorescence microscopy
7.3 FRET microscopy
Agar plate of fluorescent bacteria colonies 30

31. 7. Fluorescence microscopy
7.3 FRET microscopy
Förster Resonance Energy Transfer (very weak coupling):
RET as „optical nano ruler“ in biochemistry and cell biology : GFP-mutants
FRET
R0 = 4.7 – 4.9 nm
no FRET
protein folding
protein-protein interaction 31

32. 7. Fluorescence microscopy
7.3 FRET microscopy
Resolution of a light microscope is limited to several hundred nanometers (< organelles)
FRET allows detection of molecule-molecule interactions on a nanometer scale by means of a light microscope
Decrease of donor-emission
Increase of acceptor emission
Reduction of donor fluorescence life-time
Energy transfer (FRET-efficiency) depends strongly on donor-acceptor distance
R0 = Förster-radius (distance for which energy transfer is half maximal)
sensitized
emission 32

33. 7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging = acceptor emission at donor excitation (sensitized emission SAkzeptor) divided by donor emission at donor excitation (SDonor)
Advantages: Since both donor decrease as well as acceptor increase contribute to the signal the signal-to-noise ratio is better than for solely recording the acceptor fluorescence
SDonor
SAkzeptor 33

34. Excitation wavelength
7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – problems:
Correction for direct excitation of the acceptor when exciting donor (control measurement with YFP only) = correction factor rDE
Correction for bleedthrough : Portion of CFP in yellow channel for blue excitation in absence of FRET (acceptor) = bleedthrough of CFP in YFP-channel (rBT,CY)
or bleedthrough of YFP in CFP-channel (rBT,YC)
Excitation wavelength
FRET-detection channel 34

35. 7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – 3-filter-set:
Donor excitation and emission (ICFP,430)
Acceptor excitation and emission (IYFP,514)
Donor excitation and acceptor emission (IYFP,430)  35

36. 7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – 3-filter-set:
Model of FRET-detection of Src-Csk protein interaction (Src = protein tyrosine kinase Csk = C-terminal Src kinase)
Important signal transduction step during blood coagulation
No FRET 36

37. 7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – 3-filter-set: Visualization of Src-Csk-interaction during aIIbß3-induced fibrinogen adhesion in a thrombocyte model cell line (A5-CHO) by means of FRET
superposition 37

38. 7. Fluorescence microscopy
7.3 FRET microscopy
FRET ratio imaging – 3-filter-set: FRET for displaying Ca2+ in living cells via Yellow-Cameleon-2 (YC2) sensor
FRET-ratio image of HeLa-cells, expressing the YC2-sensor before and after adding ionomycin
FRET response of HEK/293 cells expressing YC2-seonsor after adding 1nM ionomycin and additional extracellular Ca21 (30 mM) 38

39. 7. Fluorescence microscopy
7.3 FRET microscopy
FRET fluorescence life-time microscopy: In case of FRET the donor fluorescence life-time is reduced. Determination of this donor life time reduction yields a quantitative FRET measurement which is independent of dye concentration or spectral contamination (crosstalk, bleedthrough).
Dimerization of C/EBP® – proteins in GHFT1-5 cell nuclei
(donor/acceptor CFP/YFP-C/EBP®) 39

40. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
Sample is excited by a short laser pulse
Sample molecules relax individually according to the transition probability of the different relaxation pathways to the ground state
Fluorescence intensity exhibits mono-, multi or non-exponential decay depending on nature and number of fluorescence contributions.
Laser pulse
Longer fluorescence life-time
Shorter fluorescence life-time 40

41. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
Intensity integrating measurements The determination of the fluorescence decay time ¿ or times ¿i and relative amplitudes ®i in case of multiple contributions is possible by recording the fluorescence signal for several measurement points after the excitation pulse. For a mono-exponential decay behavior or to determine the average decay time ¿ two sampling points are sufficient
For two times t1 and t2 after the excitation pulse the detector signal is integrated for a sampling window ¢ T. The ratio of the measurement signals D1 und D2 can be used to calculate the decay-time ¿ or the average decay-time ¿ : 41

42. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
Gated fluorescence detection Gated optical image intensifiers (GOI) are capable of taking pictures with high (sub-nanosecond) time resolution i.e. camera with ultrafast shutter (gate < 100 ps) which can be opened and closed for different delay-times after the sample has been excited with an ultrashort laser-pulse. By collecting a series of time-scanned fluorescence intensity images for different delay-times after excitation the fluorescence decay profile for every pixel in the field of view can be accessed and displayed as false color plot = fluorescence life-time image
gated optical image intensifiers (GOI)
Such devices are capable of taking pictures with high (sub-nanosecond) time resolution. This type of device is based on microchannel plate image intensifier technology, incorporating high speed voltage signals that effectively gate the gain of the image intensifier on very fast time scales. FIG. 1 of the accompanying drawings shows a simple schematic of such a gated image intensifier. A high speed voltage pulse is applied to an electrode mesh 2 in front of a photocathode 4 which induces a pulse on the photocathode 4 by capacitive coupling. When this pulsed voltage is present, the photoelectrons emitted by the photocathode 4 are accelerated toward the microchannel plate 6 and an amplified replica of the incident optical image is observed at the output phosphor screen 8. Typically this output image is recorded on a CCD camera and may be saved as an electronic record on a computer. Thus, by applying a series of short voltage pulses to the mesh 2, it is possible to obtain a series of time-gated images from the image intensifier 10. Because the gating voltage pulse applied to the mesh 2 can be very short, it is possible to gate this image intensifier 10 in less than 100 ps. A slightly different design, in which the gating voltage is applied directly to the photocathode 4, is able to provide gated imaging on timescales as fast as 200 ps. This mode of operation is able to run at repetition rates up to .about.1 GHZ, and is described as a high rate imager (HRI). 42

43. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
Gated fluorescence detection
(a) (b) (c)
(Top) FLIM image of an unstained human pancreas section (tissue autofluorescence) with an endocrine tumor
(below) Brightfield image of the same section after conventional histopathological staining
Tissue section of a rat ear: (a) Brightfield microscopy image stained with orcein (b) Fluorescence intensity- and (c) FLIM images of an unstained parallel sample (tissue autofluorescence) (excitation 410 nm; FLIM false color plot from 200 ps (blue) to 1800 ps (red) 43

44. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting In case of intensive excitation light many electrons of the dye are getting excited for every laser pulse i.e. the average life-time can be deduced from the fluorescence decay-time after every pulse (multiple photon emission).
A common FLIM method is the measurement of the life-time for single fluorescence photons. In doing so the dye is excited by light pulses of extremely low intensity in a way that at most one electron per pulse gets excited. The individual life- time of every photon is measured and the average life-time is determined staistically. 44

45. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting
Detection of single photons of a periodic light signal
Light intensity is so weak, that the probability to detect a photon within one period is very small.
Periods with more than one photon are extremely rare
For every detected photon its delay time with respect to the excitation pulse is determined
A delay distribution builds up over many pulse
Time resolution up to 25 ps 45

46. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting
Probe
Stop watch = TAC: Time-to-Amplitude Converter converts time between a start and a stop pulse by charging a capacitor with constant current
Start can be reference (from laser) and photon is stop -> Problem is loss of much time (due to reset time) -> Reverse counting (start = photon, stop=next laser pulse)
Histogram of arrival times after excitation -> fluorescence life-time. 46

47. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Time-resolved measurements
TCSPC = time-correlated single photon counting
Fluorescence intensity image of a vacuole which is labeled by
fluorescent phospholipids
FLIM image and corresponding distribution of life-times. Long life-times (red) are found in the cell membrane while the cytoplasma exhibits shorter life-times pointing towards a less ordered environment. 47

48. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Steady state measurements
Phase modulation Intensity of a continuous wave (CW) source is modulated at high frequency by a standing wave acousto-optic modulator (n= 2¼ ¼ 50 MHz) which will modulate the excitation intensity at double frequency. Detected fluorescence is modulated at the same frequency. The observed phase shift with respect to the excitation and the modulation depth M (ratio of Ac signal to DC signal) depends on the fluorescence life-time of the excited fluorophores. Fluorescence lifetimes tphase and tphase can be calculated and should be identical for single exponential decays.
The intensity of a continuous wave source is modulated at high frequency, by an acousto-optic modulator for example, which will modulate the fluorescence. Since the excited state has a lifetime, the fluorescence will be delayed with respect to the excitation signal, and the lifetime can be determined from the phase shift. Also, y-components to the excitation and fluorescence sine waves will be modulated, and lifetime can be determined from the modulation ratio of these y-components. Hence, 2 values for the lifetime can be determined from the phase-modulation method. 48

49. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Steady state measurements
Phase modulation
Measurement values: Demodulation (modulation depth) M Phase shift DF
Modulation of excitation light with n, which is characterized by modulation depth ME = a/d and FE :
leads to an accordingly modulated fluorescence signal F(t) with demodulation MF = A/D und phase FF
Excitation light
Intensity
Fluorescence
Time 49

50. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Steady state measurements
Phase modulation
Rate equation of change of number of excited molecules
F(t) ~ N(t)  Relationship between fluorescence life-time and fluorescence emission behavior upon intensity modulated excitation light
Relationship between measurement parameters: M = MF / ME as well as DF = FE - FE and life-time t :
Absorption rate: 50

51. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Steady state measurements
Phase modulation Continuous intensity modulated excitation of fluorescence transforms the determination of fluorescence decay-times to measurements of phase shifts and demodulation of the fluorescence signal
Demodulation M and phase shift DF of the fluorescence depend on the fluorescence life-time t as well as on the modulation frequency w = 2p n of the excitation light. The simulation shows the dependency of M and DF for a decay time of t = 4 ns and a frequency of n = 40 MHz
 Choosing frequency at wt  1 51

52. 7. Fluorescence microscopy
7.4 Fluorescence Life-Time Imaging Microscopy (FLIM)
Methods of time-resolved fluorescence diagnostics
Steady state measurements
Phase modulation
Frozen section of portio biopsies in the spectral region of lEm>500 nm (lexc = 457 nm; n = 40 MHz) Top right: Fluorescence intensity
Bottom right: corresponding HE stain image. 52