3.1.

Stray Light Suppression

Non-descanned camera detection in multifocal multiphoton microscopy offers the advantage of excellent fluorescence photon statistics, as many excitation foci contribute to the generation of a fluorescence image, while excitation energy remains below the damage threshold of the sample. However, when fluorescence light is generated in deep optical planes inside the sample, it can be scattered on its detection path within the sample. A high ratio of ballistic to scattered fluorescence photons is crucial for crisp non-descanned camera images, as scattered photons are responsible for image haze, which eventually can mask faint sample details.

In our parallel descanned concept, scattered fluorescence is blocked by detection pinholes, allowing only ballistic photons to reach the PMT segments. Figure 2 shows two-photon induced autofluorescence images of the identical optical plane $45\mathrm{\mu }\mathrm{m}$ inside a Ficus benjamini leaf and provides a typical example of the blur effects described earlier. Whereas Fig. 2a has been taken in non-descanned camera detection, Fig. 2b was measured in parallel descanned detection mode, using 0.4-mm-diam detection pinholes for optimal scattered fluorescence light suppression. In order to quantify the gain in contrast, two vertical intensity profiles (Fig. 3 ) have been extracted from both images, as indicated by the two lines in Fig. 2. Both profiles have been normalized to show the actual image dynamics. From the profiles in Fig. 3, it is evident that the parallel descanned detection image [Fig. 2b] yields a higher pixel dynamics (i.e., granular chloroplasts), a larger absolute minimum-maximum ratio, and steeper edge slopes at large, distinct image features. (Note the two organelles in Fig. 2 indicated by arrows and their corresponding fluorescence intensities located at 70 to $75\mathrm{\mu }\mathrm{m}$ and at 225 to $240\mathrm{\mu }\mathrm{m}$ in Fig. 3.)

Fig. 2

Multifocal two-photon (800-nm) excited autofluorescence of Ficus benjamini leaf $45\mathrm{\mu }\mathrm{m}$ inside the sample in minimum-maximum representation: (a) non-descanned camera detection; (b) 32-fold parallel descanned detection.

Fig. 3

Fluorescence intensity profile along vertical lines in Fig. 2.

In conclusion, descanned detection in combination with pinholes sufficiently suppresses sample scattered fluorescence and therefore allows high-contrast multifocal induced fluorescence imaging of deep²⁵ optical planes. However, this is achieved by blocking unwanted fluorescence light so that the absolute fluorescence intensity is generally much lower than compared to high Q.E. CCD cameras. It is interesting to note that the depth limitation of this imaging concept is determined by the complete loss of ballistic fluorescence signal and not by the ratio of scattered to ballistic photons as in non-descanned camera detection (see Fig. 2).

3.2.

Multiphoton Confocal Detection

In order to measure point spread functions (PSFs), we have imaged fluorescent beads with a diameter below the instrument’s resolution (FluoSpheres 505/515, Invitrogen, $d=175\mathrm{nm}$ ) that have been immobilized on a glass coverslip and submerged in water. Special care has been taken to properly adjust the coverslip correction of the UplanApo objective lens. In the confocal parallel descanned detection, the pinhole size was set to $0.4\mathrm{mm}$ , significantly smaller than the magnified diameter of the airy disk for a 515-nm point source $(d=0.985\mathrm{mm})$ to ensure comparability with the theoretical values calculated in Table 1 . Typical results of these measurements are presented in Fig. 4 . Both axial and lateral PSFs of the confocal parallel descanned detection clearly show improved full width at half maximum (FWHM) when compared to a theoretical two-photon excitation PSF as well as to an experimental PSF in the non-descanned detection mode. Measurement data have been fitted to a Gauss profile,²⁶ and their FWHM has been calculated from the Gauss fit. The optical resolutions have been calculated for an emission wavelength of $515\mathrm{nm}$ , assuming Gauss profiles and applying the Rayleigh criterion (contrast 26.4%). The corresponding numbers are listed in Table 1. From Table 1, it can be concluded that experimental and corresponding theoretical FWHM values match very well. Therefore, in this measurement configuration (excitation: $800\mathrm{nm}$ ; emission: $515\mathrm{nm}$ ; NA: 1.2; and refractive index: 1.33), axial and lateral resolutions of the confocal parallel non-descanned detection mode are improved by 35% and 21% (theoretically possible: 36% and 37%), respectively, compared to the non-descanned PMT detection mode. This improvement to $735\mathrm{nm}$ (theoretical: $716.5\mathrm{nm}$ ) in the axial and to $316.4\mathrm{nm}$ (theoretical: $255\mathrm{nm}$ ) in the lateral case is due to the use of pointlike detectors rather than finite-size detectors, which to our best knowledge has not been experimentally implemented in multifocal or line scanning setups to date. The slight broadening of the PSFs’ experimental values in comparison to the theoretical ones is probably due to imperfect overfilling of the back aperture of the objective lens, aberrations in the magnifying optics in the parallel confocal detection case, and imperfect image shift vectors, which especially effect lateral PSFs.

Fig. 4

Axial (a) and lateral (b) point spread functions, theoretical curves calculated for high aperture objective lens^{27, 28} (NA 1.2), experimental PSF measured with two-photon excitation $\left(800\mathrm{nm}\right)$ of fluorescent beads (emission maximum $515\mathrm{nm}$ ) in water (refractive index $n=1.33$ ).

