TCSPC for FLIM and FRET in - Boston Electronics Corporation

Therefore, the fluorescence light need not be fed back through the scanner and through the pinhole.It can be diverted by a dichroic mirror directly behind the microscope objective. This setup is called‘non-descanned detection’ in contrast to the ‘descanned detection’ shown above.Two-photon excitation in conjunction with non-descanned detection can be used to image tissuelayers as deep as 500 µm. Since the scattering and the absorption at the wavelength of the twophotonexcitation are small the laser beam penetrates through relatively thick tissue. Even if there issome loss on the way through the tissue it can easily compensated by increasing the laser power.The increased power does not cause much photodamage because the power density outside thefocus is small. However, as long as there are enough ballistic (non-scattered) excitation photons inthe focus the fluorescence is excited. Of course, the fluorescence photons are heavily scattered ontheir way out of the tissue and therefore emerge from a relatively large area of the sample surfacewhich is out of the focus of the objective. However, for non-descanned detection there is no need tofocus the fluorescence light anywhere. Therefore the fluorescence photons can be efficientlytransferred to the detector.It is sometimes believed that lifetime imaging is somehow connected to two-photon excitation. Thisis, of course, not correct. Depending on the signal processing technique used, lifetime imagingrequires a pulsed or modulated laser. Although a Ti:Sa laser is the ideal source lifetime imaging ispossible with the frequency doubled Ti:Sa laser, with pulsed diode lasers, or with modulated CWlasers.Optical Near-Field MicroscopyThe optical near-field microscope (SNOM or NSOM) combines the principles of the atomic forcemicroscope and the laser scanning microscope [24-28]. A sharp tip is scanned over the sample andkept in a distance comparable to the diameter of a single molecule. The tip can be the end of atapered fibre through which the laser is fed to the sample (fig. 7, left). Or, the tip is illuminated byfocusing the laser through the microscope objective on it and the evanescent field at the tip is usedto probe the sample structure (fig. 7, right). In any case, the fluorescence photons are collectedthrough the microscope objective.leverFibreLaserleverscanSamplescanSampleDetectionMicroscopeObjectiveDetectionMicroscopeObjectiveLaserFig. 7: Optical near-field microscopeThe optical near-field microscope reaches a resolution of a few 10 nm, i.e. about 10 times less thanthe laser scanning microscope. Imaging cells with this technique is difficult and possibly restrictedto special cases [27,28]. The realm of the optical near-field microscope are certainly applicationswhere fluorescing molecules or nano-particles are fixed on a flat substrate.Generally, the SNOM principle can be combined with flourescence lifetime imaging in the sameway as the normal laser scanning microscope. Since only a small number of photons can beobtained from the extremely small sample volume photon counting techniquesare used to obtainlifetime information [78]. The problem to be expected is that the proximity of the tip changes the7