To demonstrate the resolution improvements, we performed three-dimensional (3-D) fluorescence measurements of a fixed pollen sample. Especially when examining optical planes above the massive core body of the pollen (see Fig. 5 ), improved resolution of the confocal parallel descanned detection became apparent: while the non-descanned detection mode [Fig. 5b] suffers from significant fluorescent background of the pollen body, the confocal parallel descanned detection filters out this background due to its improved z-resolution. This becomes obvious when comparing the two normalized intensity profiles in Fig. 6 , which have been derived from the fluorescence intensity data presented in Fig. 5a and 5b. For the confocal parallel descanned detection mode in this measurement, fluorescence signal-to-noise and signal-to-background are improved by 200% and up to 350%, respectively.

Fig. 5

Two-photon (800-nm) excited fluorescence of pollen. Optical planes (a) and (b) are located $1.5\mathrm{\mu }\mathrm{m}$ above the massive central pollen body; spikes of pollen penetrate the optical plane. The dashed line indicates the direction of the profile in (a) confocal parallel descanned detection and (b) single beam non-descanned detection. (c) Voxel representation of complete 3-D confocal parallel descanned detection dataset; black line indicates the direction of the profiles in Fig. 6.

Fig. 6

Normalized fluorescence intensity profiles of pollen as indicated in Fig. 5. Note the higher minimum-maximum ratio and the steeper slopes in the confocal parallel descanned detection mode.

Table 1

Comparison of point spread functions of fluorescent beads (diam 175nm , emission maximum 515nm , two-photon excitation 800nm , NA 1.2, n=1.33 ).

3.3.

Detection Efficiency

In order to quantify improvements in the photon statistics, we measured fluorescence intensities of $1\mathrm{mM}$ Rhodamin 6G dissolved in water in descanned and non-descanned detection modes upon using the identical detector (H-7422-40) and experimental parameters ( $60\times$ objective lens, acquisition time, laser power, etc.). In both cases, detection efficiencies are similar when placing the detector in the focal plane of the first $(f=300\mathrm{mm})$ imaging lens, resulting in a 360-fold magnification of the exciting focus, which means that descanned detection in our setup does not result in a loss of signal intensity for ballistic photons.

When comparing descanned detection between the previously mentioned 360-fold magnification and the necessary 1880-fold (627-fold for the $20\times$ objective lens) magnification, we measured a loss in signal of approximately 23% with the multi-anode PMT (H7260-01) for a single beam. This loss is due to aberrations in the magnifying optics that image outside the active detection area $(0.8\mathrm{mm}\times 8\mathrm{mm})$ of the PMT. Further losses in fluorescence intensity occur when introducing the pinhole array in front of the PMT, i.e., a total loss of a factor of 4 for pinholes with a diameter of $0.8\mathrm{mm}$ . These losses are reciprocally dependent on the size of the pinholes. However, pinholes are necessary to block out scattered light from all the other fluorescent foci. This means that choosing a particular set of pinholes is a trade-off between signal intensity and suppression of scattered light. Therefore, it is also possible to choose pinholes with a larger diameter (e.g., $1\mathrm{mm}$ ), which would increase the throughput significantly at the expense of resolution but still keep a comparable level of scattering suppression.

Furthermore, when comparing parallel descanned detection to single-beam non-descanned detection, it has to be noted that the high-efficiency H-7422-40 PMT single-beam detector’s Q.E. is roughly twice the Q.E. of the H7260-01 multi-anode PMT.

Therefore, the advantage of the 32-fold generation of fluorescence intensity in parallel descanned detection with 0.8-mm-diam pinholes and the $60\times$ objective lens is compromised by a factor of 8 (factor 4 due to pinhole blocking and factor 2 due to Q.E.). This results in a four-fold increase in photon statistics for ballistic photons in parallel descanned detection compared to non-descanned single-beam detection.

The efficiency for the two detection methods can be compared when assuming that the detected fluorescence intensity ${\mathbf{I}}_{\mathbf{sb}}={\mathbf{I}}_{\mathbf{s}}+{\mathbf{I}}_{\mathbf{b}}$ for non-descanned single-beam detection is composed of all scattered ${\mathbf{I}}_{\mathbf{s}}$ and ballistic ${\mathbf{I}}_{\mathbf{b}}$ fluorescence light, while the detected fluorescence intensity ${\mathbf{I}}_{\mathbf{pd}}$ for parallel descanned beam detection was measured to be ${\mathbf{I}}_{\mathbf{pd}}=4\bullet {\mathbf{I}}_{\mathbf{b}}$ . Hence, parallel descanned detection provides better photon statistics than non-descanned single-beam detection, as long as the ratio of scattered to ballistic photons remains smaller than a factor of 3.

Due to the higher Q.E. of the single-beam detector and losses due to the pinholes for parallel descanned detection, single-beam non-descanned detection is more appropriate for weak fluorescence applications, when the signal-to-noise ratio becomes the limiting factor of the measurement. Furthermore, this detection method is relatively insensitive to scattered fluorescence, so especially low fluorescence detection in scattering samples is best performed by non-descanned single-beam detection.

Therefore, one can conclude that parallel descanned detection is best suited for samples with a relatively high fluorescence level so that blocking of scattered fluorescence from deep imaging planes does not result in a complete loss of signal. In this operative range, parallel descanned detection has advantages over both single-beam non-descanned detection, because of improved photon statistics, which can be utilized to reduce image acquisition time, and multibeam non-descanned camera detection, because of drastically reduced scattering haze.